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Lactic Acid Bacteria

Copyright © 2004 by Marcel Dekker, Inc. All Rights Reserved.

Food Science and Technology
EDITORIAL BOARD Senior Editors Owen R. Fennema—University of Wisconsin–Madison Y. H. Hui—Science Technology System Marcus Karel—Rutgers University (emeritus) Pieter Walstra—Wageningen University John R. Whitaker—University of California–Davis

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A Series of Monographs, Textbooks, and Reference Books

Additives: P. Michael Davidson—University of Tennessee–Knoxville Dairy science: James L. Steele—University of Wisconsin–Madison Flavor chemistry and sensory analysis: John H. Thorngate III—University of California–Davis Food engineering: Daryl B. Lund—University of Wisconsin–Madison Food lipids and flavors: David B. Min—Ohio State University Food proteins/food chemistry: Rickey Y. Yada—University of Guelph Health and disease: Seppo Salminen—University of Turku, Finland Nutrition and nutraceuticals: Mark Dreher—Mead Johnson Nutritionals Phase transition/food microstructure: Richard W. Hartel—University of Wisconsin–Madison Processing and preservation: Gustavo V. Barbosa-Cánovas—Washington State University–Pullman Safety and toxicology: Sanford Miller—University of Texas–Austin

1. 2. 3. 4.

5. 6. 7.

Flavor Research: Principles and Techniques, R. Teranishi, I. Hornstein, P. Issenberg, and E. L. Wick Principles of Enzymology for the Food Sciences, John R. Whitaker Low-Temperature Preservation of Foods and Living Matter, Owen R. Fennema, William D. Powrie, and Elmer H. Marth Principles of Food Science Part I: Food Chemistry, edited by Owen R. Fennema Part II: Physical Principles of Food Preservation, Marcus Karel, Owen R. Fennema, and Daryl B. Lund Food Emulsions, edited by Stig E. Friberg Nutritional and Safety Aspects of Food Processing, edited by Steven R. Tannenbaum Flavor Research: Recent Advances, edited by R. Teranishi, Robert A. Flath, and Hiroshi Sugisawa

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8. Computer-Aided Techniques in Food Technology, edited by Israel Saguy 9. Handbook of Tropical Foods, edited by Harvey T. Chan 10. Antimicrobials in Foods, edited by Alfred Larry Branen and P. Michael Davidson 11. Food Constituents and Food Residues: Their Chromatographic Determination, edited by James F. Lawrence 12. Aspartame: Physiology and Biochemistry, edited by Lewis D. Stegink and L. J. Filer, Jr. 13. Handbook of Vitamins: Nutritional, Biochemical, and Clinical Aspects, edited by Lawrence J. Machlin 14. Starch Conversion Technology, edited by G. M. A. van Beynum and J. A. Roels 15. Food Chemistry: Second Edition, Revised and Expanded, edited by Owen R. Fennema 16. Sensory Evaluation of Food: Statistical Methods and Procedures, Michael O’Mahony 17. Alternative Sweeteners, edited by Lyn O’Brien Nabors and Robert C. Gelardi 18. Citrus Fruits and Their Products: Analysis and Technology, S. V. Ting and Russell L. Rouseff 19. Engineering Properties of Foods, edited by M. A. Rao and S. S. H. Rizvi 20. Umami: A Basic Taste, edited by Yojiro Kawamura and Morley R. Kare 21. Food Biotechnology, edited by Dietrich Knorr 22. Food Texture: Instrumental and Sensory Measurement, edited by Howard R. Moskowitz 23. Seafoods and Fish Oils in Human Health and Disease, John E. Kinsella 24. Postharvest Physiology of Vegetables, edited by J. Weichmann 25. Handbook of Dietary Fiber: An Applied Approach, Mark L. Dreher 26. Food Toxicology, Parts A and B, Jose M. Concon 27. Modern Carbohydrate Chemistry, Roger W. Binkley 28. Trace Minerals in Foods, edited by Kenneth T. Smith 29. Protein Quality and the Effects of Processing, edited by R. Dixon Phillips and John W. Finley 30. Adulteration of Fruit Juice Beverages, edited by Steven Nagy, John A. Attaway, and Martha E. Rhodes 31. Foodborne Bacterial Pathogens, edited by Michael P. Doyle 32. Legumes: Chemistry, Technology, and Human Nutrition, edited by Ruth H. Matthews 33. Industrialization of Indigenous Fermented Foods, edited by Keith H. Steinkraus 34. International Food Regulation Handbook: Policy • Science • Law, edited by Roger D. Middlekauff and Philippe Shubik 35. Food Additives, edited by A. Larry Branen, P. Michael Davidson, and Seppo Salminen 36. Safety of Irradiated Foods, J. F. Diehl 37. Omega-3 Fatty Acids in Health and Disease, edited by Robert S. Lees and Marcus Karel 38. Food Emulsions: Second Edition, Revised and Expanded, edited by Kåre Larsson and Stig E. Friberg 39. Seafood: Effects of Technology on Nutrition, George M. Pigott and Barbee W. Tucker 40. Handbook of Vitamins: Second Edition, Revised and Expanded, edited by Lawrence J. Machlin 41. Handbook of Cereal Science and Technology, Klaus J. Lorenz and Karel Kulp

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42. Food Processing Operations and Scale-Up, Kenneth J. Valentas, Leon Levine, and J. Peter Clark 43. Fish Quality Control by Computer Vision, edited by L. F. Pau and R. Olafsson 44. Volatile Compounds in Foods and Beverages, edited by Henk Maarse 45. Instrumental Methods for Quality Assurance in Foods, edited by Daniel Y. C. Fung and Richard F. Matthews 46. Listeria, Listeriosis, and Food Safety, Elliot T. Ryser and Elmer H. Marth 47. Acesulfame-K, edited by D. G. Mayer and F. H. Kemper 48. Alternative Sweeteners: Second Edition, Revised and Expanded, edited by Lyn O’Brien Nabors and Robert C. Gelardi 49. Food Extrusion Science and Technology, edited by Jozef L. Kokini, Chi-Tang Ho, and Mukund V. Karwe 50. Surimi Technology, edited by Tyre C. Lanier and Chong M. Lee 51. Handbook of Food Engineering, edited by Dennis R. Heldman and Daryl B. Lund 52. Food Analysis by HPLC, edited by Leo M. L. Nollet 53. Fatty Acids in Foods and Their Health Implications, edited by Ching Kuang Chow 54. Clostridium botulinum: Ecology and Control in Foods, edited by Andreas H. W. Hauschild and Karen L. Dodds 55. Cereals in Breadmaking: A Molecular Colloidal Approach, Ann-Charlotte Eliasson and Kåre Larsson 56. Low-Calorie Foods Handbook, edited by Aaron M. Altschul 57. Antimicrobials in Foods: Second Edition, Revised and Expanded, edited by P. Michael Davidson and Alfred Larry Branen 58. Lactic Acid Bacteria, edited by Seppo Salminen and Atte von Wright 59. Rice Science and Technology, edited by Wayne E. Marshall and James I. Wadsworth 60. Food Biosensor Analysis, edited by Gabriele Wagner and George G. Guilbault 61. Principles of Enzymology for the Food Sciences: Second Edition, John R. Whitaker 62. Carbohydrate Polyesters as Fat Substitutes, edited by Casimir C. Akoh and Barry G. Swanson 63. Engineering Properties of Foods: Second Edition, Revised and Expanded, edited by M. A. Rao and S. S. H. Rizvi 64. Handbook of Brewing, edited by William A. Hardwick 65. Analyzing Food for Nutrition Labeling and Hazardous Contaminants, edited by Ike J. Jeon and William G. Ikins 66. Ingredient Interactions: Effects on Food Quality, edited by Anilkumar G. Gaonkar 67. Food Polysaccharides and Their Applications, edited by Alistair M. Stephen 68. Safety of Irradiated Foods: Second Edition, Revised and Expanded, J. F. Diehl 69. Nutrition Labeling Handbook, edited by Ralph Shapiro 70. Handbook of Fruit Science and Technology: Production, Composition, Storage, and Processing, edited by D. K. Salunkhe and S. S. Kadam 71. Food Antioxidants: Technological, Toxicological, and Health Perspectives, edited by D. L. Madhavi, S. S. Deshpande, and D. K. Salunkhe 72. Freezing Effects on Food Quality, edited by Lester E. Jeremiah 73. Handbook of Indigenous Fermented Foods: Second Edition, Revised and Expanded, edited by Keith H. Steinkraus 74. Carbohydrates in Food, edited by Ann-Charlotte Eliasson

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75. Baked Goods Freshness: Technology, Evaluation, and Inhibition of Staling, edited by Ronald E. Hebeda and Henry F. Zobel 76. Food Chemistry: Third Edition, edited by Owen R. Fennema 77. Handbook of Food Analysis: Volumes 1 and 2, edited by Leo M. L. Nollet 78. Computerized Control Systems in the Food Industry, edited by Gauri S. Mittal 79. Techniques for Analyzing Food Aroma, edited by Ray Marsili 80. Food Proteins and Their Applications, edited by Srinivasan Damodaran and Alain Paraf 81. Food Emulsions: Third Edition, Revised and Expanded, edited by Stig E. Friberg and Kåre Larsson 82. Nonthermal Preservation of Foods, Gustavo V. Barbosa-Cánovas, Usha R. Pothakamury, Enrique Palou, and Barry G. Swanson 83. Milk and Dairy Product Technology, Edgar Spreer 84. Applied Dairy Microbiology, edited by Elmer H. Marth and James L. Steele 85. Lactic Acid Bacteria: Microbiology and Functional Aspects: Second Edition, Revised and Expanded, edited by Seppo Salminen and Atte von Wright 86. Handbook of Vegetable Science and Technology: Production, Composition, Storage, and Processing, edited by D. K. Salunkhe and S. S. Kadam 87. Polysaccharide Association Structures in Food, edited by Reginald H. Walter 88. Food Lipids: Chemistry, Nutrition, and Biotechnology, edited by Casimir C. Akoh and David B. Min 89. Spice Science and Technology, Kenji Hirasa and Mitsuo Takemasa 90. Dairy Technology: Principles of Milk Properties and Processes, P. Walstra, T. J. Geurts, A. Noomen, A. Jellema, and M. A. J. S. van Boekel 91. Coloring of Food, Drugs, and Cosmetics, Gisbert Otterstätter 92. Listeria, Listeriosis, and Food Safety: Second Edition, Revised and Expanded, edited by Elliot T. Ryser and Elmer H. Marth 93. Complex Carbohydrates in Foods, edited by Susan Sungsoo Cho, Leon Prosky, and Mark Dreher 94. Handbook of Food Preservation, edited by M. Shafiur Rahman 95. International Food Safety Handbook: Science, International Regulation, and Control, edited by Kees van der Heijden, Maged Younes, Lawrence Fishbein, and Sanford Miller 96. Fatty Acids in Foods and Their Health Implications: Second Edition, Revised and Expanded, edited by Ching Kuang Chow 97. Seafood Enzymes: Utilization and Influence on Postharvest Seafood Quality, edited by Norman F. Haard and Benjamin K. Simpson 98. Safe Handling of Foods, edited by Jeffrey M. Farber and Ewen C. D. Todd 99. Handbook of Cereal Science and Technology: Second Edition, Revised and Expanded, edited by Karel Kulp and Joseph G. Ponte, Jr. 100. Food Analysis by HPLC: Second Edition, Revised and Expanded, edited by Leo M. L. Nollet 101. Surimi and Surimi Seafood, edited by Jae W. Park 102. Drug Residues in Foods: Pharmacology, Food Safety, and Analysis, Nickos A. Botsoglou and Dimitrios J. Fletouris 103. Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection, edited by Luis M. Botana 104. Handbook of Nutrition and Diet, Babasaheb B. Desai 105. Nondestructive Food Evaluation: Techniques to Analyze Properties and Quality, edited by Sundaram Gunasekaran 106. Green Tea: Health Benefits and Applications, Yukihiko Hara 107. Food Processing Operations Modeling: Design and Analysis, edited by Joseph Irudayaraj

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108. Wine Microbiology: Science and Technology, Claudio Delfini and Joseph V. Formica 109. Handbook of Microwave Technology for Food Applications, edited by Ashim K. Datta and Ramaswamy C. Anantheswaran 110. Applied Dairy Microbiology: Second Edition, Revised and Expanded, edited by Elmer H. Marth and James L. Steele 111. Transport Properties of Foods, George D. Saravacos and Zacharias B. Maroulis 112. Alternative Sweeteners: Third Edition, Revised and Expanded, edited by Lyn O’Brien Nabors 113. Handbook of Dietary Fiber, edited by Susan Sungsoo Cho and Mark L. Dreher 114. Control of Foodborne Microorganisms, edited by Vijay K. Juneja and John N. Sofos 115. Flavor, Fragrance, and Odor Analysis, edited by Ray Marsili 116. Food Additives: Second Edition, Revised and Expanded, edited by A. Larry Branen, P. Michael Davidson, Seppo Salminen, and John H. Thorngate, III 117. Food Lipids: Chemistry, Nutrition, and Biotechnology: Second Edition, Revised and Expanded, edited by Casimir C. Akoh and David B. Min 118. Food Protein Analysis: Quantitative Effects on Processing, R. K. OwusuApenten 119. Handbook of Food Toxicology, S. S. Deshpande 120. Food Plant Sanitation, edited by Y. H. Hui, Bernard L. Bruinsma, J. Richard Gorham, Wai-Kit Nip, Phillip S. Tong, and Phil Ventresca 121. Physical Chemistry of Foods, Pieter Walstra 122. Handbook of Food Enzymology, edited by John R. Whitaker, Alphons G. J. Voragen, and Dominic W. S. Wong 123. Postharvest Physiology and Pathology of Vegetables: Second Edition, Revised and Expanded, edited by Jerry A. Bartz and Jeffrey K. Brecht 124. Characterization of Cereals and Flours: Properties, Analysis, and Applications, edited by Gönül Kaletunç and Kenneth J. Breslauer 125. International Handbook of Foodborne Pathogens, edited by Marianne D. Miliotis and Jeffrey W. Bier 126. Food Process Design, Zacharias B. Maroulis and George D. Saravacos 127. Handbook of Dough Fermentations, edited by Karel Kulp and Klaus Lorenz 128. Extraction Optimization in Food Engineering, edited by Constantina Tzia and George Liadakis 129. Physical Principles of Food Preservation: Second Edition, Revised and Expanded, Marcus Karel and Daryl B. Lund 130. Handbook of Vegetable Preservation and Processing, edited by Y. H. Hui, Sue Ghazala, Dee M. Graham, K. D. Murrell, and Wai-Kit Nip 131. Handbook of Flavor Characterization: Sensory Analysis, Chemistry, and Physiology, edited by Kathryn D. Deibler and Jeannine Delwiche 132. Food Emulsions: Fourth Edition, Revised and Expanded, edited by Stig E. Friberg, Kåre Larsson, and Johan Sjöblom 133. Handbook of Frozen Foods, edited by Y. H. Hui, Paul Cornillon, Isabel Guerrero Legarreta, Miang H. Lim, K. D. Murrell, and Wai-Kit Nip 134. Handbook of Food and Beverage Fermentation Technology, edited by Y. H. Hui, Lisbeth Meunier-Goddik, Åse Solvejg Hansen, Jytte Josephsen, Wai-Kit Nip, Peggy S. Stanfield, and Fidel Toldrá 135. Genetic Variation in Taste Sensitivity, edited by John Prescott and Beverly J. Tepper 136. Industrialization of Indigenous Fermented Foods: Second Edition, Revised and Expanded, edited by Keith H. Steinkraus

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137. Vitamin E: Food Chemistry, Composition, and Analysis, Ronald Eitenmiller and Junsoo Lee 138. Handbook of Food Analysis: Second Edition, Revised and Expanded: Volumes 1, 2, and 3, edited by Leo M. L. Nollet 139. Lactic Acid Bacteria: Microbiological and Functional Aspects, Third Edition, Revised and Expanded, edited by Seppo Salminen, Atte von Wright, and Arthur Ouwehand 140. Fat Crystal Networks, Alejandro G. Marangoni 141. Novel Food Processing Technologies, edited by Gustavo V. Barbosa-Cánovas, M. Soledad Tapia, and M. Pilar Cano

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Additional Volumes in Preparation

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Lactic Acid Bacteria
Microbiological and Functional Aspects Third Edition, Revised and Expanded

edited by

Seppo Salminen
University of Turku Turku, Finland

Atte von Wright
University of Kuopio Kuopio, Finland

Arthur Ouwehand
University of Turku Turku, Finland

Marcel Dekker, Inc.

New York • Basel

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The previous edition of this book was published as Lactic Acid Bacteria: Microbiology and Functional Aspects, Second Edition, Revised and Expanded, edited by Seppo Salminen and Atte von Wright, 1998 (Marcel Dekker, Inc.), ISBN: 0-8247-0133-X. Although great care has been taken to provide accurate and current information, neither the author(s) nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage, or liability directly or indirectly caused or alleged to be caused by this book. The material contained herein is not intended to provide specific advice or recommendations for any specific situation. Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress. ISBN: 0-8247-5332-1 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc., 270 Madison Avenue, New York, NY 10016, U.S.A. tel: 212-696-9000; fax: 212-685-4540 Distribution and Customer Service Marcel Dekker, Inc., Cimarron Road, Monticello, New York 12701, U.S.A. tel: 800-228-1160; fax: 845-796-1772 Eastern Hemisphere Distribution Marcel Dekker AG, Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-260-6300; fax: 41-61-260-6333 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright # 2004 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

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Preface to the Third Edition

This third edition of Lactic Acid Bacteria covers the progress in this field of research over the past five years. The pace of development, already impressive when the second edition was being compiled, has shown no signs of slowing down. Consequently, most chapters in this new edition have been completely rewritten, several new contributors have been recruited, and some totally new chapters have been included, covering topics such as mathematical modeling, vegetable fermentation, methods for analysis of the gut microbiota, and probiotics for fish. It has been more and more of a challenge to keep the volume comprehensive, up-to-date, and concise. While it remains up to the reader to judge how well these goals have been achieved, we feel that the present volume gives a valuable overview of the present status of this rapidly expanding interdisciplinary area of research. As in the previous editions, a special emphasis has been placed on the health aspects of lactic acid bacteria, although, as can be seen in the table of contents, other relevant applications have also been covered. The intended audience includes, among others, microbiologists, food technologists, nutritionists, clinicians, product development experts, and regulatory experts. Seppo Salminen Atte von Wright Arthur Ouwehand

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Preface to the Second Edition

The first edition of Lactic Acid Bacteria was a profound success and well received. This, together with the very rapid progress in the field of research on lactic acid bacteria, has created a need for the second edition of the volume sooner than we had thought. Most of the material has been completely rewritten, and some totally new chapters have been included, although few changes have been made in some technical chapters. Understanding of the scientific basis of probiotic research, which was anticipated in the previous edition, has advanced in great strides. Consequently, new data on immunology, animal probiotics, and the role of propionic acid bacteria have been added. The importance of bacteriophages, both as a practical problem and as a molecular biological tool, has merited a special chapter. Other chapters have been updated for the most recent research findings and regulatory developments. We feel that this book provides the reader with a concise overview of a rapidly progressing field. Thus, it is an invaluable aid in guiding the reader through the web of accumulating new research data and in summarizing the fragmentary information available in specialist publications. Also, recent rapid developments in the area of functional foods should make the present edition valuable to an even wider audience. Seppo Salminen Atte von Wright

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Preface to the First Edition

Lactic acid–producing fermentation is an old invention. Many different cultures in various parts of the world have used it to improve the storage qualities, palatability, and nutritive value of perishable foods such as milk, vegetables, meat, fish, legumes, and cereals. The organisms that produce this type of fermentation, lactic acid bacteria, have had an important role in preserving foods, preventing food poisoning, and indirectly feeding the hungry on every continent. In the developed world, lactic acid bacteria are mainly associated with fermented dairy products such as cheese, buttermilk, and yogurt. The use of dairy starter cultures has become an industry during this century. Because of this, the technological aspects of lactic acid fermentation have been well covered in both research and training in food sciences. Since the days of the Russian scientist Metchnikoff, lactic acid bacteria have also been associated with beneficial health effects. Today an increasing number of health foods and so-called functional foods as well as pharmaceutical preparations are promoted with health claims based on the characteristics of certain strains of lactic acid bacteria. Most of these strains, however, have not been thoroughly studied, and consequently the claims are not well substantiated. Moreover, the accepted standards of clinical protocols, including double-blind randomized study designs, have not been applied in most “healthclaim” studies—health benefits are judged mainly using subjective criteria. Additionally, the specific bacterial strains used in the studies are often poorly identified. Most information about the health effects of lactic acid bacteria is thus anecdotal. There is a clear need for critical study of the effects on health of strain selection and the quality of fermented foods and their ingredients. Clinical studies should be properly conducted as doubleblind, placebo-controlled randomized trials. Both the defined bacterial strains and the proposed products should be studied to verify results. Only such studies produce the solid data that can back up health claims. This book reviews current developments in the study of lactic acid bacteria using the above-mentioned criteria. An overview of the taxonomy and general physiology of lactic acid bacteria is given. A discussion of the genetics of lactic acid bacteria as a future area of interest is included as well as a chapter on the technological aspects of manufacturing functional lactic acid bacteria starters. Many chapters consider our present
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Readers are referred to most of the recent literature in the area. Outr aim has been to give an overview of a rapidly changing and extremely important area of food and nutrition research. All Rights Reserved. the anecdotal evidence on the health benefits of specific strains of lactic acid bacteria is slowly being replaced by a more scientific outlook. covering the subject well from various aspects. One chapter of particular interest describes the development of individual lactic acid microflora. It was written by an Estonian research group that worked in association with the former Soviet space program. In particular.Hoang-Dung TRAN and friends knowledge of the effects of lactic acid bacteria in human health and disease and as animal probiotics. It will also be helpful to dairy scientists and technologists. Inc. especially when health claims are concerned. This book should serve as an important introduction to any student or scientist interested in these developments. These results have not been previously published in the West. consumer groups interested in the effects of lactic acid bacteria may benefit from the comprehensive reviews in this volume. both as a textbook and as a handbook for product development. It will be useful to government organizations developing regulatory policies for products based on lactic acid fermentation and bacteria. As new techniques as well as new interest in these organisms develop. Finally. Thus. Seppo Salminen Atte von Wright Copyright © 2004 by Marcel Dekker. those working with lactic acid bacteria and fermented foods or feed products within universities and the food industry should find this book most interesting. this book attempts to shed light on little-known and controversial aspects of lactic acid bacteria and their applications. .

8. Ouwehand Industrial Use and Production of Lactic Acid Bacteria ¨ ¨ ¨ Annika Mayra-Makinen and Marc Bigret The Genus Enterococcus: Biotechnological and Safety Issues Charles M. Vogensen. A. Inc.Hoang-Dung TRAN and friends Contents Preface to the Third Edition Preface to the Second Edition Preface to the First Edition Contributors 1. Copyright © 2004 by Marcel Dekker. P. Finn K. and Atte von Wright Bacteriophage and Antiphage Mechanisms of Lactic Acid Bacteria Jytte Josephsen and Horst Neve 2. . Franz and Wilhelm H. 6. Lactic Acid Bacteria: Classification and Physiology Lars Axelsson Bifidobacteria and Probiotic Action Jean Ballongue An Update on Probiotic Bifidobacteria Ross Crittenden The Probiotic Potential of Propionibacteria Arthur C. 3. 4. All Rights Reserved. 7. Holzapfel Genetics of Lactic Acid Bacteria Lorenzo Morelli. 5.

17. 13. 18. 15. Ouwehand and Satu Vesterlund Lactic Acid Bacteria as a Tool for Enhancing Food Safety by Removal of Dietary Toxins ¨ Hani El-Nezami. Yuan-Kun Lee. Methods for Analyzing Gut Microbiota Miguel Gueimonde and Seppo Salminen Antimicrobial Components from Lactic Acid Bacteria Arthur C. 19. 16. Robert Rastall. Jouko Setala. 20. and Charles Daly 11. Seppo Salminen. 22. Ouwehand. and Heidi Annuk Lactic Acid Bacteria and Intestinal Drug and Cholesterol Metabolism Alice H. and Eeva Salminen Prebiotics and Lactic Acid Bacteria Thea Scantlebury Manning. Hannu Mykkanen. Carolyn Haskard. Hoang-Dung TRAN and friends Mathematical Modeling of Intestinal Bacteria–Host Interactions Yuan-Kun Lee 10. 21. and Glenn Gibson Lactic Acid Bacteria in Vegetable Fermentations ¨ Maarit Maki Lactic Acid Bacteria in Cereal-Based Products Hannu Salovaara Human Lactic Acid Microflora and Its Role in the Welfare of the Host ¨ Marika Mikelsaar. Epp Sepp. 14. Copyright © 2004 by Marcel Dekker. S.9. Donohue Lactic Acid Bacteria as Animal Probiotics ¨ ¨ ¨ Juha Nousiainen. Paivi Javanainen. Atte von Wright. Lichtenstein and Barry R. Gorbach. Arthur C. and Atte von Wright Lactic Acid Bacteria in Fish and Fish Farming Einar Ringø Future Directions of Research and Product Development of Lactic Acid Bacteria Seppo Salminen. and Y. Goldin Human Studies on Probiotics: What Is Scientifically Proven Today? Seppo Salminen. Benno Safety of Novel Probiotic Bacteria Diana C. Reet Mandar. Inc. 12. . All Rights Reserved.

University of Turku. and Centre de ´ Recherche International Andre Gaillard. P. Gorbach Tufts University. Finland Charles M. U. Boston. Ivry-sur-Seine. School of Medicine. Norwegian Food Research Institute. Norway ´ Jean Ballongue Universite de Nancy 1. Franz Federal Research Center for Nutrition and Food. Massachusetts. Melbourne. Inc. Turku.Hoang-Dung TRAN and friends Contributors Heidi Annuk Lars Axelsson University of Tartu. Donohue Hani El-Nezami University of Kuopio. Vandoeuvre-les-Nancy. Estonia ˚ MATFORSK.S. Germany Glenn Gibson The University of Reading. Cork. Victoria. U. Saitama. As. Montpellier. Barry R.A.S. France Food Science Australia. . Massachusetts. Benno Japanese Collection of Microorganisms. Australia Diana C. Tartu. Institute of Hygiene and Toxicology. Boston. Ireland RMIT University. England Tufts University School of Medicine. Goldin S. Japan BioSav LTD. A. RIKEN.A. All Rights Reserved. Kuopio. Reading. Finland Miguel Gueimonde Copyright © 2004 by Marcel Dekker. Werribee. Australia Marc Bigret Ross Crittenden Charles Daly University College Cork. Karlsruhe. France Y.

A. Lichtenstein USDA Human Nutrition Research Center on Aging at Tufts University. Singapore Alice H. Wilhelm H. Estonia Valio LTD. Boston. Kiel. Finland University of Turku. Thea Scantlebury Manning ¨ Maarit Maki ¨ Reet Mandar The University of Reading. Turku. Finland The Royal Veterinary and Agricultural University. Turku. Tartu. Piacenza. Helsinki. Institute of Hygiene and Toxicology.Carolyn Haskard Australia Hoang-Dung TRAN and friends Australian Water Quality Centre. Estonia Catholic University of Sacred Heart. Inc. Ouwehand Robert Rastall Einar Ringø The University of Reading. Frederiksberg. Finland Juha Nousiainen Arthur C. Germany ¨ Paivi Javanainen Jytte Josephsen Denmark Yuan-Kun Lee Valio Ltd. South Australia. Finland Federal Research Centre for Nutrition and Food. Italy University of Kuopio. Salisbury. Finland Eeva Salminen Seppo Salminen Hannu Salovaara Epp Sepp University of Tartu. Tartu. Finland University of Turku. Finland ¨ ¨ ¨ Annika Mayra-Makinen Marika Mikelsaar Lorenzo Morelli ¨ Hannu Mykkanen Horst Neve University of Tartu. Turku. Estonia Copyright © 2004 by Marcel Dekker. Finland University of Helsinki. Norway Turku University Hospital. U.S. Holzapfel Federal Research Center for Nutrition and Food. Tartu. . All Rights Reserved. England MTT Agrifood Research Finland. Kuopio. Reading. National University of Singapore. Jokioinen. Karlsruhe. Massachusetts. Helsinki. England The Norwegian School of Veterinary Science. Tromsø. Finland University of Tartu. Helsinki. Germany Valio Ltd. Reading. Helsinki.

Finland University of Turku. Kuopio. Turku. All Rights Reserved. Inc. Helsinki.Hoang-Dung TRAN and friends ¨ ¨ Jouko Setala Valio Ltd. Satu Vesterlund Finn K. Frederiksberg. Finland Royal Veterinary and Agricultural University. Finland Copyright © 2004 by Marcel Dekker. Vogensen Denmark Atte von Wright University of Kuopio. .

The LAB term is intimately associated with bacteria involved in food and feed fermentation. and Streptococcus form the core of the group. However. nonrespiring cocci or rods. Streptococcus. Chemotaxonomic markers such as fatty acid composition and constituents of the cell wall are also used in classification. Leuconostoc. Lactobacillus. often considered in the same context as the genuine lactic acid bacteria and sharing some of their typical features. Enterococcus. Lactococcus. configuration of the lactic acid produced. ability to grow at high salt concentrations. The genus Bifidobacterium. Tetragenococcus. metabolic. The boundaries of the group have been subject to some controversy. which produce lactic acid as the major end product during the fermentation of carbohydrates. and acid or alkaline tolerance. and physiological characteristics. the present taxonomy relies partly on true phylogenetic relationships. from a practical. The classification of lactic acid bacteria into different genera is largely based on morphology. Carnobacterium. All Rights Reserved. . In addition. food-technology point of view. Leuconostoc. Pediococcus. Oenococcus. comprise around 20 genera. is phylogenetically unrelated and has a unique mode of sugar fermentation. Norwegian Food Research Institute. Inc. Pediococcus. The general description of the bacteria included in the group is gram-positive. in their broad physiological definition. but historically the genera Lactobacillus.Hoang-Dung TRAN and friends 1 Lactic Acid Bacteria: Classification and Physiology LARS AXELSSON ˚ MATFORSK. the following genera are considered the principal LAB: Aerococcus. including related bacteria normally associated with the (healthy) mucosal surfaces of humans and animals. Vagococcus. mode of glucose fermentation. Copyright © 2004 by Marcel Dekker. and Weissella. nonsporing. As. Taxonomic revisions of these genera and the description of new genera mean that LAB could. Norway I. SUMMARY Lactic acid bacteria (LAB) constitute a group of gram-positive bacteria united by a constellation of morphological. growth at different temperatures.

Some of the newly described genera are most easily determined with oligonucleotide probes.2] However.or heterofermentation). or direct sequencing of the 16S rRNA gene. and range of sugar utilization. mode of glucose fermentation (homo. Transport of amino acids and other nutrients is generally mediated by proton-motive force– dependent systems. The proton-motive force drives the uphill transport of metabolites and ions into the cell. and the metabolism is referred to as homolactic fermentation. Inc. thus sparing ATP.” Significantly.. and the metabolism is referred to as heterolactic fermentation. the phylogenetic clusters do not correlate with the current classification based on phenotypic characters. . As will be seen later in this chapter. tetrad formation).Hoang-Dung TRAN and friends which have been revealed by extensive work on determining rRNA sequences. The 6-phosphogluconate/phosphoketolase pathway results in significant amounts of other end products such as ethanol. acetate. growth at certain “cardinal” temperatures (e. PCR-based fingerprinting techniques and soluble protein patterns. This work had a large impact on the systematics of LAB and. II. and CO2 in addition to lactic acid. although revised to a considerable extent. rather than the existance of an unequivocal definition of the term. The most promising for routine use are 16S rRNA gene sequencing. GENERAL INTRODUCTION What is a lactic acid bacterium? Asking this question of scientists in the field would probably result in a fairly uniform answer. antiport systems. these characteristics Copyright © 2004 by Marcel Dekker. Two main sugar fermentation pathways can be distinguished among lactic acid bacteria.g. the first pure culture of a bacterium was “Bacterium lactis” (probably Lactococcus lactis). Glycolysis (Embden-Meyerhof-Parnas pathway) results almost exclusively in lactic acid as the end product under standard conditions. the classification basis remains remarkably unchanged. The historic tradition goes back to before the turn of the twentieth century. Important progress in the classification of these bacteria was made when the similarity between milk-souring bacteria and other lactic acid – producing bacteria from other sources was recognized[1. Most genera in the group form phylogenetically distinct groups. Lactic acid bacteria create a proton-motive force mainly by means of a membranelocated Hþ-ATPase at the expense of ATP. obtained by J. Sugar transport is mediated mainly by proton-motive force–dependant permease systems or phosphoenolpyruvate : sugar phosphotransferase systems. beyond any function in sugar transport. This is more because of a historic tradition. 108C and 458C). The term lactic acid bacteria was then used to mean “milk-souring organisms. Certain components of the phosphoenolpyruvate : sugar phosphotransferase system appear to hold key positions in the global regulation of the sugar metabolism in general. All Rights Reserved. Lister in 1873. or organic compounds. polymerase chain reaction (PCR)– based technologies using these sequences. New tools for classification and identification of LAB are currently replacing and/or complementing the traditional phenotype-based methodologies. but for some. Various growth conditions may significantly alter the end product formation by some lactic acid bacteria. Orla-Jensen used the following characteristics as a basis for classification: morphology (cocci or rods. in particular Lactobacillus and Pediococcus. in both a respiratory and a nonrespiratory mode. These changes can be attributed to an altered pyruvate metabolism and/or the use of external electron acceptors such as oxygen. or ATP-driven systems. End-product efflux and electrogenic transport of certain compounds may contribute to the formation of a proton-motive force. confusion was still prevalent when the monograph of Orla-Jensen[3] appeared.

All Rights Reserved. In the era of genomics.[4.[6] and this method is still used in defining what constitutes a species in the prokaryotic world. etc. has been one way to achieve more controlled processes. nutrient utilization. macromolecules of the cell. Pediococcus. since it makes them excellent model systems for the study of energy transduction.5] but that subject will not be dealt with here. However. of course. Today we have the means to examine. efforts in this direction may not be fruitful unless there is a sound understanding of the physiology of these bacteria. and Streptococcus. Increased knowledge of LAB physiology. Fortunately. Still. resulting in one or a few fermentation end products. It is now a relatively easy task to determine the sequence of rRNA from bacteria. the nucleic acids. Today. in detail. After the work of Orla-Jensen. Some emphasis will be placed on the variations of the general “theme” of metabolism that may occur under certain conditions.19] and many more are underway. At that time. In addition. The fermentative nature of LAB is also of considerable academic interest. modern genetic techniques are considered to be promising in this regard. such as carbohydrate metabolism and bioenergetics. it is clear that LAB have a very diverse metabolic ability to adapt to a variety of conditions. There has always been controversy as to the boundaries of the group. since the sequence contains both well-conserved and less conserved regions. . such data are meaningless without their elucidation in a physiological context. Orla-Jensen regarded LAB as a “great natural group.Hoang-Dung TRAN and friends are still very important in the classification of LAB. such as metabolism. This may be the case in the laboratory environment that we often impose on them.[11 – 13] The classification section of this chapter will deal with both the “classical” classification schemes and the current phylogenetic status of LAB. most comprising strains previously included in one of the four mentioned above. where some LAB genomes already have been published[18.[10] With this technique. a more clear picture of the phylogeny of LAB has become evident. The classification section of this chapter will concentrate on what historically constituted these four genera. Inc. Leuconostoc. rRNA sequencing is an important aid in the classification of LAB. These are. as exemplified by the descriptions of new genera.[20] we will have access to a wealth of genetic data with enormous potential to improve our understanding of these bacteria. Initially this was done with the reverse transcriptase technique. solute transport. The attempts at metabolic engineering of Lactococcus lactis [14 – 17] would not have been possible without understanding of the physiology and biochemistry of this organism. Close relationships (at species and subspecies levels) can be determined with DNA-DNA homology studies. However. believed to be more accurate in defining relationships and phylogenetic positions.[7] For determining phylogenetic positions of species and genera. the view emerged that the LAB comprised four genera: Lactobacillus. The physiology section of this chapter will describe the main features of LAB.” indicating a belief that the bacteria included were phylogenetically related and separate from other groups.[21 – 26] The designation “lactic acid bacteria” perhaps implies that these bacteria have a somewhat simple metabolism. only phenotypic characters could be examined and evaluated as phylogenetic markers. Since 1985 many new genera have been described. Copyright © 2004 by Marcel Dekker. ribosomal RNA (rRNA) is more suitable..[9] Comparisons of these sequences provide the most powerful and accurate technique for determining phylogenetic relationships of microorganisms. and membrane biology. nature has provided us with different kinds of nucleic acids for different types of taxonomic studies.[8] but it is now being done by sequencing the corresponding genes using polymerase chain reaction (PCR) technology. The physiology of LAB has been of interest ever since it was recognized that these bacteria are involved in the acidification of food and feed products.

for generating energy.[29] the lack of cytochromes may be a more reliable characteristic for preliminary diagnosis than the commonly used catalase test. vegetables).g. It is really only the gram-positive characteristic that cannot be challenged. In this chapter I will follow the historical tradition not to include spore-formers in the LAB group. The above definition. A. there are no strong scientific arguments for excluding spore-forming bacteria. LAB do not possess the mechanism of an electron transport chain and rely on fermentation. it is important to note that the situation may be totally different if heme (or hemoglobin) is added to the growth medium. etc. is useful as a core or center around which the actual descriptions of genera or species are formulated.. since some of the genera we consider “genuine” LAB are not clearly separated from these phylogenetically..[4] However. non– spore-forming. but are also fish pathogens. intestine. The main emphasis in this chapter will be on LAB normally associated with food manufacture and positive health aspects. III. This reflects the intimate association of the term with food and feed manufacture. which is gram-positive. nutritional. Variations of this general theme are common..[30 – 37] Copyright © 2004 by Marcel Dekker. acid-tolerant.g. beverages. All Rights Reserved. A true catalase and even cytochromes may be formed. catalase-negative. and strictly fermentative.Hoang-Dung TRAN and friends This volume concerns the technological. with lactic acid as the major end product during sugar fermentation. Sporolactobacillus). despite its limitations.. Since catalase activity. A key feature of LAB that must be emphasized is the inability to synthesize porphyrin groups (e. Inevitably. in some cases resulting in respiration with a functional electron transport chain. of nonaerobic habit but aerotolerant. Again. which otherwise resemble LAB (i. For instance.[28] There are more examples of the “dual” nature of LAB as a group. Furthermore. mediated by a nonheme “pseudocatalase. heme). This makes LAB devoid of a “true” catalase and cytochromes when grown in laboratory growth media. In addition.[27] carnobacteria are normal inhabitants in meat.” can occur in some LAB. LAB are generally associated with habitats rich in nutrients.. some streptococci (e. considered normal in most studies of these bacteria. This is the actual physiological background for some of the characteristics mentioned above.e. many streptococci). and health aspects of LAB. which lack heme or related compounds. and vagina of mammals. this is perhaps more of a historical tradition than a scientifically reached position. fastidious. since the group includes bacteria that are highly pathogenic and therefore undesirable in food (e.e. some lactobacilli and other LAB generally associated with food have been implicated in disease. sugar fermentation may result in very little lactic acid under certain conditions. Under these conditions. it is more appropriate to describe the typical lactic acid bacterium. Inc. . but some are also members of the normal flora of the mouth. such as various food products (milk.g. catalase and cytochromes may be formed by some LAB on certain media (see below). substrate-level phosphorylation. most characteristics used in such a definition are subject to more or less qualification. devoid of cytochromes. Therefore. Streptococcus bovis) have quite limited nutritional requirements. i. CLASSIFICATION OF LACTIC ACID BACTERIA General Description and Included Genera An unequivocal definition of the term lactic acid bacteria is not possible to give.[4] meaning that they are accurate only under “normal” or “standard” conditions and that exceptions to the definition can be found. meat.

and Tetragenococcus. were suggested to form a separate genus. Copyright © 2004 by Marcel Dekker. LAB can be divided into rods (Lactobacillus and Carnobacterium) and cocci (all other genera). cell division in two perpendicular directions in a single plane [previously incorrectly described as “division in two planes”[53]]. in particular of the streptococci. Oenococcus (O. One exception is the relatively recently described genus Weissella.g. including species previously assigned to either Lactobacillus or Leuconostoc. Major revisions of the taxonomy of LAB. by definition. Leuconostoc (Ln.). Eremococcus. Dolosicoccus.). the “wine leuconostocs.[45 – 52] These represent special cases and will not be dealt with further. both physiologically and phylogenetically. . The genera that then fit the general description of the typical LAB were Aerococcus (A. were anticipated in Bergey’s Manual of 1986[38. therefore. Helcococcus. However. The tetrad-forming genera are Aerococcus. Facklamia. previously included in Lactobacillus. not be considered in this general overview of LAB.[44] New genera. and Streptococcus sensu stricto.). they have a special pathway for sugar fermentation. and Pediococcus have largely remained unchanged. Thus. Bifidobacteria will.). Lactococcus (Lc. the former genus Streptococcus was first divided into three: Enterococcus (E. otherwise resembling lactococci. Inc.39] and to some extent already realized by the year of that issue. which clearly separates them from the LAB group. some motile LAB.).).[10] it is still important in the current descriptions of the LAB genera.). The revisions made since 1986 are supported by extensive chemotaxonomic and genetic data.). the general basis for the classification of LAB in different genera has remained largely unchanged since the work of Orla-Jensen. Although Bifidobacterium species do fit the general description above. In addition. forming the genus Tetragenococcus (T. Dolosigranulum. e. is used as a key characteristic in the differentiation of the cocci. Vagococcus (V. Alloiococcus. Pediococcus (P. Globicatella.[11] A distinct phylogenetic cluster of heterofermentative LAB. Classification at the Genus Level As mentioned. leading to tetrad formation.[43] The genera Lactobacillus. which is the first genus in the LAB group that. Ignavigranum.Hoang-Dung TRAN and friends It is appropriate to start the introduction to the LAB genera with Bergey’s Manual of 1986. All Rights Reserved.[28] and the former species Pediococcus halophilus has been raised to genus level. Still. Lactobacillus (Lb. but some rod-shaped LAB. Although morphology is regarded as questionable as a key characteristic in bacterial taxonomy. has been suggested to form a separate genus. Weissella (W. and Lactosphaera. Thus. due to their significance in the gastrointestinal tract of animals and humans and possible probiotic action. since this edition essentially is the last in a continuoum reflecting the historical tradition dating back to the work of Orla-Jensen (1919).). can include both cocci and rods.[13] Leuconostoc oenos.[40 – 42] Later. they are phylogenetically more related to the Actinomycetaceae group of gram-positive bacteria. with the description of new genera and species it is becoming increasingly difficult to use these classical tests for reliable genus identification. The genus Bifidobacterium is historically also considered to belong to the LAB group.). bifidum. have also been described to include some strains that were shown to be related to the LAB group. B. unique to the genus.” has been proposed to form a genus of its own. now form the genus Carnobacterium (C.[13] Furthermore. the bifidobacteria were designated Lb. and Streptococcus (S. these phenotypic characteristics are useful as a starting point for more sophisticated tests.[3] However.)..). Pediococcus. In the 7th edition of Bergey’s Manual of 1957. bifidobacteria are described in more detail elsewhere in this book. Leuconostoc.

. Aerococci. The genus Carnobacterium is indistinguishable from Lactobacillus with these tests. and nucleic acid precursors) and limited oxygen availability. while growth at 458C is dependent on the species. concentrating on the means by which classification within a genus can be done and mentioning some of the most interesting species from a food technology point of view. although variable reactions can be found among streptococci. converting glucose almost quantitatively to lactic acid and the heterofermentative. lactococci/vagococci.43] In general.5% NaCl) may also be used to distinguish between enterococci. Classification at Species Level It is impossible within the scope of this review to describe the classification of all species of LAB. All Rights Reserved. For example. enterococci.[60] Therefore. of which direct rRNA sequencing is the most accurate at the genus level.. and CO2. Tolerance to acid and/or alkaline conditions may also be useful. fermenting glucose to lactic acid. Streptococci generally do not grow at 108C. Pediococci can be confused with aerococci. Under these conditions LAB can be divided into two groups: the homofermentative. all other LAB are homofermentative. a test for gas production from glucose will distinguish between the groups. since the morphology is similar.[56] Weissella species can easily be confused with leuconstocs or heterofermentative lactobacilli. In practice. Oenococci fall into the Leuconostoc group with the classical tests but are easily distinguished by their extreme acid and ethanol tolerance. Salt tolerance (6. although not all can grow at the standard test pH of 9. the genus Enterococcus contains many species that do not conform to the classical tests.[55] Extreme salt tolerance (18% NaCl) is confined to the genus Tetragenococcus. For the most recent. Vagococcus and Carnobacterium have unique fatty acid compositions. comprehensive review on Copyright © 2004 by Marcel Dekker. contrary to the more microaerophilic nature of aerococci. The genus Lactobacillus alone includes about 80 recognized species. carnobacteria. Formation of the different isomeric forms of lactic acid during fermentation of glucose can be used to distinguish between leuconostocs and most heterofermentative lactobacilli. tetragenococci.A. as is Vagococcus from Lactococcus. lactococci and vagococci at 108C.58] Classification of LAB is becoming dependent on more sophisticated methods. but Weissella strains may cause confusion in this regard. but not at 458C.[13. and vagococci are characterized by growth at relatively high pH.0 and inability to grow on acetate media selective for lactobacilli.[44] It should be noted that there are phenotypic overlaps between genera and exceptions to the general rules outlined in Table 1 can be found. which separate these genera from most other LAB. A summary of the differentiation of LAB genera with classical phenotypic tests is shown in Table 1.Hoang-Dung TRAN and friends An important characteristic used in the differentiation of the LAB genera is the mode of glucose fermentation under standard conditions. and a subgroup of lactobacilli are heterofermentative.e. see Sec. vitamins. Growth at certain temperatures is used mainly to distinguish between some of the cocci.6. the following section will only be a summary. Known rRNA sequences have also been used to develop genus-specific probes. i. pediococci are more acid-tolerant than aerococci and grow well anaerobically.[57. weissellas. The “classical” enterococci grow at both 108C and 458C.59] C. Inc. carnobacteria can be distinguished from lactobacilli by their ability to grow at pH 9. nonlimiting concentrations of glucose and growth factors (amino acids. IV. as the former produce only D -lactic acid and the latter a racemate (DL -lactic acid).) Leuconostocs. ethanol/acetic acid.[28. oenococci. and streptococci. However.[54] (For a more detailed discussion concerning the metabolic pathways.

2 2c þ 2 NDd 2 ND 2 L Enteroc.or heterofermentation of glucose. þ 2 þ 2 þ 2 2 þ L Character Tetrad formation CO2 from glucoseb Growth at 108C Growth at 458C Growth in 6. depending on media. Inc. 2 2 þ 2 2 2 + 2 L Leucon.5% NaCl Growth in 18% NaCl Growth at pH 4. DL þ . Table 1 Differential Characteristics of Lactic Acid Bacteria Rods Cocci Lactob. 2 + + + + 2 + 2 f D . Vagoc.Hoang-Dung TRAN and friends Copyright © 2004 by Marcel Dekker. response varies between species. negative. L -. ND.6 Lactic acide a Carnob. Oenoc. b Test for homo. 2 2 2 + 2 2 2 2 L Tetragenoc. þ 2 þ 2 þ þ 2 þ L Weissella a 2 þ þ 2 + 2 + 2 f D . or DL -lactic acid varies between species. DL Streptoc. negative and positive denotes homofermentative and heterofermentative. c Small amounts of CO2 can be produced. DL Aeroc. Weissella strains may also be rod-shaped. e Configuration of lactic acid produced from glucose.4 Growth at pH 9. L . 2. respectively. þ 2 + + + 2 þ 2 f L . 2 þ þ 2 + 2 + 2 D Pedioc. f Production of D -. not determined. All Rights Reserved. +. 2 2 þ þ þ 2 þ þ L Lactoc. positive. . d No growth in 8% NaCl has been reported.

presence of certain enzymes (e. presence and type of teichoic acid. the genera Enterococcus. All Rights Reserved. This is perhaps more true regarding classification at the species than at the genus level.63 – 66] and will certainly be an even more valuable resource in the future as more chapters are updated. the classical phenotypic/biochemical characterization is important for a preliminary classification as well as learning about the properties of the strains. can be causative agents of dental caries. e. The sequence of 16S Copyright © 2004 by Marcel Dekker. and molecular characteristics of the genera. including type of diamino acid in the peptidoglycan. there is undoubtedly some correlation between the presence of the group D antigen and the “classical” enterococci.[38] As a general rule.[67] which summarizes the phenotypical. e.g. the reader is referred to a review ¨ by Schleifer and Kilpper-Balz. some streptococci. and “other” streptococci. Inc. The method is now considered to be less important in classification.. Similarily. guanine þ cytosine (G þ C) ratio of the DNA.” in the mid-1980s. but note that the vagococci also possess the group N antigen. Wood. the online publication “The Prokaryotes”[62] contains recently updated chapters for lactococci. Streptococcus. salt and pH tolerance. acetoin formation (Voges-Proskauer test). pyogenes and S. Lactococcus. Still. others of infective endocarditis. the reader is referred to Volume 2 in the Lactic Acid Bacteria series edited by B. fatty acid composition. arginine hydrolysis. A review by Stiles and Holzapfel[61] is also very useful. while the oral streptococci are a. bgalactosidase and b-glucuronidase). The genus is broadly divided into three groups: pyogenic. S. was earlier included in this group. type of hemolysis. enterococci. and serological typing.42.. growth characteristics in milk. presence and type of menaquinones.g. bile tolerance.”[38] were shown to be unrelated to all other streptococci and have been excluded. growth at certain temperatures.[38.54. proper classification of LAB is beginning to rely on molecular biology methods.g. and the “leuconostoc-like” LAB[58. agalactiae. J.. and configuration of the lactic acid produced.[42] The pyogenic group contains several famous pathogens. At the time of this writing. production of extracellular polysaccharides. mutans. growth factor requirements. Further characterization includes more molecular/chemotaxonomic approaches.[43] Despite the formation of new genera. and electrophoretic mobility of the lactate dehydrogenase (LDH). but has been transferred to the oral group. Enterococcus. but still very useful in the rapid identification of major pathogens. the pyogenic streptococci are b-hemolytic. Some of the characteristics listed in Table 1 are useful also in the classification at species level. oral. although some of Orla-Jensen’s concepts are still viable. serological typing with the Lancefield grouping[68] has been very important in the classification of streptococci. the genus Streptococcus sensu stricto is still very large and the classification is difficult. Historically.g. S. S.[69] Some anaerobic cocci. pneumoniae. e. . Streptococcus. and Vagococcus were earlier included in one genus. As indicated previously. biochemical.. and Vagococcus As mentioned. only an analysis at the nucleic acid level will resolve classification problems. Streptococcus. Other characteristics used in the phenotypic/biochemical characterization of strains are range of carbohydrates fermented. Some oral streptococci.64] However.or nonhemolytic. previously included in the genus as the group “anaerobic streptococci. which contains species mostly associated with the oral cavity of humans and animals. Another pathogen. In some cases. 1. Lactococcus.Hoang-Dung TRAN and friends the taxonomy of LAB. For details regarding the major taxonomic revision of the “streptococci. the group N antigen is correlated with lactococci. but exceptions can be found. B.[5] which also includes descriptions of the individual species.

and (d) ferment ribose. the large phenotypic differences would justify two separate species. respectively. lactis subsp. since DNA-DNA homology values of greater than 70% were determined. III. Only the first two are important in dairy manufacture. the suggestion was later rejected. Biochemical characteristics.[69] A close relationship at the DNA level with S. faecalis (previously S. such as carbohydrate fermentation. lactis subsp. salivarius subsp.[71] Heat resistance. (b) grow in 4% NaCl. lactis can be distinguished: Lc. S.E). lactis subsp. lactis includes species formerly designated S. and Lactobacillus xylosus.73] Farrow and Collins[73] proposed that S.Hoang-Dung TRAN and friends rRNA has been determined for most species.e. thermophilus has been developed.g. lactis have also been shown to be genetically distinct by DNA-DNA homology studies[75] and comparison of 16S rRNA sequences. S. thermophilus from most other streptococci. (c) hydrolyze arginine. and the rather limited number of carbohydrates attacked distinguish S. hydrolysis of arginine. lactis subsp. which is used in the manufacture of yogurt (in coculture with ¨ Lb. MG1363 and LM0230. cremoris. garviae strains have been associated with bovine mastitis and fish lactococcosis. Lc. cremoris is distinguished from Lc. lactis subsp.[70. faecalis). hordniae.[76] However. Inc. vestibularis.E) have also been employed in the identification and classification of streptococci. lactis. in particular E.[72] but is now in the oral group. All Rights Reserved. lactis subsp. species of the newly described genus Vagococcus are easily confused with lactococci. lactis subsp. also belongs to this closely related group of streptococci. lactis subsp. salivarius.[42] since an investigation of a large number of strains revealed lower DNA-DNA homology values for some strains. are used in classification schemes. Lc. lactis subsp. cremoris or S. However. Enterococci are not considered to be of particular importance in food technology. S.[77] Common biochemical characteristics (e. and RAPD (see Sec. but genetic methods are also available[76 – 80] (see Sec. REP-PCR. salivarius has been established. is actually used in dairy technology. Lc. sugar utilization) can be used to distinguish between the species of lactococci. but out of the five species currently recognized.[65] Essentially the only streptococcal species associated with food technology is S.and species-specific oligonucleotide probes are available for vagococci. Thus. the distinction between cocci and rods is not always an easy task. cremoris. Some. such as RFLP.. In addition. Lc. lactis subsp.[66] Three subspecies of Lc. As noted.81] Genus. the ability to grow at 528C.[41] The lactis and cremoris subspecies of Lc. A third species. III. Genetic fingerprinting techniques. it should be noted that some strains of the subspecies lactis phenotype are genetically subspecies cremoris.[72. among them the laboratory strains commonly used worldwide.[72] A species-specific probe for S. and the mutans groups. bulgaricus) and certain cheeses. and Lc. thermophilus was included in ¨ the group “other streptococci” by Schleifer and Kilpper-Balz[67] and Hardie. lactis by the inability to (a) grow at 408C. thermophilus. i. cremoris includes species previously designated S.. . for some time the name S. delbruckii subsp.[66] only one. Lc. Lc. but not all strains of vagococci are motile. lactis. can be opportunistic Copyright © 2004 by Marcel Dekker.[74] Lactococci are intimately associated with dairy products. the mitis.[41] The latter illustrates the fact that morphology can be deceptive in classification of LAB. thermophilus was valid.71] and this has clarified the intrageneric phylogeny. the salivarius.[82] which make a reliable identification of these bacteria feasible. the bovis. thermophilus be considered a subspecies of S. lactis. diacetylactis. and certain enzyme activities.[43. Some species. but the genera are clearly distinguished by fatty acid composition. These studies suggest that the oral group can be divided in five phylogenetic subgroups: the anginosus.

Pediococcus. All Rights Reserved.[91] As mentioned. arginine and hippurate hydrolysis. faecium) and E. one probably has to identify strains to species level to unambigously show that they are enterococci. E.[100] As mentioned. acidilactici and P. halophilus to Tetragenococcus. muriaticus. tetragenococci generally require salt Copyright © 2004 by Marcel Dekker.24] 2.86] Vancomycin resistance and the conjugative transfer of the trait have received special attention in this regard due to the “last resort” status of vancomycin in the treatment of patients infected with multiresistant staphylococci.”[95] Pediococci are important in food technology in both a negative and positive sense. preparations of E. since growth may lead to diacetyl/acetoin formation. and presence and/or type of menaquinones. Only two species. for instance.[90] The probiotic approach is not far-fetched. to definitely assign a strain to the genus with the tests shown in Table 1. lactic acid bacteria that divide alternately in two perpendicular directions to form tetrads. hirae (in older publications: S. However. which is involved in the ripening of cheese. hydrolysis of arginine. Useful additional tests for which a majority of enterococci are positive are the Voges-Proskauer test and fermentation of ribose. the genus Tetragenococcus contains strains previously regarded as P. P. faecalis and E.97] Pediococci may also be important constituents of the complex known as the nonstarter lactic acid bacteria (NSLAB). Aerococcus.[101] In addition to extreme salt tolerance (. bioenergetics and membrane biology. faecalis have been used as probiotics[88. faecium) have been of great value for general LAB research in being model organisms in physiological studies of.[55] Species of enterococci have also been shown to be present in some local types of cheeses in southern Europe. halophilus. growth at different pH levels (7.[95.[58] With only phenotypic tests.84] and are. halophilus and T.[85. has been shown to be related to Tetragenococcus phylogenetically. faecium (previously S. damnosus is a major spoilage organism in beer manufacture. but one enterococcal species. resulting in a buttery taste. therefore. T.18% NaCl).[93] Information on the genus Aerococcus is given in a review by Weiss.[87] However. which distinguishes them from other LAB. and Tetragenococcus Aerococci. .89] and as silage inoculants. are currently recognized.58] E.[98. generally undesirable in food.[96] P. acidilactici are difficult to distinguish using these characteristics but have been shown to be distinct species with DNA-DNA homology studies. and the configuration of lactic acid produced. pentosaceus and P.92] For further information on identification of enterococci see Devriese et al.23. the genus Pediococcus can be described as “the only acidophilic.99] The main characteristics for distinguishing between the species are the range of sugars fermented.[94] With the transfer of P. homofermentative. There is also some concern about the propensity of fecal enterococci to be resistent to antibiotics and to transfer such traits by means of mobile genetic elements. it is difficult. solitarius. if not impossible.[90. and tetragenococci constitute the tetradforming LAB.96] P. pediococci.5). since the natural habitat of many enterococci is the intestines of humans and animals. urinae-equi to Aerococcus and P.[57. pentosaceus are used as starter cultures for sausage making and as silage inoculants. a recent report suggests that some aerococci could be responsible for greening of cooked meat products.[21. Aerococci are generally of minor interest in food technology.0 and 4.”[29] Genetic fingerprinting methods are also available for distinguishing pediococci. Inc.[58.[67] A number of genetic methods are also available. Species are differentiated mainly by carbohydrate fermentation patterns.Hoang-Dung TRAN and friends pathogens[83. The genus Aerococcus currently contains five species.[96] These species are also similar in that they may produce a nonheme “pseudocatalase. feacalis or S.

cremoris. a bacterium isolated from garden soil was shown to represent a new species of Weissella.63. It is important to note that although these taxonomic revisions were necessary from a phylogenetic point of view. but classification is difficult. Ln. oeni.[13] Recently. care must be taken that malate is not present in the growth medium when the configuration of the lactic acid (from glucose fermentation) is to be determined. paramesenteroides together with some heterofermentative lactobacilli (e. It was. Oenococcus. e.Hoang-Dung TRAN and friends in the range of 5% NaCl for growth.[13. This method employs both PCR with genus-specific primers and restriction pattern analysis for species determination. Oenococci are easy distinguishable by their extreme acid and ethanol tolerance.[106] Leuconostocs may form significant amounts of diacetyl from citrate in milk. Leuconostocs are also important in spontaneous vegetable fermentations. mainly Ln.[111] Hence.[63] Copyright © 2004 by Marcel Dekker. alloted to the species Ln.109.[108] Species and subspecies of Leuconostoc and Weissella are distinguished by characteristics such as carbohydrate fermentation patterns. an amplified ribosomal DNA restriction analysis (ARDRA) was recently developed. These phylogenetic studies also revealed that the so-called wine leuconostocs. confusus and Lb. were only distantly related to other leuconostocs and that this species therefore warranted a separate genus.102.[96.E) have also been used successfully for distinguishing the Leuconostoc-like LAB.[13] For rapid identification of Weissella species. and the genus Weissella was suggested to comprise these “leuconostoc-like” bacteria. III.[96] Tetragenococcus species are important in lactic fermentation of high-salt-containing food.110] The dissimilation of malate requires attention. hydrolysis of esculine. dextran formation (from sucrose). sauerkraut. where they may proliferate at low temperatures.. oenos is now designated O.[109] Ribotyping and PCR-based methods (see Sec. easy to confuse leuconostocs with some “coccoid rods” of the heterofermentative lactobacilli. and some species. .g. oenos. but separating Weissella from Leuconostoc sensu stricto and from some heterofermentative lactobacilli is still problematic.g.[44] Thus. as this metabolism results in L -lactic acid. The leuconostocs were thus separated from other cocci of the LAB by their heterofermentative metabolism and from heterofermentative lactobacilli by morphology and some key traits. since this group was separated from both other leuconostocs and lactobacilli. e.[104. Inc.. viridescens) could represent the nucleus of a new genus.105] Later. where they often initiate the lactic fermentation. the genus Oenococcus was proposed for these bacteria. and Weissella The genus Leuconostoc was previously defined as being heterofermentative. Lb. and somewhat surprisingly. they do not make the practical classification of “leuconostoc-like” LAB simpler than before. For practical reasons it is best to treat the “leuconostoc-like” LAB as a group and use confirmatory tests to definitely assign genus and species status for a particular strain. and dissimilation of citrate and/or malate. have been used in the dairy industry for this purpose. Leuconostoc. growth requirements. other heterofermentative LAB falling into this group were isolated from meat sources. Weissella therefore includes both cocci and rods. Later. growth at different pH and temperatures. The most reliable method to determine if a strain belongs to Weissella is probably to use a genus-specific rRNA probe. mesenteroides subsp. coccoid LAB producing only D -lactic acid from glucose and not producing ammonia from arginine. however. All Rights Reserved.103] 3. soy sauce.[104. Phylogenetic analysis of the leuconostocs revealed considerable heterogeneity of the genus.[107] Many Weissella species seem to be associated with meat..g.[13] As mentioned.105] It was anticipated that Ln.

buchneri Lb.Hoang-Dung TRAN and friends 4. Lactobacillus and Carnobacterium The genus Lactobacillus is by far the largest of the genera included in LAB.and heterofermentative sugar metabolism. b a Copyright © 2004 by Marcel Dekker. For example.116] (For further details regarding the division of LAB in homo.[114] the physiological division into the three groups (A. Orla-Jensen (1919) essentially tried to divide this group in a way similar to that with the cocci. and physiological properties. but each species was also assigned a suffix (a. and Betabacterium.and heterofermentative. delbruckii Lb. or c) to reflect the position in certain phylogenetic clusters (see below). which made differentiation into several genera possible.[112] The heterogeneity and the large number of species are due to the definition of the genus. It is also very heterogeneous. Inc. phenotypic traits were early recognized. although the designations have been dropped and some modifications in the definitions of the subgroups have been made. Streptobacterium.[113. among the cocci. viridescens (see above). respectively.A. Note that the cluster c contains those heterofermentative lactobacilli that now should be designated Weissella.115. and C) was kept. Lb. confusus and Lb. curvatus Lb.114] Table 2 shows a summary of the characteristics used to distinguish between the three groups and some of the more well-known species included in each group. B.6-diphosphate aldolase and phosphoketolase. fermentum Lb. Obligately heterofermentative 2 þ þa 2 þ Lb. since these species belong to different phylogenetic clusters. Remarkably.g. twice the span usually accepted for a single genus. Source: Adapted from Sharpe [117] and Kandler and Weiss [113]. . fructose-1. casei Lb. acidophilus was placed in group Aa and Lb. b. Inducible by pentoses. salivarius Group II. see Sec.) In the most recent comprehensive description of the genus. brevis Lb. Facultatively heterofermentative þ 2 þa þ þb Lb. All Rights Reserved. Thus. IV. Even if the situation was more difficult for the rod-shaped LAB. acidophilus ¨ Lb. However. Obligately homofermentative 2 2 2 þ 2 Lb. helveticus Lb. Lb. this division is still valid to a considerable degree. biochemical. plantarum Lb.. the subgenera of Lactobacillus were created: Thermobacterium. This range is 32 – 55%. salivarius in group Ab. The physiological basis for the division is (generally) the presence or absence of the key enzymes of homo. Such a definition is comparable to an arrangement where all the coccoid LAB were included in one genus.[113. reuteri Characteristic Pentose fermentation CO2 from glucose CO2 from gluconate FDP aldolase present Phosphoketolase present When fermented. The heterogeneity is reflected by the range of mol% G þ C of the DNA of species included in the genus. which essentially is rod-shaped lactic acid bacteria. Table 2 Arrangement of the Genus Lactobacillus Group I. encompassing species with a large variety of phenotypic. e. sakei Group III.

bulgaricus). They are generally the most acid-tolerant of the LAB[21] and will.. terminate many spontaneous lactic fermentations such as silage and vegetable fermentations. carnis. based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S – 23S ribosomal intergenic spacer regions (ISRs). plantarum. mol% G þ C of the DNA. that new species are constantly being described.Hoang-Dung TRAN and friends The classical ways of distinguishing between species of lactobacilli have been carbohydrate fermentation patterns. For detailed information on this topic. In the first few years of the new millenium (2000 – 2002).E).119] (see below). electrophoretic mobility of the LDH. Lb.118. Different PCR techniques and direct sequencing of PCR fragments (e.[123] D. was recently developed. pH 9). therefore. details of the classification of species of lactobacilli are not discussed further. but proper classification may also require analysis of the peptodoglycan. however. can be found in many habitats. delbrueckii subsp.[28] Carnobacteria are characteristically found in meat and meat products. divergens..116] Species-specific oligonucleotide probes (derived from rRNA sequences) for a growing number of lactobacilli are now available.[28. and growth at certain temperatures. piscicola. However. for distinguishing between C. All Rights Reserved... bulgaricus (previously Lb. the sourdough organism Lb.[28] A simple identification key. brevis. and Lb.g.g. carnobacteria grow at relatively high pH (e. C. casei. Lb. the reader is referred to the previously mentioned review by Hammes and Vogel. Lactobacilli are widespread in nature. of rRNA genes) are also offering new possibilities for rapid identification and classification of lactobacilli and LAB in general (see Sec.[107] Lactobacilli are also associated with the oral cavity. and DNA-DNA homology studies. divergens.[121] Furthermore.117] These characteristics are still useful. sanfransiscensis and the yogurt-associated Lb.[122] In addition.[10] Initially. Inc. configuration of lactic acid produced. and vagina of humans and animals. a genus-specific probe has been developed.g. Phylogeny of the Lactic Acid Bacteria Comparisons of the sequence of rRNAs is now regarded to be the optimal measure for determining true phylogenetic relations among bacteria.[54. it is not easy to envisage how a new classification system would appear.[114] Note.113] Later studies showed that these bacteria were separate from lactobacilli and warranted a separate genus[28] and that the metabolism of glucose was predominantly homofermentative. Others are more specialized and are found only in certain niches. approximately 20 new Lactobacillus species were validly published.[59] A rapid method for identification of Carnobacterium isolates from food. these comparisons were made by DNA-rRNA hybridizations or oligonucleotide cataloguing (i. gastrointestinal tract.[81] possibly providing simpler classification schemes. Due to the scope of this chapter. since it would be difficult to find properties other than rRNA sequences that would constitute a basis for a phylogenetically correct taxonomy.[120] Generally. e.. and many species have found applications in the food industry.[113. growth requirements. sequencing Copyright © 2004 by Marcel Dekker. . the fatty acid composition of carnobacteria differs from that of lactobacilli. e. piscicola. Species of the genus Carnobacterium were originally classified as group III lactobacilli under the designations Lb. where they are able to proliferate even at low temperatures.e. Lb. and typical meat-associated lactobacilli has been published. hydrolysis of arginine. and Lb. while lactobacilli do not.[113. As mentioned. confirmed by DNA-DNA hybridization.g. it has been known for some time that the genus Lactobacillus and its division into the three groups shown in Table 2 are not in accord with natural relationships revealed by phylogenetic analysis[113.117] Some species. III.

a certain clustering is evident.125] The LAB have been considered to form a “supercluster. each of which forms a coherent phylogenetic unit. The “leuconostoc-like” LAB.D.. Inc.[8] but now by direct PCR sequencing of the rRNA genes. Tetragenococcus.118] The details of the phylogenetic relationships between the LAB genera and also between LAB and other genera of the low-G þ C subdivision were revealed mainly by the extensive work of M. clostridia) and facultatively or strictly aerobic species (e.[44. Microbacterium.” which lies phylogenetically between the strictly anaerobic species (e. The genera Aerococcus and Tetragenococcus could be included in this cluster as peripheral members. although the evolutionary distance to this genus is large. and the following discussion is based on the work by Collins and coworkers. which reflects the mol% G þ C in the DNA. Staphylococus. Corynebacterium. This is the case for Aerococcus. although this tendency is not as strong as for the Enterococcus group.[10. Leuconostoc sensu stricto.70.[10] From the data obtained from both oligonucleotide cataloguing and rRNA sequencing. i. this group of genera appears somewhat closer phylogenetically to aerobes and facultative anaerobes of the low-G þ C subdivision than to the remaining LAB. pontis) clearly belonging to the low-G þ C subdivision have a G þ C content in that range.43.105. As indicated. in accord with their lifestyle. first by the use of reverse transcriptase. Streptococcus sensu stricto. but is rather an interval around 53 – 55%. together with aerobes and facultative anaerobes like Bacillus.124] both phylogenetically and physiologically. i.. and Listeria and anaerobes such as Clostridium. All Rights Reserved. and Oenococcus also belong to this branch. The genera Lactococcus and Streptococcus are also more related to each other than to other LAB.. Weisella and Leuconostoc sensu stricto. since some species (e. most genera of the LAB are now partly defined and based on phylogenetic measurements. It has been discussed whether Oenococcus represents a case of a rapidly evolving organism. The “splitpoint” is often set at 50%. Actinomyces.g. Copyright © 2004 by Marcel Dekker.[11 – 13. fermentum and Lb.119. resulting in a rapid divergence from related species. Collins with coworkers (but also others) using both reverse transcriptase and PCR sequencing techniques. Advances in molecular genetics techniques have led to methods for sequencing long stretches of rRNA. one can imagine that the adaptation to an unusual habitat (wine) has required extensive changes in the genome. Carnobacterium.124. and Vagococcus form a tight cluster and are more related to each other than to any other LAB. It is common to designate the 2 gram-positive phyla the high-G þ C and the low-G þ C subdivision. Lb.71.g. Arthrobacter. Propionibacterium. staphylococci and bacilli). Oenococcus.101.e. The schematic phylogenetic tree of the LAB as a group.[113. and Ruminococcus. Furthermore. and Streptomyces. Enterococcus. The computerized methods now available for handling large amounts of sequence data have made it possible to construct meaningful phylogenetic trees of the entire bacterial kingdom as well as details of certain parts of it. The gram-positive bacteria cluster in 2 of the 12 major eubacterial phyla (but not all bacteria in these phyla have a gram-positive cell-wall).g.. Enterococcus.e. shown in Fig. Lactococcus. . Among these. Carnobacterium. Vagococcus. 1. are clearly related.104] Since the closest relatives of Oenococcus seem to be the “leuconostoc-like” LAB. Peptococcus.Hoang-Dung TRAN and friends of cleavage products of rRNA).116] This is not entirely true.[10. and Weissella. it has been shown that the gram-positive type cell wall has a relatively strong phylogenetic relevance. Thus..118] The low-G þ C or the Clostridium subdivison includes all LAB. Micrococcus.[105. The high-G þ C or Actinomycetes subdivision encompasses genera such as Bifidobacterium. “on the threshhold of anaerobic and aerobic life”.

as the designation indicates. Lb.g. form a supercluster within LAB. casei – Pediococcus group. Note: Evolutionary distances are approximate. previously regarded as heterofermentative lactobacilli. included in the latter group are all species of Pediococcus. which is no surprise. all heterofermentative. All Rights Reserved.. acidophilus. . as shown by the phylogenetic structure of Lactobacillus-Pediococcus and perfectly illustrated by the new genus Weissella. All pediococci except P. Inc.e. of the obligately homofermentative (group I) lactobacilli (e. and Lb. Lb. comprising all other lacobacilli. now belong to the genus Weissella [13]]. helveticus. dextrinicus form a tight cluster within the Lb. the remaining obligately homofermentative. delbrueckii with subspecies. What conclusions can be drawn from the phylogenetic investigations of LAB? One obvious conclusion. jensenii) and a few facultatively heterofermentative (group II) lactobacilli and (b) the Lb. including some aerobic and facultatively anaerobic gram-positives of the low G þ C subdivision. but notably not all. possibly each worthy of genus status. and most of the facultatively heterofermentative lactobacilli [note that some species. The remaining LAB genera. which contains many. Lactobacillus and Pediococcus. casei – Pediococcus group.[125] These clusters do not correlate with the physiological subdivision of the genus Lactobacillus as described above (see Table 2). delbrueckii group..Hoang-Dung TRAN and friends Figure 1 Schematic. In addition. i. Another conclusion is that future taxonomic Copyright © 2004 by Marcel Dekker. is that morphology is a poor indicator of relatedness. The clusters are (a) the Lb. Lb. unrooted phylogenetic tree of the lactic acid bacteria. which can be divided into two subclusters.

[13. curvatus. Inc. All Rights Reserved. LAB seem to be phylogenetically intermixed with the aerobic and facultatively anaerobic genera of the low-G þ C subdivision of gram-positive bacteria.[128] and Lb. Lb. New Tools for Classification and Identification The classification of LAB. and the list of available probes published by Pot et al. many typical lactobacilli such as Lb.[78. it is not clear whether the Enterococcus group is less related to the strict anaerobes than.g. .[74] and even for distinguishing between the subspecies lactis and cremoris of Lc. acidophilus. and Lb. but would it be generally accepted? Another solution would be to define smaller phylogenetic clusters as new genera. helveticus.[76] 16S rRNA sequence data from LAB have been accumulated during recent years. direct sequencing of the 16S rRNA gene has emerged as the most powerful and relatively easy one-step method for classification of bacteria. Genus.130] lactobacilli from different niches.[19. Therefore.and group-specific probes have been developed for such purposes.82] Copyright © 2004 by Marcel Dekker. described above. Consequently. However. scattered reports including some rRNA sequences and earlier oligonucleotide cataloguing[118] suggest that the evolutionary distance between these groups is quite large. This has recently been supported by genome sequencing. Listeria. these characteristics may not be enough to definitely assign a strain to a particular species. In 1983 it was suggested that pediococci be included in an “extended” Lactobacillus genus. it is preferable that there be some correlation between the phylogenetic position of a certain strain or species and its phenotype. For certain applications (e. The problem lies in how to find the right characteristic(s) that will correlate with true relationships. thermophilus.Hoang-Dung TRAN and friends revisions are still needed.59. However. E. delbrueckii.[126] Phylogenetically this could be correct. it seems that some LAB “overlap” with some of the more aerobic genera such as Bacillus. Rather. In practice. As pointed out by Woese [10].[134] distinguishing vagococci from other LAB.[82] S. The determination of 16S rRNA sequences for the elucidation of the phylogeny of the LAB around 1990 (see above) initiated a rapid development of DNA probes for identification of these bacteria.. in the routine identification of isolates. lactis.20] No comprehensive rRNA sequence comparisons between LAB and the strict anaerobes have been made. As a group. fermentum would end up in different genera.[135] has of course expanded considerably since then. with the availability of rapid and automatic DNA sequencing technology. for instance.g..[127] Lb. casei –Pediococcus group. The main remaining problem is how to deal with the genera Lactobacilus and Pediococcus. for Lb.[129] Identification of LAB with the use of 16S (or 23S rRNA)-targeted probes was developed and used for lactococci and enterococci. although much has been done during the last 15 years to clarify natural relations and to define genera from such analyses. LAB are not clearly separated from all other groups of the low-G þ C subdivision of the gram-positives. the statement that LAB are phylogenetically positioned between the strict anaerobes and the aerobes in accordance with their lifestyle is not correct. analysis of food samples) it may also be interesting to determine the occurrence of specific groups of LAB. e. Before that there had been some attempts to develop DNA probes based on selected fragments of a DNA library. is largely based on phenotypic and biochemical characters. Today.[131 – 133] carnobacteria from meat. and Staphylococcus. In particular. the Lb. plantarum.

NotI) are used. Lb. reuteri. acidophilus group. more commonly known as ribotyping. showed heterogeneous ribopatterns. but the digests can be run on conventional gels.[152 – 154] PFGE is therefore used as the principal method for creating unique “fingerprints” of defined commercial strains by vendors for starter cultures and/or probiotics. One of the targets for such an amplification are obviously rRNA genes. With this technique it is possible to amplify a gene or a part of a gene from a very limited number of cells for subsequent DNA sequencing. while Lb.149 – 151] Other genotypic fingerprinting methods are based on restriction endonuclease cleaving of the chromosomal DNA. The PCR technique is becoming more and more useful for classification purposes. PFGE is considered the gold standard in classifying bacterial strains with genotypic fingerprinting at the strain level because of its high discriminatory power. and successful use for LAB has been reported.[145] Reproducibility is claimed to be higher than for RAPD. e.[123. it has become an easy task to determine the 16S rRNA sequence from any bacterium in a short time.[146 – 148] Intergenic spacer sequence polymorphism in variants has also been used in PCR-based methods (tDNAPCR. When rare-cutting enzymes (e. a quite homogeneous species phenotypically. which exploits conserved repetitive DNA sequences in bacterial genomes.134] or actually replace them since the oligonucleotide probes designed from 16S rRNA sequencing also can be used in PCR applications. ISR-PCR) for species identification in LAB. reuteri.Hoang-Dung TRAN and friends Bacteria typically have five to seven copies of each rRNA gene in the chromosome. but reproducible results require highly standardized conditions..82.[137] and pediococci. phenotypically heterogeneous.[144] Another fingerprinting PCR-based method with similarities to RAPD is REP-PCR (with variants known as ERIC-PCR and BOX-PCR).[137] This method.[144] The simplicity of this method makes it very attractive. and the method is often referred to as just PFGE. Automated instruments that generate large databases of reproducible ribopatterns are available. pulse field gel electrophoresis (PFGE) is used to separate the fragments. plantarum strains. lactococci.[142. which contains six species with similar phenotype that are considered important as candidates for probiotic strains.141] RAPD has been shown to be applicable for distinguishing strains belonging to the so-called Lb. This has been shown also for LAB.[138] It is interesting to note that Lb.g. The method of breaking the cells to obtain a crude DNA preparation is one of the critical steps in this regard. In a study of several Lb. has to be evaluated from case to case as to its applicability in strain or species recognition. The most commonly known is randomly amplified polymorphic DNA (RAPD). had more homogeneous ribopatterns.[140. plantarum. Inc. Using enzymes that cut more often complicates the analysis (many more fragments) and requires sophisticated statistical analysis. and this method has replaced the reverse transcriptase technique for collecting rRNA sequence data for phylogenetic analysis.[79] Lb.. e. This has been exploited in a restriction fragment length polymorphism (RFLP) molecular typing method. A number of fingerprinting techniques based on PCR have also been developed. creating large and relatively few fragments.g. All Rights Reserved.. PCR can also be used in combination with probing techniques[78.[139] possibly setting the standard for interlaboratory work with this identification method.[136] This method appears useful for species and subspecies recognition in some cases. With automated sequencing systems and convenient direct PCR sequencing methods.143] although several methods can be used for this particular cluster of species (see below). although highly standardized. plantarum. The method is often referred to as restriction endonuclease analysis Copyright © 2004 by Marcel Dekker.[100] but not in others. therefore.g. . RAPD was found to be more suitable to discriminate between different subtypes of species than ribotyping.

Inc.[137. REA. probably depending on what methods a particular laboratory starts to use. This may lead to significantly different end product patterns. One of the most extensive and informative studies is still the collective ˚ work done by M.[155 – 157] Amplified fragment length polymorphism (AFLP) is a PCR-based REA in which a selected portion of the fragments are amplified. automated PCR sequencing of rRNA. This section will describe the well-known fermentation pathways and how various sugars are fermented. and RAPD. acidophilus group. They used a set of Lb.[160] and [161]..Hoang-Dung TRAN and friends (REA) and has been successfully used for classifying LAB with very high discriminatory power.[163] i. reuteri and Lb.[135] and the similarity clusters clearly correlate with results based on genetic data. A database of digitized and normalized patterns from a large number of LAB was constructed.e. In contrast to methods analyzing patterns derived from DNA. however. chemotaxonomic. METABOLISM OF LACTIC ACID BACTERIA The essential feature of LAB metabolism is efficient carbohydrate fermentation coupled to substrate-level phosphorylation.e. M. Stahl.159] This work elegantly shows that each method has advantages and disadvantages and that one single method is not the solution for all applications. visualized. Therefore. It is clear. see Refs. and genotypic methods. the predominant end product is. a number of alternatives to classical phenotypic/biochemical identification of LAB have emerged since 1990. interlaboratory comparisons of patterns may not be reliable. that LAB adapt to various conditions and change their metabolism accordingly. Johansson.[143] In several studies the genetic methods described above have been compared in classifying LAB. The method was considered very useful in classifying LAB of the Lb. which may be of importance in their natural habitat. growth conditions are extremely important since the protein composition of the cells may vary depending on media.[158] The method has good resolution at the strain level and has the potential to replace PFGE as a standard fingerprinting method.[135. but rather that the methods complement each other. As for other methods involving a statistical analysis of banding patterns. Another technique that has proven to be very useful in the classification of LAB is soluble protein patterns.-L. The technique involves polyacrylamide gel electrophoresis of whole cell proteins and statistical analyses of the patterns obtained (for reviews. ribotyping. For thorough identification/classification in bacterial systematics. i. Each method has its advantages and disadvantages.155 – 157. and the banding pattern analyzed as for other fingerprinting methods. Copyright © 2004 by Marcel Dekker. etc.138. All Rights Reserved.160. IV.162] The method can be used directly as a screening method to assign a particular strain to a species when the pattern is compared with those in the database. . It will also show some of the more unusual features of LAB metabolism.50% of sugar carbon).144. and coworkers. Which one to use is often a matter of taste. using several phenotypic. standardization is the key to reproducible results. of course. lactic acid (. it is still recommended to apply a polyphasic approach. plantarum strains and systematically characterized these with phenotypic tests.[161] To summarize. The generated ATP is subsequently used for biosynthetic purposes. temperature.. DNA-DNA homology. LAB as a group exhibit an enormous capacity to degrade different carbohydrates and related compounds. rRNA sequences and DNA-DNA homologies. Generally.

ethanol) in addition to lactic acid. In general. However. whereas “heterofermentative LAB” are those that use the 6-PG/PK pathway. and the metabolism is referred to as a homolactic fermentation. a high-energy phosphate bond is required for activation of the sugar. and CO2 and 1 mol ATP/mol glucose. oenococci.g. The remaining pentose-5-phosphate is split by phosphoketolase into GAP and acetyl phosphate. The transport and phosphorylation of glucose may occur as outlined. Since this metabolism leads to significant amounts of other end products (CO2. ethanol.6-diphosphate (FDP).e. A conversion factor of 0. it is referred to as a heterolactic fermentation. 2). glucose) fermentation within LAB (Fig. followed by decarboxylation. the hexose monophosphate shunt and. Glycolysis (Embden-Meyerhof-Parnas pathway). Hexose Fermentation As mentioned in the classification section in this chapter. and it is perhaps appropriate to add a note of caution. homolactic fermentation of glucose results in 2 mol of lactic acid and a net gain of 2 ATP per mol glucose consumed. excess sugar and limited access to oxygen. Some species use the phosphoenolpyruvate : sugar phosphotransferase system (PTS). which is split by a FDP aldolase into dihydroxyacetonephosphate (DHAP) and glyceraldehyde-3-phosphate (GAP). it should be noted that glycolysis may lead to a heterolactic fermentation (meaning significant amounts of end products other than lactic acid) under certain conditions and that some LAB regarded as homofermentative use the 6-PG/PK pathway when metabolizing certain substrates. When no additional electron acceptor is available (see Sec. Under normal conditions. thereby reoxidizing the NADH formed during the earlier glycolytic steps. All Rights Reserved.. IV.[164] It is characterized by initial dehydrogenation steps with the formation of 6-phosphogluconate. which also involves phosphoketolase but does not have 6-phosphogluconate as an intermediate.. V. I will refer to it as the 6-phosphogluconate/phosphoketolase (6-PG/PK) pathway. there are two major pathways for hexose (e. The terminology regarding these pathways and the bacteria that use them is rather confusing. pyruvate is reduced to lactic acid by a NADþ-dependent lactate dehydrogenase (nLDH). This will be discussed in more detail later. i. the 6-phosphogluconate pathway.9 from sugar to end-product carbon is common and probably reflects an Copyright © 2004 by Marcel Dekker. In theory. and weissellas. GAP (and DHAP via GAP) is then converted to pyruvate in a metabolic sequence including substrate-level phosphorylation at two sites. resulting in lactic acid formation. acetyl phosphate is reduced to ethanol via acetyl CoA and acetaldehyde.e. In practice. in which phosphoenolpyruvate is the phosphoryl donor (see Sec.Hoang-Dung TRAN and friends A. lactic acid is virtually the only end product. such as the pentose phosphate pathway. used by all LAB except leuconostocs. i.. Heterolactic fermentation of glucose through the 6-PG/PK pathway gives 1 mol each of lactic acid. used by Kandler and Weiss[113] in Bergey’s Manual. The other main fermentation pathway has had several designations. thereby recognizing a key step in the metabolic sequence (the phosphoketolase split) and at the same time distinguishing it from the bifidum pathway. the term “homofermentative LAB” refers to those in the group that use the glycolytic pathway for glucose fermentation.D). is characterized by the formation of fructose-1.B). Inc. A redox balance is thus obtained. In either case. Major Fermentation Pathways 1. group III lactobacilli. the pentose phosphoketolase pathway. . GAP is metabolized in the same way as for the glycolytic pathway. these theoretical values are seldom obtained. transport of free sugar and phosphorylation by an ATP-dependent glucokinase.

6-diphosphate aldolase. 4. 7. Copyright © 2004 by Marcel Dekker. pyruvate kinase. 10. Embden-Meyerhof-Parnas pathway). 9. All Rights Reserved. 6. Inc. lactate dehydrogenase.Hoang-Dung TRAN and friends Figure 2 Major fermentation pathways of glucose: (A) homolactic fermentation (glycolysis. 6-phosphogluconate dehydrogenase. Selected enzymes are numbered: 1. alcohol dehydrogenase. 8. fructose-1. 3. 5. (B) heterolactic fermentation (6-phosphogluconate/phosphoketolase pathway). acetaldehyde dehydrogenase. glucose-6-phosphate dehydrogenase. glyceradehyde-3-phosphate dehydrogenase. Glucokinase. 2. . phosphoketolase.

but separate enzymes are required for the metabolism of the tagatose derivatives. 3A). Copyright © 2004 by Marcel Dekker. These complex media may also contribute to other fermentation balances and to the formation of other end products. Hexoses other than glucose. which uses a PTS for uptake of this sugar. 165 and Sec. One important exception is galactose metabolism in LAB. E. and Lb. are fermented by many LAB. In these species. even though most growth factors (e. Tagatose is a stereoisomer of fructose.Hoang-Dung TRAN and friends incorporation of sugar carbon into the biomass. nucleotides. such as mannose. and fructose. IV. lactis. casei. since compounds like organic acids. galactose.g. amino acids.g. and sugar residues can alter the fermentation.[115] The presence of oxygen may also have a significant effect on the metabolism (see Ref. (B) Leloir pathway. Lc. Inc. All Rights Reserved. The sugars enter the major pathways at the level of glucose-6-phosphate or fructose-6-phosphate after isomerization and/or phosphorylation. . The tagatose pathway coincides with Figure 3 Galactose metabolism in lactic acid bacteria: (A) tagatose-6-phosphate pathway.. e. the galactose-6-phosphate formed by the PTS is metabolized through the tagatose-6-phosphate pathway[166] (Fig. amino acids. faecalis. and vitamins) are supplied in excess in the rich media frequently used. in particular acetic acid..C).

. lactis.178] An interesting feature of maltose metabolism in Lc.g. S. only metabolize the glucose moiety after transport of lactose and cleavage by b-gal. lactose enters the cytoplasm as lactose phosphate.[178] Sucrose fermentation mediated by a permease system is initiated by the cleavage of the sugar by sucrose hydrolase to yield glucose and fructose. Lb.174] However.[172. when sugar PTS are involved.Hoang-Dung TRAN and friends glycolysis at the level of GAP.. In some lactococci. However. delbrueckii subsp. while galactose is excreted into the medium. This knowledge is relevant for the understanding of sugar metabolism in general[171] and of the processes of milk fermentation in particular.172. thermophilus has also been thoroughly studied. 3B). thermophilus. which is cleaved by phospho-b-D -galactosidase (P-b-gal) to yield glucose and galactose-6-phosphate. which enter the major pathways. disaccharides enter the cell either as free sugars or as sugar phosphates. Lb. lactis is one of the most well-studied systems of sugar fermentation occurring in LAB. whereas b-glucose-1-phosphate probably is a precursor for cell wall synthesis. the free disaccharides are split by specific hydrolases to monosaccharides. whereas galactose-6-phosphate is metabolized through the tagatose-6-phosphate pathway. sucrose is transported by sucrose PTS and a specific Copyright © 2004 by Marcel Dekker.[176] Galactose excretion has been attributed to a low galactokinase activity[174.[98.[174. at least those used as dairy starters.115] 2. and Lb. Inc. A permease system for transport seems to be operational.e. The enzyme system of the lactose PTS and P-b-gal are generally inducible and repressed by glucose. bulgaricus.[22.175] Lactose transport and metabolism in the economically important species S.98. casei is also well characterized. which then enter the major pathways described above. Disaccharide Fermentation Depending on the mode of transport.[98. contain a lactose PTS[168. since both P-b-gal and b-gal activity were found in the same strains. All Rights Reserved.[115] The lactose metabolism of Lc.177] but is also energetically favorable and a feature of the lactose transporter. . delbrueckii subsp. lactis 65. Only the glucose moiety is used in glycolysis. i.173] which may then enter the major pathways. Many strains in this category also have the capacity to transport galactose with a permease and convert it to glucose-6-phosphate via the Leloir pathway (Fig. an equally common way to metabolize lactose among LAB is by means of a lactose carrier (permease) and subsequent cleavage by b-galactosidase (b-gal) to yield glucose and galactose.174] Some of the thermophilic LAB. In the former case. e. lactis. since the artificial substrate used for P-b-gal (ortho-nitrophenylgalactose phosphate) may be hydrolyzed by b-gal or by a phosphatase yielding the substrate for b-gal.169] A lactose PTS from Lb. Some reports on this subject would suggest that many LAB contain both a lactose PTS and a lactose permease system for lactose metabolism.[167] This pathway is also used by galactose-fermenting LAB that transport galactose with a permease and lack a galactose PTS. By far the most studied disaccharide metabolism in LAB is lactose fermentation. acidophilus. In the latter case.[170] In these strains. Most strains of Lc. specific phosphohydrolases split the disaccharide phosphates into one part free monosaccharides and one part monosaccharide phosphates.[22.[176] Maltose fermentation among LAB has been studied most extensively in lactococci. Glucose is phosphorylated by glucokinase and metabolized through the glycolytic pathway.1 is that maltose is cleaved by a maltose phosphorylase into glucose and b-glucose-1-phosphate. low P-b-gal activity in strains with high b-gal activity may represent an artefact.

which enter the common pathways. but this is not the case.[182] Many group II and group III lactobacilli also ferment gluconate. All Rights Reserved. Presumably. the reduction of acetyl phosphate to ethanol becomes redundant. yielding a heterolactic fermentation by species regarded as homofermentative. The metabolism has been studied in E.[180] Fermentation of other disaccharides.[115] These compounds can then be metabolized by the lower half of the 6-PG/PK pathway (Fig. all genera of LAB are pentose positive with one exception. Instead. 3. acetyl phosphate is used by the enzyme acetate kinase in a substrate-level phosphorylation step yielding acetate and ATP. In dextran production by Ln. mesenteroides. . Fermentation of pentoses thus leads to production of equimolar amounts of lactic acid and acetic acid. gluconate is fermented by the 6-PG/PK pathway. Fermentation of Pentoses and Related Compounds Pentoses are readily fermented by many LAB. resulting in the respective monosaccharides (or monosaccharide phosphates). casei. gluconate is transported by an inducible gluconate-PTS and the resulting 6-phosphogluconate enters the 6-PG/PK pathway.Hoang-Dung TRAN and friends sucrose-6-phosphate hydrolase cleaves the sucrose-6-phosophate to glucose-6-phosphate and fructose. Like pentoses.[113] presumably in a metabolic sequence similar to that for E. 2B). this is an appropriate place to mention gluconate fermentation. Lb. Although gluconate is not a pentose. such as cellobiose.. Copyright © 2004 by Marcel Dekker. These are translocated through the membrane by specific pentitol PTS. can grow at the expense of pentitols.[179] Sucrose may also act as a donor of monosaccharides for exopolysaccharide formation in certain LAB. Homofermentative LAB that utilize pentoses generally do so in the same way as heterofermentative LAB.[179] The sucrose PTS and sucrose-6-phosphate hydrolase are induced by the presence of sucrose in the medium. the pentoses are phosphorylated and converted to ribulose-5-phosphate or xylulose-5-phosphate by epimerases or isomerases. the metabolism is mediated by specific transport systems and hydrolases. faecalis. faecalis. although transport may be mediated by a permease in some cases. dextransucrase.[115. Since a dehydrogenation step is necessary before the phosphoketolase reaction. Inside. but a similar pathway may also exist in Lb. and trehalose. A few species of LAB.[182] In E. some acetyl phosphate has to be reduced to ethanol in order to maintain the redox balance. disregarding some strain and species differences. faecalis. sucrose is cleaved by a cell wall – associated enzyme. and since no dehydrogenation steps are necessary to reach the intermediate xylulose-5-phosphate. This would imply that only heterofermentative LAB can utilize pentoses. has not been studied to any large extent.g.181] The heterolactic fermentation of pentoses results in a different end product pattern compared to glucose fermentation. melibiose. the group I lactobacilli. The resulting pentitol phosphates are oxidized to pentose phosphates by dehydrogenases and subsequently metabolized through the 6-PG/PK pathway. casei. In general. The glucose moiety is used for dextran synthesis and fructose is fermented in the usual manner. The ability to ferment these sugars differ between different species of LAB. e. which can be performed by some LAB. The phosphoketolase of these species is induced by substrates fermented by the 6-PG/PK pathway and repressed by glucose. some ethanol is produced from acetyl phosphate because of the need to reoxidize the NADH formed during the initial dehydrogenation. No CO2 is formed.[182] Similar to gluconate fermentation. Inc. specific permeases are used to transport the sugars into the cells. In fact.

Inc. As mentioned previously. resulting in the use of glycolysis for hexose fermentation. including the remaining LAB (i. probably by the glycolytic pathway. streptococci. FDP aldolase and phosphoketolase. Some lactobacilli regarded as obligately heterofermentative may in fact possess the apparent missing key enzyme. respectively. All Rights Reserved.[114] The first category includes the group I lactobacilli and some individual species from other genera. brevis (group III) was shown to ferment fructose by the glycolytic pathway including a fructose-inducible FDP aldolase. the borders between the metabolic categories of LAB may not be as absolute as previously thought.115] This leads to the obvious difference in end product formation from glucose. Obligately homofermentative species possess a constitutive FDP aldolase and lack phosphoketolase. it is possible to divide the group broadly into three metabolic categories. different species of LAB produce either exclusively acid. These LAB are thus homofermentative with regard to hexoses and heterofermentative with regard to pentoses and some other substrates and should. pediococci. pentoses (and presumably gluconate and pentitols when fermented) induce the synthesis of phosphoketolase. . resulting in a heterolactic fermentation. exclusively D -lactic acid. At any rate.[184] It was suggested that transaldolases and transketolases of the classical pentose phosphate pathway were involved in converting the substrate for normal glycolysis. or predominantly one form but measurable amounts of the other. which are obligately homofermentative. Recent reports suggest that there exists some deviation from the normal patterns described above.113] they should probably be regarded as facultatively heterofermentative.[113. They resemble the obligately homofermentative LAB in that they possess a constitutive FDP aldolase. 5.115] The position of species in the genus Carnobacterium is somewhat unclear.[39. whereas the opposite holds for obligately heterofermentative species. meaning that only the 6-PG/PK pathway is available for sugar fermentation. group III lactobacilli. The third category. and weissellas.[183] The fermentation of a pentose by Lc. but also to the inability of the group I lactobacilli to attack pentoses (and gluconate).Hoang-Dung TRAN and friends 4. oenococci.185] This depends on the L -lactic Copyright © 2004 by Marcel Dekker. lactis IO-1 was shown to result almost exclusively in lactic acid under certain conditions. and vagococci). A strain of Lb.[113. approximately equal amounts of both.113.[28. lactococci.[113] Later. since gas and acetic acid were produced in significant amounts. tetragenococci. more detailed studies showed that glucose is fermented almost entirely to lactic acid.. Whether these examples represent some exceptional cases or something more general is not clear at the moment. be termed facultatively heterofermentative. which are obligately heterofermentative. and this is in fact the basis for the division of the genus into three groups. divergens). the other products arising from metabolism of components in the medium other than glucose and/or some deviation of the pyruvate formed by glycolysis. These bacteria were first classified as heterofermentative (then under the designation Lb. holds an intermediate position. The genus Lactobacillus contains species that can be placed in all three categories. The second category includes leuconostocs.[120] Since carnobacteria generally ferment ribose and gluconate. Configuration of Lactic Acid During the fermentation of sugars. meaning that sugars only can be fermented by glycolysis. The apparent difference on an enzyme level between these two categories is the presence or absence of the key enzymes of glycolysis and 6-PG/PK pathway. Sugar Fermentation and Metabolic Categories of LAB From the descriptions of sugar fermentation patterns of LAB presented above.e. therefore. group II lactobacilli and most species of enterococci.

L -lactic acid is the major form produced in the early growth phase and D -lactic acid in the late to stationary phase.and L -lactic acid is not clear. the use of external electron acceptors. the formation of acetyl CoA can be required for lipid biosynthesis. this metabolism proceeds to a significant degree only if there is a pyruvate surplus in the cell relative to the need for NADþ regeneration. but then serving an anabolic role. Pyruvate holds a key position in many fermentations in serving as an electron (or hydrogen) acceptor for this regeneration step. 4. termed a racemase. plantarum did not change the growth rate in laboratory media or the global lactic acid concentration at any stage of the culture. Fates of Pyruvate It is well known that LAB may change their metabolism in response to various conditions. The alternative fates of pyruvate are depicted in Fig. This will be discussed below. IV. resulting in a different end-product pattern than seen with glucose fermentation under normal conditions. Only a few species. Thus. e. if both D . All Rights Reserved. bavaricus. Enterococci and streptococci also possess this type of LDH. The absence of L -lactic acid was simply accompanied by an increase in D -lactic acid production. as these may be connected to each other. or both. The oxidation results in the formation of NADH from NADþ. In most of these cases.[185] The pH and internal pyruvate concentration have been thought to influence the activities of the LDH and thus the ratio of the isomers at different growth phases.[185] In this case. the change can be attributed to an altered pyruvate metabolism.Hoang-Dung TRAN and friends presence of specific NADþ-dependent lactate dehydrogenases (nLDH) and their respective activities. which subsequently can be used for ATP production by substrate-level phosphorylation. The essential feature of most bacterial fermentations is the oxidation of a substrate to generate energy-rich intermediates.and L -lactic acid. Inctivation of L-nLDH by mutation in a strain of Lb. For instance.g.[188] 1. this is true in both major fermentation pathways used by LAB (Fig. the L -lactic acid initially produced induces the racemase. but they represent a summary of the LAB group as a whole.B). A pyruvate surplus can be created in two ways: (a) a source of pyruvate other than the fermented Copyright © 2004 by Marcel Dekker.[186] indicating that the reduction of pyruvate to lactate is not a rate-limiting step in glycolysis.[185] B. sakei. depending on conditions and enzymatic capacity.[115] Different species may use different pathways. pentosaceus and many lactobacilli change the ratio of the isomers during batch growth. produce an enzyme.3-butanediol (Fig. Lb. Inc. 4) is common among LAB[115] and is very significant technologically in the fermentation of milk. Worthy of note in this regard is the species Lb.[168. which has to be regenerated in order for the cells to continue the fermentation. Generally. LAB use alternative ways of utilizing pyruvate than the reduction to lactic acid. which is activated by FDP (see Sec.[185] but not much is known in this regard.189] However. which converts L -lactic acid to D -lactic acid. Not all of these reactions are used by a single strain. which genetically appears to be a variant of Lb. 2). . casei and lactococci possess an allosteric L-nLDH. there are generally one D-nLDH and one L-nLDH present.and L -lactic acid are formed.. The Diacetyl/Acetoin Pathway The pathway(s) leading to diacetyl (butter aroma) and acetoin/2. sakei with no racemase activity and therefore only produces L -lactic acid[187] P. Under certain circumstances. which results in a mixture of D . Indeed. Lb. The reason for or possible advantage to producing a mixture of D . curvatus and Lb. Some of these reactions may be operational even under normal glucose fermentation.

diacetyl synthase. Dashed arrow denotes a nonenzymatic reaction. 2. pyruvate-formate lyase. acetate kinase. 6. 3. pyruvate dehydrogenase. Important metabolites and end products are framed.Hoang-Dung TRAN and friends Figure 4 Pathways for the alternative fates of pyruvate. 5. . Copyright © 2004 by Marcel Dekker. acetolactate synthase. pyruvate oxidase. Selected enzymatic reactions are numbered: 1. Inc. All Rights Reserved. 4.

nLDH. and ethanol. fermentation.3-butanediol is produced in much larger amounts than diacetyl. cremoris (former name Ln.190. All Rights Reserved. These species may vary somewhat in induction pattern and pH optimum of the enzymes of citrate dissimilation and diacetyl/acetoin production. 4. This topic has been studied extensively. and a review by Hugenholtz[190] covers most of the aspects of citrate metabolism in connection with diacetyl/acetoin production in LAB. The nLDH:s of Lc.[14. By overexpressing NADH oxidase. Oxaloactetate is further decarboxylated to pyruvate and CO2 by oxaloacetate decarboxylase. and ethanol) are formed with decreasing growth rate. The enzyme pyruvate-formate lyase catalyzes the reaction of pyruvate and CoA to formate and acetyl CoA. resulting ultimately in ethanol formation. lactis ferment galactose[167] or maltose. formate.[190] Diacetyl is formed by chemical decomposition of a-acetolactate (nonenzymatic). thus sparing the pyruvate formed by carbohydrate fermentation. and pyruvate-formate lyase. lower intracellular levels of FDP are found in cells of Lc. lactis and Lb.[195.[196] A similar end product pattern is found when some strains of Lc.196] End products formed are lactate. both biochemically and genetically. formate. Citrate is transported into the cell by a citrate permease and cleaved by citrate lyase to yield oxaloacetate and acetate. . lactis (biovar. one of the approaches was to create a pyruvate surplus according to the second possibility. Larger amounts of the products of the pyruvate-formate lyase system (acetate. The former is what occurs in milk. consists of the pyruvateformate lyase system.[178] It has been proposed that the change to a mixed acid fermentation by these strains is due to a reduction of the glycolytic rate. Evidence now clearly favors the route via a-acetolactate since a-acetolactate can be detected as an intermediate in cultures producing diacetyl and an a-acetolactate synthase has been identified in several LAB.. in this case mixed acid. This reaction is favored by aeration and low pH.5 mg/mL). casei are allosteric enzymes with a specific requirement for the key glycolytic intermediate FDP (see Fig. mesenteroides subsp. Historically. lactis subsp.15. 4) was the most important. which is present in significant amounts (1. The Pyruvate-Formate Lyase System Another “branch” of pyruvate metabolism. is now being exploited in metabolic engineering strategies to control diacetyl production.[193] The extensive knowledge obtained in recent years of this system. lactis. oxygen could efficiently act as an electron acceptor and the surplus pyruvate could subsequently be diverted to diacetyl by several strategies. there was some debate as to which of the two possible pathways (Fig. 2) for activity. This affects the levels of glycolytic intermediates and subsequently the activities of the enzymes that compete for pyruvate. casei and Lc. Acetyl CoA may be used either as an electron acceptor. i. but does not contribute to the aroma. Inc. cremoris or Ln.Hoang-Dung TRAN and friends carbohydrate exists in the growth medium and (b) another compound acts as electron acceptor. or both. grown in anaerobic continous culture under substrate limitation. acetate.[115] Most notably. citrovorum). as a precursor for substrate-level phosphorylation via acetyl phosphate. diacetylactis) and Ln. Acetoin and/or 2.e. low sugar concentrations and low pH favor diacetyl/acetoin formation.[185] In general.194] Interestingly.[189. lactis performing mixed acid fermentation compared to those found in cells during homolactic Copyright © 2004 by Marcel Dekker. as mentioned above. where additional pyruvate originates from the breakdown of citrate.191 – 193] In general. shown in Fig.[15] 2. resulting in a change from a homolactic to a heterolactic. The system has been shown to be operational in several species of LAB. the pathway is used by some strains of Lb. Species used in the dairy industry for the purpose of diacetyl production are Lc. with lowering of the dilution rate in the continuous culture system.

202. All Rights Reserved.. Exposed to air. during a semistarved state. The enzyme has been suggested to be involved in aerobic metabolism of Lb. a possible explanation of the elevated pyruvate oxidase level could be that the levels of glycolytic intermediates in some way regulated enzyme synthesis. since pyruvate-formate lyase is inactivated by oxygen.[188. mediated by the enzyme pyruvate oxidase (Fig. lactis can perform a homoacetic fermentation under substrate limitation.[167] Thus. but then primarily under aerobic conditions. or indirect through reactions of oxygen with the flavin-containing enzymes NADH : H2O2 oxidase and NADH : H2O oxidase. The Pyruvate Oxidase Pathway Oxygen has a profound effect on the fate of pyruvate in LAB. 4).[188] indicated that pyruvate dehydrogenase has an anabolic role in producing acetyl CoA for lipid synthesis under aerobic conditions. The Pyruvate Dehydrogenase Pathway Evidence have been obtained that a pyruvate dehydrogenase enzyme complex is active in lactococci. however.[167. the pyruvate dehydrogenase can also play a role in catabolism. 4). Similar to the pyruvate-formate lyase system. This effect may be direct. which forms significant amounts of acetic acid aerobically. Instead.[196] indicating that more ATP/glucose was formed during mixed acid fermentation. Recently.[201] A certain similarity to the regulation of the pyruvate-lyase system in Lc. plantarum. plantarum had much lower growth rate on lactose than on glucose.[201] pyruvate oxidase had the highest activity in the early stationary phase of growth and lactose-grown cells had generally higher levels of the enzyme than glucose-grown cells. this role is probably played by pyruvateformate lyase. 4) and thus resembles the pyruvate-formate lyase system. The excess NADH formed can be reoxidized by NADH oxidases. . 4. energy can be gained by using the pyruvate-formate lyase pathway.[198. The pyruvate-formate lyase system is only active anaerobically. Anaerobically. increased molar growth yield for glucose (Yglc) was obtained in the continuous culture system at low compared to high growth rates. The study by Cogan et al. can thus be noted. known inhibitors of pyruvate-formate lyase.[165] Pyruvate oxidase converts pyruvate to CO2 and acetyl phosphate with the formation of H2O2 (Fig. which has the advantage of not reducing NADþ in the process. Aerated cultures of nongrowing cells of Lc. 3.[197] are also lower in heterolactic cells. the cells respond by regulating certain enzyme activities so as to partly prevent pyruvate from being reduced to lactic acid. caused by either substrate limitation or the nature of the substrate.200] and presumably inactivated when cells are exposed to air.203] The enzyme complex produces acetyl CoA (Fig.199] FDP could nevertheless have an indirect role by stimulating components involved in global control of carbon metabolism (see Sec. all the pyruvate generated from glycolysis is channeled through the pyruvate dehydrogenase complex with acetic acid (and presumably CO2) as Copyright © 2004 by Marcel Dekker. Indeed.178. since there is a substrate-level phosphorylation site involving acetyl phosphate and acetate kinase (Fig. lactis. 4).Hoang-Dung TRAN and friends fermentation. the role of FDP in the shift to heterolactic fermentation has been questioned and the effect of the NADH/NADþ ratio on glyceraldehyde-3-phosphate dehydrogenase and nLDH has been suggested as the main factor. described above. The effect can be rather dramatic. the cells are dependent on a functional pyruvate dehydrogenase for acetyl CoA production.196] Similarily. Inc. which is consistent with the enzyme being extremely oxygen sensitive[197. VI). the levels of triose phosphates.[201] In the study by Sedewitz et al. Since this strain of Lb.[203] In this case.

and carnobacteria. brevis.[203] C. The active role of oxygen as electron acceptor in the metabolism of LAB is further illustrated by the fact that certain substrates are fermented only when oxygen is available. . Reduction of acetyl phosphate (via acetyl CoA) constitutes the ethanol branch of the pathway. additional ATP could be produced (by the acetate kinase reaction) and as a consequence Yglc increased. whereas most of the homofermentative LAB did. are almost unable to ferment glucose Copyright © 2004 by Marcel Dekker. and the systems are often induced by oxygen. This may create a situation similar to the breakdown of citrate described earlier. Inc. oxygen acts as an external electron acceptor. This is especially true for the fermentation of reduced compounds. since this compound can be used in substrate-level phosphorylation with the production of ATP. Examples are oxygen-dependent glycerol fermentation by P. Under these conditions.[207] It is also well known that some heterofermentative lactobacilli. Lucey and Condon[205] showed that strains of Leuconostoc sp.or four-electron transfer. The products of the reactions are either NADþ and H2O2 or NADþ and H2O. i. the NADH oxidases do not compete with nLDH.[188] homofermentative lactobacilli. thus supporting the role of NADH oxidase in aerobic metabolism. NADH oxidases seem to be widespread among LAB. However. Oxygen as Electron Acceptor In reactions with NADH oxidases. a pyruvate surplus is not created and lactic acid remains the main product of pyruvate metabolism. has been shown for Lc. A prerequisite for this metabolism to occur is that the NADH formed during both glycolysis and in the pyruvate dehydrogenase reaction can be reoxidized by NADH oxidases. A NADH oxidase – deficient mutant did not shift from ethanol to acetate production and Yglc was the same aerobically and anaerobically. pentosaceus [206] and mannitol fermentation by Lb. which efficiently prevented ethanol formation. acetate was formed from acetyl phosphate. most notably strains of Lb. depending on whether the enzyme mediates a two. Increased production of acetoin in aerated cultures.[203] NADH oxidases may compete efficiently with nLDH in homofermentative LAB. oxygen can play an active role. The effect was attributed to an active NADH oxidase. In many cases this can be advantageous to LAB. In addition. This reaction may mask initial NADH : H2O2 oxidase activity. a pyruvate surplus available for metabolism through the diacetyl/acetoin pathway. but rather with acetaldehyde dehydrogenase and alcohol dehydrogenase. the enzymes of the ethanol “branch” of the 6-PG/PK pathway (Fig. compared to unaerated.[204] The probable explanation is that in heterofermentative LAB. lactis. All Rights Reserved. acetyl CoA) as an electron acceptor in addition to pyruvate. This route is in a way a waste of acetyl phosphate.e. the nLDH has probably very low activity and cannot effectively compete for pyruvate. 2 and see below). the growth rate was higher with aeration. which use H2O2 as electron acceptor with the formation of H2O. Most LAB also possess a NADH peroxidase. Accordingly.[204] Worthy of note is that heterofermentative LAB (leuconostocs and group III lactobacilli) did not respond to aeration with acetoin production. Heterofermentative LAB may circumvent this waste by using external electron acceptors. LAB that ferment glucose by the 6-PG/PK pathway use acetyl phosphate (or more accurately.[204] The reason for this is not clear. Instead.Hoang-Dung TRAN and friends the final product. such as polyols. The shut-off of the ethanol branch of the 6-PG/PK pathway in the presence of oxygen seems to be very common among heterofermentative LAB.. doubled the Yglc in aerated cultures compared to unaerated. casei. an increase of Yglc is not always seen. As was hinted above.

165.214] an opinion that has some support. The true extent of this will certainly be revealed in the near future as many more LAB genomes will be available. involving NADþ-dependent and/or NADþ-independent LDH.[31 – 33] but in fact also observed in other LAB such as lactococci and leuconostocs. Inc. they are unable to synthesize porphyrins. both with regard to the actual relationship to oxygen as described above and in the phylogenetic position of these bacteria (see Sec. III. lactobacilli. All Rights Reserved.[19] Some of them have been characterized to some extent.g.. 215). oxygen interacts with LAB metabolism in a very active way.[29] Apparently.[210] The ATP yield would be 1 ATP per mol lactate consumed. see Ref.[210. several organic compounds can serve the same purpose. e. lactis IL1403 genome[18.[33. Other Electron Acceptors LAB are not restricted to oxygen as an external electron acceptor. but will ferment it aerobically. which have a scavenging effect on superoxide (for a review. the use of oxygen as terminal electron acceptor and increased ATP production. Lb.[209] The deficiency creates an absolute requirement for an external electron acceptor (e. accumulation may reach autoinhibitory levels depending on strain.[212] These observations were long overlooked.211] A pathway for this oxygen-dependent lactate fermentation of Lb.217] It appears that other LAB associated with plant material (rich in Mn2þ). pyruvate oxidase.[216.[20] In summary. Even lactate. and it has been argued that it is perhaps inaccurate to designate them mere aerotolerant anaerobes. it should be noted that LAB generally do not have the same potential to protect themselves against the toxic effect of oxygen as genuine aerobic organisms. many LAB have the capacity to synthesize the apoenzymes of catalase and/or cytochromes. LAB are generally devoid of catalase.[215] D. plantarum is the most studied species in this regard and was recently shown to harbor genes for several transport systems for cations. some LAB may change their otherwise fermentative mode of metabolism to a respiratory one. and absent in others. but the theme was revived with the complete sequencing of the Lc.. Data have now accumulated indicating that the respiratory mode of growth of lactococci could be preferred to the “normal” fermentative mode.g.g. This was first noted and to some extent investigated in enterococci.[37] It is also quite common among LAB to synthesize a “true” catalase in heme-containing media. including the formation of cytochromes. and acetate kinase. presumably by oxidative phosphorylation. Since H2O2 is produced by NADH oxidases. . Anaerobically.Hoang-Dung TRAN and friends anaerobically. oxygen) in the fermentation of glucose.[208] This is due to a deficiency in the ethanol branch of the 6-PG/PK pathway. but as mentioned previously.D). e. This is especially true for the Copyright © 2004 by Marcel Dekker. Some lactobacilli have developed a unique system for protection against superoxide. also possess manganese-accumulating systems. the end product of “normal” metabolism. more specifically a lack of acetaldehyde dehydrogenase. plantarum has been proposed. such as leuconostocs and pediococci.[165] Superoxide dismutase is present in some LAB.. In the presence of heme or hemoglobin in the growth medium.213] and the discovery that this strain harbored the genes for cytochrome oxidase (bd). can be fermented to acetate and CO2 by some LAB. lactococci and enterococci. However. This system is based on specific accumulation of Mn2þ to high intracellular concentrations (30 – 35 mM). although H2O2 may be decomposed by a pseudocatalase in some strains[29] or by a heme-catalase under certain conditions by others (see above).

MRS-like media.B). It may even explain some of the confusing results with regard to carbon recoveries and fermentation balances in studies of LAB and citrate dissimilation. do not have a counterpart with homofermenters. but acts as a precursor to one. plantarum mannitol fermentation would suggest. mucosae. All Rights Reserved.221] This way of utilizing citrate may be more common among LAB.[228] Although enzymatic evidence is missing. However. Lb. Under normal growth conditions. especially heterofermentative. Different LAB use different pathways in the further metabolism of these products. Decarboxylation of oxaloacetate. a-ketoacids.[225] The products of the citrate lyase reaction are used differently in other LAB. citrate. fructose. it was initially noticed that some unidentified heterofermentative strains produced succinic acid in normal. it was shown that this was due to a utilization of citrate in a cofermentation with glucose[227] and that these strains belong to a new species of lactobacilli.[226] Later. This spares acetyl phosphate from being reduced to ethanol. The fact that some heterofermentative LAB have a defect ethanol branch (see above) and are. In studies of intestinal lactobacilli. since this (in theory) can double the amount of ATP produced per glucose consumed. dependent on an external electron acceptor (at least for glucose fermentation) may support this theory. and glycerol.[219. only minor deviations of the homolactic fermentation may occur as a result of the use of external electron acceptors. therefore.[224. . Such a deviation may be production of some acetoin as a result of a build-up of excess pyruvate. the excess pyruvate is reduced to lactic acid. the Copyright © 2004 by Marcel Dekker. 1. Examples of organic external electron acceptors that can be used by LAB are acetaldehyde. fumarate. compared to in the absence. Growing cells of heterofermentative LAB dissimilating citrate in a cofermentation with carbohydrate do not form significant amounts of diacetyl or acetoin. organic electron acceptors can play an essential role for homofermentative LAB in anaerobic metabolism of certain substrates. than the special case of Lb. The essential step is a cleavage of citrate by citrate lyase to form acetate and oxaloacetate. A pathway for succinic acid formation from oxaloacetate was proposed and proven by Chen and McFeeters. acetyl phosphate holds a key position as intermediate in the 6-PG/PK pathway. The presence or absence of an external electron acceptor will decide whether ethanol (no ATP) or acetate (1 ATP) is formed.[209. most studies on this subject have been aimed at the understanding of acetoin and diacetyl formation. plantarum.[224] and other fates of citrate may have been overlooked. IV. and more ATP can be formed through the acetate kinase reaction.” permitting growth when an external electron acceptor is missing. The changes that occur in the end product formation of heterofermentative LAB in the presence of an external electron acceptor. even under “normal” glucose fermentation.Hoang-Dung TRAN and friends heterofermentative LAB. Lucey and Condon[205] have suggested that the ethanol branch of the 6-PG/PK pathway in no more than a “salvage route. yielding pyruvate. resulting in a more efficient glucose utilization and increased growth rate.225] Rather.[224] Due to its industrial importance. in which an external electron acceptor was required for growth.218 – 223] Further details on the metabolism of some of these will be discussed below. has already been mentioned in connection with the diacetyl/acetoin pathway (see Sec. Inc. Citrate Citrate is not used directly as an electron acceptor.[219] This metabolism was found in the anaerobic fermentation of mannitol by Lb. In this regard. It is perhaps a fair statement to say that this group of LAB has a more obvious use for external electron acceptors.

Hoang-Dung TRAN and friends end product formation for one of these strains. but only in the presence of a cosubstrate.g. pentosus. formate. Inc. glucose. unpublished). OD600 increased from 0. mucosae Lbp 1031 during growth on glucose and citrate in a modified MRS medium. Samples were withdrawn from the culture and subjected to HPLC analysis. A hypothetical pathway of this metabolism is depicted in Fig. but cells performing the metabolism (as evidenced by HPLC) have significantly higher ATP content than control cells (S.[230] This metabolism is very slow and can only be observed after prolonged incubation. e. X. acetate. 4. with the formation of succinic acid. Lindgren. unpublished). which may lead to an altered end product pattern. A. plantarum.7 (5 h). The products of the cometabolism of lactate and citrate are succinic acid. indicating increased ATP production (L. and CO2. which can be performed by some strains of Lb. coli could dissimilate citrate.[231] The differences between LAB regarding the use of citrate may depend on the presence or absence of the enzyme oxaloacetate decarboxylase. shown in Fig. nitrate. 5. no growth is evident. (citrate fermenting) and Escherichia coli (citrate nonfermenting) when it was shown that E. O. acetate.[232] End product formation by Lb.[230] This metabolism has been further studied in a strain of Lb. is consistent with the operation of a citrate lyase and the succinic acid pathway. Axelsson. unpublished). possibly acting on the pyruvate-formate lyase. If oxaloacetate decarboxylase is absent. Lindgren and L. Axelsson. indicating the operation of both the succinic acid pathway and a pyruvate-formate lyase. citrate. ethanol. lactate. Symbols: B. an alternative route is the succinic acid pathway. It was noticed that succinic acid production from citrate was fairly common among heterofermentative lactobacilli isolated from the intestine of several animals (L. A similar comparison was made between Enterobacter sp. the typical stimulation of growth rate and glucose utilization occurred. 6. Figure 5 Copyright © 2004 by Marcel Dekker. W. This may point to an importance of this metabolism for survival and maintenance. In fact. All Rights Reserved. Axelsson and S. and nitrite were shown to inhibit formate production.. During the sampling time. .2 (0 h) to 1. The property seems to be common in plant-associated heterofermentative lactobacilli as well. Oxygen. succinate. acetoin production. In addition. citrate utilization (the primary step being the lyase reaction) results in an increase in the pyruvate pool. If present.[229] Citrate is also used as an electron acceptor in an anaerobic degradation of lactate.

Glycerol is first dehydrated to 3-hydroxypropionaldehyde (3-HPA) and further reduced to 1.3-propanediol as for Lb. 2. brevis was shown to be functional in Lb. however. all heterofermentative.3-propanediol by a NADþ : 1. reuteri ferments glucose alone with lactate. Lb. brevis ferments glucose poorly anaerobically.[218] whereas Lb. many strains of Lb. and Lb. reuteri. initially termed reuterin. since cells grown on pentoses do not contain any glycerol dehydratase activity. This compound is a potent antimicrobial substance.[218] The products of the cofermentation are lactate. As mentioned previously. The same pathway for glycerol reduction to 1. Lb. reuteri under these conditions accumulates and excretes the intermediate 3-HPA. acetate. All Rights Reserved. .3-propanediol. can use glycerol as an electron acceptor in an anaerobic cofermentation with glucose. Glycerol Strains of Lb. Lb.3-propanediol dehydrogenase. brevis metabolizing glycerol accumulate 1. and CO2 as end products. Resting cells of Lb.233] Some differences in response to glycerol between these species can be noted. Again. Some strains do ferment glucose. Inc. brevis. but rather by using glycerol as electron acceptor. ethanol. but changes to more acetate/less ethanol when glycerol is added. brevis. if glycerol is added. buchneri.[237] Copyright © 2004 by Marcel Dekker.2-propanediol. collinoides. reuteri.[234 – 236] The enzymes of the glycerol pathway seem to be under some kind of regulation in Lb.[223] The addition of glycerol also stimulates growth rate and Yglc is increased. CO2 and 1.[223.Hoang-Dung TRAN and friends Figure 6 Proposed pathway for succinic acid production in heterofermentative lactobacilli growing on glucose and citrate. the NADH formed during glucose fermentation is not reoxidized by the ethanol pathway.

Lb. In the presence of glycerol. the overall equation for fructose fermentation would be: 3 fructose þ 2 ADP þ 2 Pi ! 1 lactate þ 1 acetate þ 1 CO2 þ 2 mannitol þ 2 ATP (1) As can be seen. This has been due to its technological significance. The reduction of some of the fructose to mannitol could play a role. this enables the cells to produce ATP through the acetate kinase reaction. Dobrogosz. Axelsson. Assuming no ethanol formation. However. . mainly in wine manufacture.g.[241] Heterofermentative LAB can use glucose as energy source and fructose as electron acceptor. but some of the sugar is reduced to mannitol by a NADþ : mannitol dehydrogenase. and 1. Fiuzat. The NADþ-dependent malic enzyme catalyzes the decarboxylation of malate to pyruvate Copyright © 2004 by Marcel Dekker.[243. unpublished). collinoides also ferment the initial lactate formed from glucose with acetate.Hoang-Dung TRAN and friends This is logical.244] E. strains of Lb.[240] This metabolism represents an interesting example of a case where the same compound acts as both the growth substrate and electron acceptor.[209] Fructose is fermented by the normal 6-PG/PK pathway. is very similar to that observed when oxygen is used as electron acceptor by Ln. since more ATP per glucose can be formed through the acetate kinase reaction. faecalis and Lb. fructose. considering that pentose fermentation does not generate excess NADH. Axelsson. Fructose It was early noted that fructose fermentation by heterofermentative LAB resulted in mannitol formation. thus obtaining optimal growth rate.” i. unpublished). L. this is less efficient than glucose fermentation in terms of ATP formed per sugar consumed. but Lucey and Condon[205] suggested that ATP formation is the rate-limiting step in these bacteria. On the contrary. CO2.239] The growth response of Lb. ethanol.e. casei. can use malate as the sole energy source. where glucose. but also because it has presented some interesting physiological problems with regard to metabolism and bioenergetics. Inc. Few LAB. and sucrose are the main sugars. mesenteroides. The metabolism may be of great importance in natural plant fermentations.3-propanediol as end products. reuteri has about the same levels of this enzyme whether grown on pentose or glucose. brevis and Lb..[205] The increase in Yglc is easy to understand. All Rights Reserved. But why is the growth rate increased? No clear-cut answer can be given.. a priority is given to growth rate rather than efficiency of substrate utilization.[242] Renewed interest in this metabolism of LAB has recently occurred as a result of the suggestion to use mannitol as a “nutraceutical. e. A maltose-fructose cofermentation has also been suggested to improve sourdough fermentation. an active ingredient in functional foods. and W. 3. despite the fact that the glycerol pathway is not used when the cells ferment pentoses (M. the growth rate is higher on fructose than glucose (L.[238. The Malo-Lactic Fermentation The metabolism of L -malic acid (the D form is not attacked) by LAB has been extensively studied. This indicates that under conditions of substrate excess. reuteri with the addition of glycerol (increased growth rate and Yglc).[209] Similar to the use of other external electron acceptors. E. since the cells may be able to form more ATP per time unit. for some heterofermentative lactobacilli.

but was subsequently shown to be mainly a consequence of so-called electrogenic precursor/product exchange (for further discussion on energetics.[247] This may be of importance. and more common in LAB. The stimulatory effect was first attributed to the deacidification of the external medium. This fermentation. depending on the wine variety.[111] The pathway for malate dissimilation was first believed to be a sequence of reactions.D). where malate is present in fairly high concentrations. III. This would indicate that hydrogen exchange reactions occurred within the enzyme and that oxaloacetate and/or pyruvate may be intermediates. since leuconostocs only possess a D -LDH (see Sec. purification to near homogeneity of the enzyme catalyzing the complete reaction was achieved:[111] Malate À ! lactate þ CO2 NADþ (3) To distinguish the enzyme from the malic enzyme and malate dehydrogenase. which proceeds in a cofermentation with a fermentable carbohydrate. since apparently no potential electron acceptors such as oxaloacetate or pyruvate are produced by the reaction. i.C and IV. Subsequently. Later studies have shown that the reaction is not stoichiometrically complete and that small. in particular that required for generating and maintaining a proton gradient. . IV. it was given the trivial name malo-lactic enzyme [the proper name being L -malate : NADþ carboxylyase[245]]. although NADþ was required for enzymatic activity.[246] This is difficult to explain. but significant amounts of pyruvate and NADH are released. (2)..[164] LAB performing the MLF in cofermentation with a carbohydrate generally benefit from this in a way that resembles the use of external electron acceptors (see Secs. This could be achieved by the “energy recycling model” of lactate efflux. The more interesting. ethanol. Copyright © 2004 by Marcel Dekker. V. see Sec.[111] Pyruvate could thus not be an intermediate. resulting in pyruvate as a free intermediate. no NADH was detected in early studies of the enzyme. where it may be desirable or undesirable. increased growth rate and a higher Yglc compared to growth on solely glucose. malate metabolism is the conversion of malate to lactate and CO2. Pyruvate would subsequently be reduced to lactic acid by the LDH.[248] These authors suggested that this effect is indirect in that MLF relieves the cells from some of the energy requirements. often referred to as malo-lactic fermentation (MLF). the first being identical to Eq.[107] The conversion of malate to lactate in the late stages of wine-making is well known.[111] which is a consequence of the reaction since malic acid has a lower pKa than lactic acid. but cannot fully account for the stimulatory effect. MLF is significant in the fermentation of vegetables and fruits. and CO2 with ATP generation presumably through the acetate kinase reaction.e. All Rights Reserved. can be performed by many LAB. Inc. An indication of another mechanism was that MLF-leuconostocs produced exclusively L -lactic acid from malate. but bound to the enzyme.[245] The reaction can thus provide additional electron acceptors.A). Measurements of internal ATP concentrations in cells performing the MLF have also indicated that the reaction confer benefits in the form of energy.B). Curiously.Hoang-Dung TRAN and friends and CO2[182] as follows: Malate þ NADþ ! pyruvate þ CO2 þ NADH (2) Pyruvate is converted to acetate.

Hoang-Dung TRAN and friends F. while Lc.[249. the Opp system was shown to accept oligopeptides up to 18 residues.g. Several peptidases with different specificities have been identified in lactococci. helveticus strains require 13 – 15 amino acids. in cheese manufacture) have proteolytic activity.. Earlier studies noticed a “missing link” in the proteolytic pathway. such a peptidase has never been convincingly identified. These are amino acid transport systems.[255] The problem was then as follows: since the products of casein degradation appear to cross the membrane as di. They are. 7. Refined tools for analyzing the products of casein degradation by PrtP. Refs. but to date all well-characterized peptidases have been found to be intracellular. one study also suggested that a broadspecificity di-/tripeptide transport system was essential for casein utilization. All dairy lactococci used for acidification of milk (e. inactivation by chromosomal integration).[256] Second. and thorough studies of the Opp system subsequently solved this problem. An extracellular. however. lactis subsp. with oligopeptides smaller than 9 residues being a large fraction. further strengthening the role of this transport system.[249] The requirement for a particular amino acid may be the result of mutations in the genes for amino acid biosynthesis and/or the downregulation of these genes or the involved enzymes. First.[259] A model for the complete proteolytic pathway can now be constructed and is schematically shown in Fig. it was calculated that oligopeptides small enough to be accepted by the oligotransport system represent 98% of the nitrogen source for growth of lactococci in milk. It should be noted. while the di-/tripeptide transport system was not.[257] Third. performing Copyright © 2004 by Marcel Dekker. Inc. Mutants defective in the gene of this protein grow to only very low densities in milk. lactis subsp. using well-defined mutants.[254] Using mutants. and it is clear that LAB have adapted to rich environments by developing systems to efficiently exploit the nitrogen sources present there.or tripeptides and PrtP only produces larger oligopeptides. . the advanced knowledge of the genetics of the system. therefore. PrtP exists in at least two variants in lactococci with somewhat different specificities in the degradation of milk casein. The gene for PrtP. Nitrogen Metabolism: The Proteolytic System It is a general belief that LAB have a very limited capacity to synthesize amino acids using inorganic nitrogen sources. by analyzing the peptide fraction in milk during growth. in particular that of Lc. it was clearly shown that the Opp system was essential for growth on casein. One of the most extensively studied systems in this regard is the proteolytic system of dairy LAB.250] Growth on chemically defined minimal media is generally slow. methodologies for constructing well-defined mutants (e. Some strains of Lc.g.and tripeptide transport systems. as it has been shown that a proteolytic system is necessary for appreciable and rapid growth in milk.[253] and an oligopeptide transport system (Opp) accepting 4. dependent on preformed amino acids being present in the growth medium as a nitrogen source. casein was shown to be degraded by PrtP to a much larger extent than previously thought. lactis are in fact prototrophic for most amino acids. The reason is of course the technological significance in milk fermentation. All Rights Reserved. cremoris and Lb.. that the requirement for amino acids differs among the species and strain variations exist within species. As mentioned.[22] two di. lactis. membrane-anchored serine proteinase (PrtP) was identified as being essential for this activity. There are several reviews covering this topic in more depth (e. Below follows a short summary. since the peptides produced by the activity of PrtP were too large to be directly transported by the transport systems identified. 251.[258] Fourth.g.252).. there should be an extracellular peptidase involved in the pathway.to 8amino acid residue peptides.

Opp. lactis.and tripeptides and free amino acids. plantarum contains all genes necessary for protein degradation. oligopeptide transport system. since detailed studies in other species are scarce.[261] Copyright © 2004 by Marcel Dekker.[260] presumably produced by the action of intracellular peptidases from transported oligopeptides in the in vivo situation. but note that these contribute very little to the total growth of lactococci in milk (see text).. As far as they do exist. membrane-anchored proteinase. the extracellular proteinase. The extent to which this knowledge of the lactococcal proteolytic system can be transferred to other LAB is not known at present. Included is transport of di. they seem to indicate that the proteolytic systems are indeed similar to lactococci. the prime step in the pathway. All Rights Reserved. amino acid transport system(s).[19] Other lactobacilli do possess a PrtP protein very similar to the lactococcal counterpart. D.e. M. has been shown to be regulated by specific dipeptides. Inc. the genome of Lb. cytoplasmic membrane. PrtP. i. . genes encoding for the Opp system and and array of peptidases of different specificity. A. di-/tripeptide transport system(s).[251] Except for the primary enzyme in the pathway.Hoang-Dung TRAN and friends Figure 7 Model of the proteolytic pathway in Lc.

and a cell wall without outer membrane are valuable properties in this regard. Lactococcus.e.266] It was also quite early established that the Hþ-ATPases of E.[24.. and a pH gradient (DpH). which use these bacteria. Aerotolerance. present in respiring organisms. Instead.. 263]). The flow of electrons through the system via different carriers in effect pumps protons out of the cell. The most important energy-requiring events in cells are the synthesis of macromolecules and the transport of essential solutes against a concentration gradient. Model systems. are easy to control and manipulate. The so-called chemiosmotic theory in bioenergetics[264. The proton gradient across the cytoplasmic membrane is composed of two components. inside negative. DC and DpH exerts an inwardly directed force termed the proton-motive force (PMF). LAB do not possess an electron transport chain (at least not in the absence of preformed heme) and are hence not able to form ATP in this way. is used to form ATP from ADP and phosphate. and the presentation here can only be a short summary. in particular species of the genera Enterococcus. but the major role of this enzyme is the reverse reaction.265] is now generally accepted.V. an efficient fermentative metabolism with no oxidative phosphorylation. and Internal pH The metabolism of LAB is aimed at the generation of ATP. the Hþ-translocating ATPase. but the discussion on the subject has been placed here because transport systems in general are tightly coupled to the bioenergetics of the cells. i. There are excellent reviews covering this vast field (e. Refs. Cellular metabolism leads to an electrochemical proton gradient across the cytoplasmic membrane. however.g. ATP is generated by substrate-level phosphorylation. ATP. or in this function also known as the ATP synthase. faecalis. an electrical potential (DC).23] LAB (and fermentative bacteria in general) thus establish a PMF. ATP. Bioenergetics of LAB 1. LAB do. lactis is capable of acting as an ATP synthase under certain conditions.37. . Hoang-Dung TRAN and friends ENERGY TRANSDUCTION AND SOLUTE TRANSPORT Members of LAB have been extensively studied with regard to mechanisms of transport and energetics. the Proton Motive Force. This is accomplished by a membrane-located enzyme. The most commonly known system for creating a proton gradient is the membrane-linked electron transport chain. The results obtained with these model systems have been important for the understanding of living cells in general. The difference in function between the Hþ-ATPase of LAB and the ATP synthase of respiring organisms is merely a reflection of the different modes of metabolism. A.[23] Sugar transport is of course connected to the carbohydrate metabolism described in previous sections.[22. the hydrolysis of ATP with concomitant pumping of protons out of the cells. into the cell. possess an enzyme very similar to the ATP synthase. is needed for the thermodynamically unfavorable “reaction” of building cells. ATP. which can drive energy-consuming reactions such as the uphill transport of metabolites and ions. All Rights Reserved. this force is large enough to be converted into chemical energy.e. inside alkaline. i. or high-energy compounds interconvertible with ATP. The Hþ-ATPase of Lc. The energy of the reversal flow of protons “through” the enzyme. Copyright © 2004 by Marcel Dekker.171.262. In organisms with an electron transport chain.. the universal energy carrier in all living cells. and Streptococcus. [21 – 26. which is characteristic for all fermentative organisms. Inc.

The question is whether this process. and respiring or photosynthesizing eubacteria. i.10 mM). casei have the same basic structure as the ATP synthase from mitochondria. Based on theoretical calculations.. Studies of E. since an initial high growth rate (which presumably would be the result of more ATP available for biosynthesis) could be advantageous in competition with other microorganisms. In practice. The conditions for maximium advantage of lactate efflux Copyright © 2004 by Marcel Dekker. In contrast. since they tolerate lower and a wider range of internal pHs. . due to massive acid production. 8B? Here. faecalis system. In the case of lactate.[274. a net charge leaves the cell together with the end product.2– 4.3 and low external lactate concentrations (. lactobacilli are significantly more tolerant to low pHi than enterococci. lactis subsp. leuconostocs. this energy-saving process is only operational at the initial stage of growth in a batch culture. a model was proposed as to how energy could be conserved by end-product efflux. lactis subsp. chloroplasts. It appears that some lactobacilli have developed a very “relaxed” system. cremoris was grown in coculture with a lactate-consuming organism. where solute Y (Fig. Both the activity and the synthesis of the Hþ-ATPase are regulated by pHi. Inc. lactis subsp. however. There are.22] Since the external pH (pHo) falls well below these values. 8A. and Lb. The answer to the question is yes. a PMF is generated. and streptococci rarely tolerate a pHi lower than 5. A dramatic effect was observed when Lc. the so-called “energy recycling model”. which works even at a pHi of 4.4. 8B) was represented by a fermentation end product (such as lactate). “Energy Recycling” and Electrogenic Precursor/Product Exchange The maintainance of a pHi above the threshold level and the generation of a PMF require the use of a substantial part of the ATP generated by substrate-level phosphorylation.[272] The extrusion of protons by the Hþ-ATPase and the electrogenic uptake of Kþ maintain the cytoplasm more alkaline than the outside medium. a mechanism must exist to maintain pHi above the threshold levels. Any other means of generating or maintaining the PMF than the Hþ-ATPase would save energy. is reversible.Hoang-Dung TRAN and friends Lc.[273] If the efflux is electrogenic. the outwardly directed gradient of solute Y drives the efflux in symport with a proton.[270] LAB are to a certain degree exceptions to this.[21. In general. which to some degree compensate for the drain of ATP. enterococci. differences within the LAB group.[22] 2.[267 – 269] It is well known that bacteria attempt to maintain the cytoplasmic pH (pHi) at a certain level or interval. faecalis suggest that the Hþ-ATPase plays a crucial role and that this role is the main function of the enzyme. This makes less ATP available for biosynthetic purposes. Experimental results with Lc. In an ecological context.275] A carrier-mediated electrogenic lactate efflux was shown to occur at pHo above 6. cremoris.[21.271] It is logical that the enzymes and the general machinery of the cells have threshold levels below (and above) which they cannot function. All Rights Reserved. in its principles. under certain conditions. more than one proton (on average) per lactate molecule has to be exported to obtain an electrogenic efflux.e. thus creating a PMF. A general scheme for PMF-driven transport is depicted in Fig. cremoris have supported the model. lactococci. as shown in Fig. The mechanism of pH homeostasis in lactococci seems to be very similar to the E. which enters the cell together with a proton (proton symport). Some LAB have developed such systems.0. At lower pHo and higher external lactate concentrations. Pseudomonas stutzeri. the significance of the process may be substantial. the lactate efflux was electroneutral and subsequently not contributing to the generation of a PMF. and streptococci. The inwardly directed gradient of protons is the driving force for the influx of a solute X.

M denotes the cytoplasmic membrane.Hoang-Dung TRAN and friends Figure 8 Schematic representation of proton-motive force (PMF) formation by a Hþ-ATPase and PMF-driven transport (A). Copyright © 2004 by Marcel Dekker. and electrogenic end product efflux (B). All Rights Reserved. . Inc.

another mechanism for creating a PMF has been identified in LAB.E). All Rights Reserved.[276] The energy recycling model has been shown to be applicable also for acetate efflux in Lb. and it is perhaps not surprising that the lactate export system differs.[277] This species responds to a shift from anaerobic to aerobic conditions by producing some acetate at the expense of lactate and by increasing the molar growth yield for glucose.or trivalent anion (citrate) is exchanged for monovalent (acetate) and neutral (acetoin) products concomitant with decarboxylation..0. or indirect.24] Much of the discussion below is based on these results. It has been shown that MLF in Lb. Inc. but may under normal conditions. 9. e. generally a carbohydrate. together with additional ATP formation via the acetate kinase reaction. The benefits of citrate metabolism in Lc. A carriermediated. These findings stem from the attempts to explain the energy benefits of the malo-lactic fermentation (see Sec. electroneutral export of lactate was shown for Lb.e. i.200 mM) and the pHo drops to levels well below 4.e. For this species. ATP-driven). The transport of solutes is highly connected to the bioenergetics of the cells. and this. The key is the compartmentalization of the pathway. . and ions has emerged. nucleotides or nucleotide precursors. lactis functions as an indirect proton pump.. plantarum and Lc. amino acids. The system of energy recycling by lactate efflux is not general for all LAB. i.[280.[278] An electrogenic acetate efflux was demonstrated.Hoang-Dung TRAN and friends (low external lactate concentration and high pHo) were thus maintained throughout growth and a 60– 70% increase of the molar growth yield for lactose was obtained. It is not known if the system of Lb. cofermentation of sugar malate. was suggested to contribute to the energy economy of the cells. plantarum. (b) secondary transport (chemiosmotic. 9A). respectively.281] The precursor (malate) is exchanged for a product (lactate) with higher charge.. be a mechanism to conserve rather than directly generate energy. helveticus is typical for the more acid-tolerant LAB. mediated by one exchange protein (Fig. where essentially a di. helveticus is very different from lactococci. the decarboxylation occurring inside the cell and consuming one proton. lactis. and (c) group translocation (chemical modification concomitant with transport)..e. IV.[24] The PMF formed by the MLF alone is high enough to drive ATP synthesis by the Hþ-ATPase. which can be broadly divided into three categories based on differences in the form of energy used in the translocation process:[26] (a) primary transport (chemical energy. LAB use different types of transport systems. e. presumably by the same general mechanism. lactis can be explained by the same mechanism. Transport of Solutes LAB are generally very fastidious and require amino acids. via one precursor uptake protein and one product exit protein (Fig. phosphoenolpyruvate : sugar phosphotransferase system (sugar PTS).[22.[282] Certain lactobacilli can produce biogenic amines such as histamine and tyramine by decarboxylation of the amino acids histidine and tyrosine. a nearly complete picture of the transport of sugars. making the overall reaction electrogenic.g. Recently. PMF-driven).g.[279] This species is able to produce very large amounts of lactic acid (. and vitamins in addition to an energy source. The massive lactic acid production and acid tolerance of Lb. peptides.[24] A generalization of these so-called electrogenic precursor/product exchange reactions is shown in Fig. i. This leads to the generation of a PMF.. helveticus. The exchange reaction may be direct.[24] B. Copyright © 2004 by Marcel Dekker. A prerequisite for rapid growth is efficient transport systems for the uptake of essential nutrients. 9B). It should be emphasized that the most extensive research on transport systems among LAB has been done with Lc.

ATP-Driven) The most common class of primary transporters in LAB are members of the ABC (ATP-binding cassette)-transporter superfamily.[283] They are represented by systems for accumulating substrates and compatible solutes. such as the glutamate/glutamine transporter. The product can be exported via a precursor/product antiport (A) or by a separate carrier (B). drugs) such as LmrA of Lc. The reaction contributes to the formation of the PMF. but also by systems for excreting unwanted compounds (e. and the OpuA (Lc. plantarum) or QacT (Lb. All Rights Reserved. Copyright © 2004 by Marcel Dekker.g. 1. Primary Transport (Phosphate Bond – Linked Transport. Inc. The precursor (X-COO) with a certain charge (n) is transported into the cell and decarboxylated to yield the product (X-H) with higher charge (n þ 1). the oligopeptide (Opp) transport system. plantarum) transporters for defense against osmotic shock.Hoang-Dung TRAN and friends Figure 9 Schematic representation of electrogenic precursor/product exchange with intracellular decarboxylation. lactis/Lb. . lactis..

permease) translocates the solute across the membrane in symport with one proton.285] The activity of the transport system decreases if internal ATP levels are lowered. and a multiprotein complex has been predicted for the former. PMF-driven lactose transport has been shown in lactococci. leucine. specificities. The PMF-driven transport systems are perhaps the most common and general among LAB for the transport of nutrients into the cell (as in most bacteria). Despite this being an exchange reaction.[24] In vivo. isoleucine. The oligopeptide (Opp) transport system. plantarum.g. thermophilus.Hoang-Dung TRAN and friends Glutamate and glutamine transport in lactococci is mediated by a system dependent on the production of metabolic energy by either glycolysis or the ADI pathway. the cells can be relieved of this effect if higher concentrations of glutamine are included in the medium as a source of glutamate. thermophilus.288] The mechanism has been studied in some detail in S.[22] Similarly.A). 8A. Presumably. The principles of this transport system have already been mentioned and are schematically drawn in Fig. but differences in affinity may allow the cells to use either system at different substrate concentrations. LAB use an ABC-transporter. The physiological characteristics of the glutamate/glutamine and the oligopeptide transport systems are similar.[284.F). As mentioned. have not been studied to any large extent. for accumulating glycine betaine. These compounds do not affect the macromolecules of the cells and may in fact stabilize the structures of some enzymes. and S. and valine. the exchange reaction seems to be the most relevant since it is faster than the symport reaction. may Copyright © 2004 by Marcel Dekker.. while galactose is excreted. 2. The system has been characterized both genetically and biochemically. Secondary Transport PMF-Driven Symport. which leads to growth limitations at alkaline pH values. crucial in the proteolytic pathway (see Sec. This feature has been mostly studied in Lc. two of which (OppD and OppF) show the typical ATP-binding domains of the ABC-transporter superfamily. most amino acids are also transported by PMF-driven symport systems. e.[289] but can also transport lactose in symport with a proton with PMF as the driving force. IV. this species only ferments the glucose moiety of lactose. halophilus. lactis and Lb.[285] The system has an absolute preference for the undissociated form of glutamate (glutamic acid).98. .263. Efflux of galactose is a result of internal accumulation (due to a defective induction of galactokinase) and the fact that the carrier also has affinity for this substrate.24. is ATP driven and unaffected by the magnitude of the PMF. is not a strict antiporter.[254] It is composed of five proteins. see Ref.[290] In certain lactococci the permease-mediated transport of lactose coexists with a lactose PTS system.[22] Stucturally similar amino acids. IV. LacS.[22. For a review on the subject. All Rights Reserved. it has been shown that glucose can be transported either by a PMF-driven system or a glucose PTS in a strain of T.[22] In lactococci.[291] The reason for having two separate transport systems for the same solute is not fully understood. membrane-associated protein (carrier. the actual carrier. OpuA or equivalent. lactobacilli. etc. 26.[286] Since glutamine transport is independent of pH. many sugars are transported in this way (see also Sec. as was suggested. although the actual mechanisms.[24] Microbial cells respond to osmotic up-shifts by accumulating certain compounds intracellularly to very high concentrations. with the exception of lactose transport. which appears to be the preferred compatible solute.287. with energetic benefits. Inc. A single transport system for glutamate and glutamine has been identified. A specific.

e. Ornithine.[22. the arginine deiminase (ADI) pathway. is shown in Fig. The stoichiometry for the arginine : ornithine exchange is strictly 1 : 1. as well as the antiporter itself is repressed by glucose and induced by arginine. and (b) the antiporter can catalyze heterologous exchange of arginine for lysine in addition to exchange of arginine for ornithine.and tripeptide transport systems has also been shown to be PMF driven. with a broad-specificity carrier. and the exchange has been shown to be mediated by a single. most arginine-metabolizing LAB cannot use arginine as sole energy source.[22] One of the di. at least for lactococci.[255] For the amino acid proline. one of the end products of the metabolism. the arginine/ornithine antiporter.[22.113] but catabolize it simultaneously with a fermentable carbohydrate.[292 – 294] The pathway of this metabolism. M denotes the cytoplasmic membrane.. that the driving force for arginine uptake and ornithine excretion is the concentration gradients of these compounds[22] with no involvement of PMF.[24] Figure 10 The arginine deiminase pathway with the arginine/ornithine antiporter (AP). which thus might be recaptured. All Rights Reserved. Inc. anabolic roles). The activity of the PMF-dependent lysine carrier is probably the answer to this question since (a) it has some affinity for ornithine.Hoang-Dung TRAN and friends share the same carrier protein. Copyright © 2004 by Marcel Dekker. except carbamate kinase (which presumably has other.[295] It has been established. while an active transport system for the free amino acid is missing (i. For unknown reasons. and the question arises as to how the biosynthetic need for arginine is supplied. peptides seems to be the preferred source. Differences between carriers with regard to affinity and pH regulation has been shown. Precursor: Product Antiport. 10. a dependence on passive diffusion). membraneassociated protein.296] The arginine metabolism leaves the cells without any net accumulation of amino acid.[296] The enzymes of the ADI pathway. Many LAB can derive energy through substrate-level phosphorylation by the metabolism of arginine. .or tripeptides (in the form of Pro-X or Pro-X-X). is excreted into the medium. This has been attributed to an efficient transport system for proline-containing di.

The energy of the phosphoryl group is transferred in a series of reactions. The sugar-specific components may also exist as a fusion protein. translocation. whereas Enzyme IIBC (EIIBC) and Enzyme IIA (EIIA) are sugar specific (denoted by a superscript in Fig. respectively.Hoang-Dung TRAN and friends 3. The suffixes represent different domains containing active sites involved in the reactions. All Rights Reserved. or as separate proteins IIA. the glucose PTS in most LAB also recognizes mannose and is often designated man-PTS to distinguish it from the glc-PTS. Group Translocation: The Phosphoenol pyruvate:Sugar Phosphotransferase System The PTS is a complex enzyme machinery. designated EIIABC. Inc. and IIC. coli. but the specificity may not be absolute. IIB. are sugar nonspecific (can be shared by several PTS). EIIBC is a membrane-located enzyme that acts in concert with the phosphorylated EIIA to mediate recognition. The first two proteins in the series. i. . first described in E. It has been argued that the production of 2 mol of PEP per hexose consumed (as in glycolysis) is needed for a functional metabolism. EIIBC and EIIA are sugar specific.[297] The distribution of PTS in bacteria has been discussed by several authors. PK. and phosphorylation. See text for details.297 – 299] There has been a general agreement that the presence of PTS is highly correlated to the ability to ferment substrates through the Embden-Meyerhof-Parnas pathway. 11. 297). which mediates transport and phosphorylation of the sugar. see Ref. which has a different substrate specificity. 11).. EIIBC and EIIA were denoted EIII and EII.[263. whose main function is to translocate a sugar across a membrane with simultaneous phosphorylation (for a comprehensive review. Enzyme I (EI) and heat-stable protein (HPr).e. Since there are two separate molecular species outside (sugar) and inside (sugar phosphate) the membrane. Copyright © 2004 by Marcel Dekker. the translocation does not involve any concentration gradients. including a PTS. to a membrane-located enzyme. The components and reactions of the system are depicted in Fig. glycolysis. The energy for the process is provided by the high-energy phosphate bond of phosphoenolpyruvate (PEP). pyruvate kinase.[299] LAB have Figure 11 Sugar transport mediated by the phosphoenolpyruvate : sugar phosphotransferase system (PTS) and relation to glycolysis. Thus. via PTS-specific proteins. Note that in older literature.

To avoid unnecessary enzyme synthesis and to achieve maximum profit from a substrate mixture. It can either donate the phosphoryl group to EI and initiate the PTS cycle. Inc. One of the foundations for this was laid with the basic knowledge obtained by research on PTS-mediated transport (for reviews. All Rights Reserved.[263. CCR involves both direct transcriptional control via trans-acting repressor proteins Copyright © 2004 by Marcel Dekker. This is schematically shown in Fig. bacteria have developed mechanisms to monitor nutritional and energy status and to respond to these conditions.[262] The transport of the sugar is directly coupled to the subsequent metabolism.e.[262] This pool of metabolites. Carbon metabolism regulation has been shown to be an interplay between several components that have roles in more than one context. or it is used by pyruvate kinase to form ATP. in which PEP holds a key position. 3-phosphoglycerate and 2-phosphoglycerate.[262] VI. i.[262] A decrease in the glycolytic rate as a result of limiting concentration of sugar will result in a decrease in FDP levels and an increase in Pi.242. PEP has two alternative fates. reports clearly show that heterofermentative LAB also can have sugar PTS activity. which in turn provides the PEP needed for a new cycle to begin. and catabolism. enables the cells to quickly resume transport and glycolysis once a sugar becomes available. see Refs. when the FDP level is high and the Pi level low. this is not sufficient as a regulatory response since all the (potential) inducers are present.300] As a generalization.302] Within the scope of this chapter. designated the PEP potential. REGULATION OF CARBON METABOLISM I found it appropriate to place this short section on the regulation of carbon metabolism in LAB not only after the general sections on metabolism. This is achieved by a mechanism known as carbon catabolite repression (CCR). However.263]). Regulation of carbon metabolism is complex and features intriguing regulatory circuits. LAB have been valuable as model systems for basic research on how carbon metabolism is regulated in bacteria. 11.[263. These compounds are fermented by the 6-PG/PK pathway and hence do not result in two PEP per substrate. but also after the section on energy transduction and transport. During complete starvation. Pyruvate kinase is subject to regulation. “Coarse control” is achieved by a substrate induction. In addition. thus connecting transport of solutes. The PEP potential may also be important in providing maintenance energy during starvation by a residual activity of pyruvate kinase.A).299] However. where FDP acts as activator and Pi as inhibitor.[183.[171. facing a substrate mixture.Hoang-Dung TRAN and friends provided some support for this in that sugar PTS were not found at first in heterofermentative LAB. 11. As depicted in Fig. The system actually constitutes a cycle. when both pyruvate kinase and the PTS are inoperative. pyruvate kinase is most active and the concentration of PEP is low. bacteria utilize carbon sources in a hierarchial manner and repress the genes necessary for catabolizing the less preferred substrate.[301] Consequently. PTSs for gluconate and pentitols have been described in LAB (see Sec. most carbon sources act as inducers for transcription of the genes encoding transport and/or catabolic systems for that particular substrate. the cells contain high concentrations of PEP and the preceding intermediates in the glycolytic pathway. . although it may be uncommon. pyruvate kinase activity decreases and the concentration of PEP increases. the sugar PTS is tightly coupled to glycolysis in most bacteria.. By global transcriptional control.[262] Under optimum glycolyzing conditions. IV. transcriptional control. only a few of those features can be mentioned.

resulting in HPr(His  P). but HPr(Ser  P) has also been implicated. usually present in or in the vicinity of the promoter region of the pertinent gene/operon.[26. As mentioned. The phenomenon of inducer expulsion is less understood. IV. which in gram-negative bacteria is performed mainly by Enzyme IIAGlc of the PTS system. respectively. see Ref. CcpA is a trans-acting repressor of several catabolic operons involved in the utilization of carbon sources other than glucose. CCR also encompass ways of keeping intracellular inducer concentrations low. HPr(His  P).Hoang-Dung TRAN and friends and mechanisms that keep the inducer concentration in the cytoplasm low. which frees HPr for its PTS function again. Interestingly. but the phenotypes are similar in both systems. The phosphorylation status of HPr and its effect on CcpA could thus be a link between the levels of glycolytic intermediates and mode of fermentation.e. HPr. CcpA acts as activator of glycolytic enzymes. All Rights Reserved.g. Escherichia coli) and in the low G þ C gram-positive bacteria. The balance of the three different forms. as mentioned.[304. as first suggested (see Sec.. Inc. thereby indirectly affecting gene expression. CcpA exerts its function through binding to the cis-acting sequence called catabolite-responsive element (cre). Thus. Lower levels of glycolytic intermediates will turn off the kinase activity of HPrK/P and instead activate its phophatase activity. However. Cells induced for the lactose-PTS do not accumulate TMG if glucose or glucose analogues are transported by the mannose-PTS.303] The phosphorylation of HPr by EI  P in the PTS cycle depicted in Fig. HPr can also be phosphorylated at serine-46 by HPrK/P. mainly Bacillus subtilis. instead of a direct allosteric effect on nLDH. also indicative of high catabolic rate. The role of HPr is emphasized by the fact that HPr(Ser  P) is the activator of the CcpA protein. . Two mechanisms can be distinguished: inducer exclusion and inducer expulsion. 303).307] Inducer exclusion has mostly been attributed to competition for HPr(His  P) by affinity differences of HPr(His  P) for the sugarspecific EIIA proteins. in LAB. and HPr(Ser  P). Global control of carbon metabolism has been extensively studied both in enteric bacteria (e. is the PTS protein HPr together with a HPr kinase/phosphatase (HPrK/ P). The mechanisms of CCR and carbon control in these two major groups of bacteria are partly different (for a review. lactis the mechanism involves stimulation of a sugar phosphatase by HPr(Ser  P).[179. and this is achieved not only for PTS sugars. stimulate the kinase. i.[306] This study also showed that a CcpA mutant performed a mixed acid fermentation. In others. reflects the physiological state of the cell (the double phosphorylated form HPr(His  P)(Ser  P) also exists.B). but also. 11 takes place at the histidine-15 residue. high ATP levels. Copyright © 2004 by Marcel Dekker. Central to CCR in gram-positive bacteria is catabolite control protein A (CcpA). to avoid uptake of one sugar when a preferential substrate is present and to excrete already accumulated inducer when a preferential substrate becomes available. The phosphorylation of HPr to HPr(Ser  P) will break the PTS cycle and ultimately reduce sugar uptake and catabolism.[308] Figure 12 summarizes the central role of HPr in global regulation of carbon metabolism in LAB. and components of the PTS system play a pivotal role. but in Lc.262.305] However. the phosphorylation state of HPr reflects the metabolic state of the cell. CcpA can also act as an activator of transcription in some instances. The inducer exclusion effect was shown quite early in lactococci using the galactoside analogue thiomethyl-b-D -galactopyranoside (TMG). lactis.. resulting in HPr(Ser  P). It has been firmly established that the link between the energy status in the cells and CCR in gram-positive bacteria. High PTS activity and high catabolic rate will increase the pool of FDP which stimulates HPrK/P kinase activity in some LAB. such as the las operon in Lc. A complex between HPr(Ser  P) and CcpA is first formed and the complex binds specifically to the cre site.

This will be important in defining the background for differences in phenotype and physiology.[313.[303] but very important contributions regarding HPr were also made in the heterofermentative species Lb. but the physiological role is not understood). Genome sequencing will give another dimension to research of the complex networks that regulate metabolism. where enzyme activities are modulated in small steps by a genetic method using artificial promoters. giving an enormous range of modulating and adjusting catabolic capacity. only two LAB genome sequences have been published.[20] For some species more than one strain will be sequenced. brevis. such as CcpA. At the time of this writing. and/or through allosteric control of different transporters. Recently. Attempts are being made to build a regulatory network based on predictions of regulators from the annotated sequence. lactis genome.[309. The analysis of the Lc. is the most advanced. Studies on the PTS system and the role of its components in carbon control in LAB have mostly been done in Lc. casei.[312] the metabolic control of glycolysis was investigated (reviewed in Ref. some of them already completed but not yet published..310] CcpA and/or its gene have been found in all LAB investigated so far. The different forms exert their functions through other global regulators. Inc.[199. i. . such studies have been initiated for the primary metabolism in Lc.19] but more than 30 are underway.[18.315] Further studies along these lines will be very valuable for understanding how the primary metabolism in LAB is controlled and regulated. lactis. phenotypic Copyright © 2004 by Marcel Dekker. phosphoenolpyruvate : sugar phosphotransferase system. lactis and Lb. glycolysis.e. All Rights Reserved. the first completed among LAB. One could therefore assume that the general picture outlined above is valid for all LAB. 313).Hoang-Dung TRAN and friends Figure 12 Schematic representation of the central role of HPr in the global regulation of carbon transport and metabolism. mutagenesis. See text for further details. Another angle in the studies of carbon metabolism and its regulation is to apply metabolic control analysis and metabolic optimization.314] since changing GAPDH levels in growing cells did not alter the glycolytic flux or change the mode of fermentation from homolactic to mixed acid. PTS. These studies have to some degree questioned the key controlling role over glycolysis attributed to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).[311] In an elegant series of studies.

dispensed with biosynthetic capability. It was natural to group them together. more adaptive than one could imagine. These relationships can probably be assessed more easily than defining common phenotypic characters for a particular natural group. a group of organisms that are diverse but physiologically similar. The author acknowledges the large amount of work needed. it is clear that the classification problems that always have been evident with regard to LAB may stem from an (historical) overemphasis on a few characteristics. if these mutants are returned to a rich medium. A group of bacteria were isolated from similar niches and turned out to be lactic acid producers. The results of genome sequencing of LAB will certainly strengthen this view and. but effective way of outcompeting other microorganisms. All Rights Reserved. indicating that some of the biosynthetic genes are not expressed.[19] LAB are perfectly adapted to environments rich in nutrients and energy sources. Although the reasoning above may be somewhat oversimplified. revealed a large region in the chromosome containing genes encoding several nutrient utilization systems and extracellular functions. but 59 were not. Inc. LAB have therefore developed strategies to efficiently compete with these organisms. Apparently genetic material for biosynthesis is still present. but early studies showed that mutagenesis can render some lactobacilli prototrophic for some amino acids. the nutrient requirements in minimal media[317] are greater than the sequence implicates. to some extent. This has not been an easy task. plantarum strain mentioned above. This cluster of genes has been termed a “lifestyle adaptation region”. before a more complete picture of the regulatory network can be established. LAB comprise a very diverse group of organisms. and postgenome methodologies such as two-dimensional gel electrophoresis (proteom analysis) and DNA microarrays (transcriptome analysis). We now have the means to determine natural relationships more objectively and more accurately than ever before.[316] An estimate of 111 regulators were putatively identified from the sequence. The overall view I would like to pass on is that LAB are more than just lactic acid producers. VII. A further 34 were proposed a function. The second LAB genome to be completed.[250] However. Some of these were unique for Lc. Lactic acid production is merely a reflection of an underlying metabolism. therefore. which have sufficient characteristics in common that some generalizations can be made. lactis. in reality (phylogenetically). as shown for the sequenced Lb. but only 18 have been characterized. Copyright © 2004 by Marcel Dekker.[19] However. . in general. since they are specialized for nutrient-rich environments (limited biosynthetic ability) and their metabolism is aimed at acid production. Another possibility is the following: the commitment to life in rich environments demands a simple. energetics. Lb. These environments are of course excellent for supporting growth of other microorganisms. who they are (classification). One important strategy is acid production and acid tolerance. plantarum strain WCFS1.Hoang-Dung TRAN and friends studies.[318] This may be why the term LAB arose. They have. but more important. we are left with. which is far more complex and. have already done so. it shows that there has been a strong selection for cells that are committed to life in rich environments. In future classification of bacteria. CONCLUDING REMARKS The aim of this review on lactic acid bacteria has been to indicate what they are (general description). and transport). Solution: acid production! Therefore. and how they do it (metabolism. This may have evolutionary implications. what they do (metabolism). The reasons for this are not known. including classical biology and physiology. they readily revert to the auxotrophic state. most importantly.

The authors (correctly) suspected that carnobacteria could be similar to enterococci because of a phylogenetic relationship. However.J. 97– 149. Zur Kenntnis der Milchsaurebakterien der Brennerei-Maische.Hoang-Dung TRAN and friends it will be necessary to do “reverse phylogenetics. essentially serve the same purpose. The study by Meisel et al..L. Carr. Electrogenic precursor/product exchange. W..G. Eds. Dworkin. Johnson. Natl. as I have tried to emphasize. Genome sequencing has confirmed this picture..de/link/service/books/10125/). Pace. Academic Press: London. Lohnis. as a generalization. Zentbl. Chapman & Hall: London. M.. Holt. By using a substrate. In The Prokaryotes: An Evolving Electronic Resource for the Microbiological Community—Release 3.J. Pace. They have developed very efficient transport systems. 1919. 1 – 13. SpringerVerlag: Berlin. 18. M. LAB have developed systems that will allow them to derive more energy from a rich medium than just carbohydrate.L.[19] However. Their extreme saccharolytic nature is another example. 7. some approaches in this direction have been taken with regard to LAB. 82. G. 6. J. Ed.. 3.springer. Lane. Sci. F. D. J.[35] in which it was shown that a species of Carnobacterium (in the study designated Lb. 8 – 11.J. ParasitKde. 1. as an electron acceptor during carbohydrate fermentation. B.g. first define the natural relationships among bacteria with..1. Bacterial classification III.. Krieg. Sogin. Defining taxonomic ranks. Copyright © 2004 by Marcel Dekker. The Genera of Lactic Acid Bacteria. rRNA sequencing and then search for the unifying phenotypic characteristics. Inc. der Presshefe.. N. The lactic acid bacteria—a broad view. M.. 1. E. Ingram. Acad. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analysis. malate and citrate metabolism.R. J. Abt. Bakt. . der Melasse. B. Interestingly. D.g. 2000 (URL: http://link.A. 1984. des Sauerkohls. the term lactic acid bacteria will probably be used in the foreseeable future.. III.. Versuch einer Gruppierung der Milchsaurebakterien.. Eds. G.e. which enable them to quickly take up the necessary solutes.B. otherwise nonfermentable.G. Wood.. have similarities in their ecological behavior. REFERENCES ¨ Henneberg. Williams and Wilkins: Baltimore. was done with the phylogenetically close relationship between carnobacteria and enterococci in mind. they indirectly derive some energy from that substrate that otherwise would be lost. maltaromicus) can synthesize cytochromes in the presence of heme. Again. Abt. Cutting. 1995. 1975. Proc. Some enterococci are known to be able to synthesize cytochromes in the presence of heme. e. der sauren Gurken und des Sauerteigs. The Lactic Acid Bacteria.R. II 1904. ParasitKde. The general metabolism and physiology of LAB reflect their adaptation to niches rich in nutrients.. As indicated above and in Sec. 5. C.H. USA 1985. Stackebrandt. since it is useful to describe a group of organisms that have many physiological properties in common and.. ¨ ¨ 2. Nucleic acids in bacterial classification. des ¨ Bieres. Vol.. Orla-Jensen.V. 11. the genome of Lb.. e.” i. 6955– 6959. Bakt. Holzapfel. Orla-Jensen’s concept of LAB being a “great natural group” may not be entirely correct. Zentbl. In Bergey’s Manual of Systematic Bacteriology..C. Host and Son: Copenhagen.. W. One of these systems involves the “cofermentations” that have been mentioned several times. Whiting. S. N. der Milch. Stahl. All Rights Reserved. II 1907. 8.. In Lactic Acid Bacteria in Beverages and Food. ¨ ¨ sowie einige Bemerkungen uber die Milchsaurebakterien des menschlishen Magens. 4. Olsen. plantarum can serve as an example as it revealed an impressive 25 complete sugar PTS systems. 154 – 170.

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Copyright © 2004 by Marcel Dekker.J. 82. Thompson. Snoep. K.H. Koebmann. X. J...V. X. W. Microbiology 2002. 5887–5890. J. USA 1994. Neal. S. 148. Van. 310. Jamet. Glyceraldehyde-3-phosphate dehydrogenase has no control over glycolytic flux in Lactococcus lactis MG1363. M. 1987. Hugenholtz.. Reizer. Bongers. Westerhoff. 1994. W. Sci. Jensen. Artificial promoters for metabolic optimization. Martens. Ye. Saier. J.. 91. Poolman. 3102– 3106. Chem. T. 149 – 163..N. M. Bacteriol.L. 1978. Hagen. Jr. completely defined medium for meat lactobacilli. Control of glycolysis by glyceraldehyde3-phosphate dehydrogenase in Streptococcus cremoris and Streptococcus lactis. 133. J. 314. 58. 87. Rev.H... P. B. J. J. J. P. Konings. Appl.W..R. B. Cui. Acad. 85. 269.R. Lindgren. 185. Solem. Cui. Microbiol.. Bacteriol. 2003. J. Inhibition of the phosphoenolpyruvate : lactose phosphotransferase system and activation of a cytoplasmic sugar-phosphate phosphatase in Lactococcus lactis by ATP-dependent metabolite-activated phosphorylation of serine 46 in the phosphocarrier protein HPr. Thomas.. 318. L. Starrenburg. Bacteriol. J. B. E. 315.H. 1998.Hoang-Dung TRAN and friends 307.. Reizer... 311. 1163 –1174. metabolic control and experimental analysis.J. P. Møretrø. C. Saier. 237 – 248. Renault.. Ye. ´ Guedon.J. E. FEMS Microbiol..J.. Saier.E.. All Rights Reserved.. J. 1994. Axelsson. Bosman. Catabolite inhibition and sequential metabolism of sugars by Streptococcus lactis.J. Cui. M. the combined approach: kinetic modelling. R. Gene regulation in Lactococcus lactis: the gap between predicted and characterized regulators. Antonie van Leeuwenhoek 2002.. Hammer... 317. 1998. A new.. . Jensen. Ye.. H. 93 – 112. J. Jr. 1564– 1571. Experimental determination of control of glycolysis in Lactococcus lactis. J.W. ATP-dependent phosphorylation of serine-46 in the phosphocarrier protein HPr regulates lactose/H þ symport in Lactobacillus brevis. 309.. Turner.E. 715 – 722. J. J..W. Biol. 169. Jr. Inc.D. M.. 3484–3492. D. 176. Jensen. J. 316.. 312. Bioeng.. Bacteriol. 191– 195. K. M.H.. J. S. Antonie van Leeuwenhoek 2002.. 308.J.. 1003– 1013. 313. T. Natl.F. Reizer. Hoefnagel. II. Regulation of the glucose : H þ symporter by metabolite-activated ATP-dependent phosphorylation of HPr in Lactobacillus brevis. Andersen. C.. 82. Dobrogosz.J. Solem. Biotechnol. Koebmann. Proc. Kiers.. M. Antagonistic activities of lactic acid bacteria in food and feed fermentations. H.R... Metabolic engineering of lactic acid bacteria. 11837– 11844. P. J. B. Kleerebezem. B. 1990. X...

Inc. REFERENCES 1. 3304– 3309. 2003.. Remon.R.. Mercenier.. 21. Turneer. Muller-Alouf. H.. Huyghebaert. Copyright © 2004 by Marcel Dekker. with targets in both food and feed development and promoting human and animal health and well-being.-C. Steidler. Vermeire. Goddeeris. and especially of their role in promoting health and combating disease. Remaut. M. Vaccine 2002. All Rights Reserved. A.. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10.. N. V. 20. D. Protection against tetanus toxin after intragastric administration of two recombinant lactic acid bacteria: impact of strain viability and in vivo persistance. L.. Nat. C. Biotechnol. J.. Geoffroy.. 785 – 789. as they did thousands of years ago. M. Neirynck... Cox. are emerging. 2. Snoeck. . Goudercourt. ¨ Grangette. B. We hope that this volume convinces the reader that new and novel applications based on a better understanding on the potential of lactic acid bacteria in biotechnology. J. The potential significance more than justifies multidisciplinary research in this field. E.Hoang-Dung TRAN and friends and precision.. A. S..

Hoang-Dung TRAN and friends 2 Bifidobacteria and Probiotic Action JEAN BALLONGUE ´ Universite de Nancy 1. the traditional microflora of yogurt—Streptococcus salivarius ssp.[1] which holds that milk fermented with Lactobacillus has a favorable influence on the endogenous intestinal flora. The frequently contradictory findings are due to the unreliability of the methods for isolating and identifying bacteria from stools.[2] “the microorganisms with the best chance of passing through the stomach and small intestine and colonizing the medium are those endogenous to the species consuming the fermented product.. which. . unlike the bacteria of yogurt. France The medical world has long been interested in the nutrient properties of yogurt. numerous studies have been carried out on Lactobacillus.” Research has been focused on the genus Bifidobacterium. who demonstrated that these microorganisms do not survive passage through the stomach and small intestine. Inc. Subsequently. are isolated from animals and humans. already alluded to when they were first discovered in 1899. thermophilus and Lactobacillus delbrueckii spp. was challenged in 1915 by Rahe.[5 – 7] Since 1986. and Centre de Recherche International ´ Andre Gaillard.[4] The therapeutic properties of this genus of bacterium led the Japanese to introduce it to their diet. which are not obtained from human ecosystems. The probiotic effects of Bifidobacterium. All Rights Reserved. Note: This chapter is reprinted from the previous edition. Vandoeuvre-les-Nancy. Ivry-sur-Seine. According to Gurr et al. who were soon followed by dairy industrialists and medical teams. 67 Copyright © 2004 by Marcel Dekker. Nutritionists subsequently turned their attention to other microorganisms. This new product with pleasant organoleptic qualities has aroused considerable interest from consumers. bulgaricus—have been enhanced by a third bacterium belonging to the genus Bifidobacterium and sometimes associated with Lactobacillus acidophilus. The theory of Metchnikoff.[3] were demonstrated by Manciaux in 1958.

taxonomy was based entirely on morphological criteria. Despite the differences between these two bacteria. Where to place this bacterium within the classification system was then addressed.[21] II. These rods. which had hitherto been based entirely on their morphology. the teams of Sebald et al. show grampositive staining. Two trends were distinguished: the French school. which he identified as belonging to the genus Lactobacillus. Inc.I. All Rights Reserved.[16] In 1965. which preferred to integrate the bifidobacteria in the genus Lactobacillus. and are immobile and nonsporulate. At the beginning of the century. which was to develop and gain precision as time passed in parallel with the progress in biology. It is. The development of instruments of analysis made it possible to obtain accurate and reproducible data. and Tissier[3] named this bacterium Bacillus bifidus communis. Research into the biochemistry of the prokaryotes has shown that analysis of the cell constituents could become an essential tool in the classification and identification of bacteria.[10] using new methods. bifidum. Hoang-Dung TRAN and friends THE BIFIDOBACTERIA: DISCOVERY AND HISTORY In 1899 at the Institut Pasteur. Lactobacillus bifidus.10.12 – 14] and the Anglo-Saxon school.[19] Today. Copyright © 2004 by Marcel Dekker. are usually concave. and above all metabolic and enzymatic characteristics of the organism. They therefore concluded that the classification of the bifidobacteria in the genus Lactobacillus is not justified. with an irregular outer wall.[18] showed that the percentage of guanine þ cytosine (G þ C%) in the DNA of Bifidobacterium differed from that of Lactobacillus. In Italy at about the same time Moro[8] discovered in similar conditions a bacterium different from that of Tissier. henceforth took into account new criteria: the physiology.[17] and Werner et al. The advent of chemotaxonomy in the 1960s marked the beginning of another period in bacterial taxonomy. Tissier observed and isolated from stools of infants a bacterium with a very unusual and hitherto unknown Y-shape. the 8th edition of Bergey’s Manual of Determinative Bacteriology recognized Bifidobacterium as a genus in its own right consisting of 11 species. and eliminate subjective judgments. was responsible for a decisive shift in the direction of the history of taxonomy. which are grouped according to their ecological origin: 15 are isolated only from animals. Table 1 summarizes the various names proposed for this bacterium since its discovery[15] (see Table 8 for species isolated to date). Orla-Jensen. minimize errors in individual research.” which may have certain ramifications. which belongs to the Actinomycetaceae family. in 1967 De Vries and Stouthamer[11] demonstrated the presence in bifides of fructose-6-phosphate phosphoketolase (F6PPK) and the absence of aldolase and glucose-6-phosphatase dehydrogenase. two enzymes found in the lactobacilli. Thus. . Holland[9] proposed a common name. MORPHOLOGY The bacteria of the genus Bifidobacterium present a globally bacillar form. and 9 colonize the natural cavities of humans.[9. Corynebacterium. In 1974. The classification and identification of microorganisms. nutritive requirements of the energy metabolism. with progress in molecular genetics.[20] includes 24 species. and Propionibacterium. which was for the separation of the genera Lactobacillus and Bifidobacterium to combine all bifid bacteria under the single classification of B. this genus. and their extremities are generally swollen to form “lumps.

8 Year 1900 1919 1923– 1934 1920 1924 1927 1929 1931 1934 1937 1938 1938 1938 1939– 1957 1944 1949 1950 1953 1957 1963 1969 1969 1972 1974 Bacillus bifidus Bacteroides bifidus Lactobacillus bifidus Bifidobacterium bifidum Bacterium bifidum Tisseria bifida Nocardia bifida Actynomices bifidus Actinobacterium bifidum Lactobacillus acidophilus var. bifidum var.[22] It would appear rather that the composition of the culture medium is responsible for these V-. Several medium constituents may influence the shape of these bacteria: The concentration of N-acetylglucosamine.Hoang-Dung TRAN and friends Table 1 Name Chronology of the Taxonomy of Bifidobacterium Author Tissier Castellani and Chalmers Bergey’s Manual. in a favorable medium the bacilli are longer. or X-shaped forms encountered in the genus Bifidobacterium. bifidus Lactobacillus parabifidus Bifidobacterium bifidum Lactobacillus bifidus Cohnistreptothrix bifidus Corynebacterium bifidum Lactobacillus bifidus Lactobacillus bifidus var. affects the shape of B. ed.[24] Various amino acids (alanine.23] However. however. ¨ Gyorgy Dehnert Reuter Mitsuoka Scardovi Holdemann and Moore Bergey’s Manual. Y-. In contrast. [15]. glutamic acid. eds.[26 – 28] The lower the levels of N-acetylglucosamine and amino acids. .[25] Ca2þ ions. All Rights Reserved.[22] Copyright © 2004 by Marcel Dekker.[22. involved in the synthesis of peptidoglycan (Fig. 5– 7 Negrovi and Fischer Olsen Norris et al. not unusual to encounter more rounded shapes as well as very long or short bacilli of varying widths. pennsylvanicus. aspartic acid. and serine). 1– 4 Holland Orla-Jensen Lehmann and Neumann Pribram Vuillemin Nannizzi Puntoni Weiss and Rettger Weiss and Rettger Prevot Bergey’s Manual. this polymorphism cannot be assimilated to degeneration since these forms can generate the initial forms once more. pennsylvanicus Five groups of bifidus bacteria Description of human species New animal species New animal species New animal species Creation of the genus Bifidobacterium constituted by 11 species Source: From Ref. Inc. which often accumulates in the bifurcations or lumps. the more highly branched are the shapes. eds. Gram staining reveals a frequently irregular distribution of chromatin. 1).

III. bifidum which is relatively aerotolerant. and for others still the presence of catalase is linked to the presence of hemin in the medium. the degree of tolerance of oxygen depends on the species and culture medium. the sensitivity to oxygen varies considerably depending on the strain.[15] No growth without the accumulation of H2O2: the strains tested require a low redox potential for growth and fermentation.[29] Three types of responses are observed during the switch from anaerobiosis to aerobic conditions: Aerobic growth without the accumulation of H2O2: a strain of B. PHYSIOLOGY Respiratory Type The bifidobacteria are strictly anaerobic microorganisms. The absence of H2O2 seen in liquid aerobic culture devoid of catalase of NADH peroxidase activity can be explained by an unknown peroxidase system. The endogenous absorption of oxygen is linked to Copyright © 2004 by Marcel Dekker. Among the strains able to develop in the presence of oxygen. Inc. others become catalase positive. However. In the presence of CO2. All Rights Reserved. . which could destroy the H2O2. forms small quantities of H2O2 by NADH oxidation.Hoang-Dung TRAN and friends Figure 1 Oxygen dissimilation in Bifidobacterium. the accumulation of hydrogen peroxide is considered to be toxic for the key enzyme in the sugar metabolism of Bifidobacterium: fructose-6-phosphate phosphoketolase (F6PPK). A. some remain catalase negative. Limited growth with the accumulation of H2O2.[20] A study of the absorption of oxygen by five strains of Bifidobacterium of human origin has shown that the partial pressure of oxygen falls in the medium during the multiplication of these strains.

B.31] but these early studies should be repeated in view of the difficulty in identifying species of Bifidobacterium at the time these studies were performed. resulting in an accumulation of toxic hydrogen peroxide.Hoang-Dung TRAN and friends the presence of NADH oxidase.1. Another possibility would be the prevention of multiplication by the presence of active oxygen such as superoxide.[15] The initial optimum growth pH is between 6. Inc. There is no growth below 208C. Temperature and pH The optimum temperature for the development of the human species is between 36 and 388C. In reality. 2). B.58C.C. No growth can occur below 5.[22.0. bifidum characterized by the loss of strictly aerobic character have been isolated.6-diphosphate.27). all strains accumulated hydrogen peroxide.[30] The mutants of some strains identified at the time as B. .[32] Aldolase and glucose-6-phosphate dehydrogenase are absent. whereas fructose-6-phosphate phosphoketolase is found[11] (Fig.[33] Small quantities of succinic acid are produced by some strains. METABOLISM Sugar Metabolism In the genus Bifidobacterium hexoses are degraded exclusively and specifically by the fructose-6-phosphate pathway described by Scardovi and Trovatelli.0 or above 8. pyruvic acid can be broken down along two pathways: the first is the reduction of the pyruvate to form L (þ)-lactate by L (þ)-dehydrogenase (E. bifidum dies at 608C. All Rights Reserved. but the activity of this enzyme varied depending on the strain investigated.[20] IV. and the bacteria of this type have no thermoresistance above 468C: B. an enzyme controlled by fructose-1.5 and 7. In contrast. which is subsequently reduced by NADH peroxidase.[33] The proportions of the final fermentation products vary considerably from one strain to another and even within the same species. about 41 –438C and may even reach 46. 1. a portion of which is subsequently reduced to form ethyl alcohol and so regenerate NAD. 1.0. A. These conclusions are summarized in Fig. tests carried out to detect phosphoclastic enzyme have been unsuccessful. Furthermore. It takes place even in the absence of glucose and appears to depend directly on the quantity of polysaccharides accumulated in the cells. that for the animal species is slightly higher. The second pathway involves the splitting of pyruvate by phosphoroclastic enzyme to form formic acid and acetyl phosphate.[20] Copyright © 2004 by Marcel Dekker. The strains most sensitive to oxygen had low NADH peroxidase activity.1. Metabolites The fermentation of two moles of glucose leads globally to three moles of acetate and two moles of lactate. and a small amount of CO2 may be produced during the degradation of gluconate. However.

Enzymes The final fermentation products are formed by the sequential action of transaldolase. 9 ¼ homofermentative pathway enzymes. 4 ¼ transketolase. .1.2). xylulose-5-phosphate phosphoketolase. 1 ¼ hexokinase and glucose-6-phosphate isomerase. is absent in the anaerobic bacteria which could be morphologically Copyright © 2004 by Marcel Dekker. 7 ¼ xylulose-5-phosphate phosphocetolase.22).1).1. 3 ¼ transaldolase. 6 ¼ ribulose-5-phosphate epimerase. transketolase. 2 ¼ fructose-6-phosphate phosphocetolase. and enzymes belonging to the Embden-Meyerhoff-Parnas pathway.Hoang-Dung TRAN and friends Metabolic pathway of Bifidobacterium. which is specific to the genus. 12 ¼ formate dehydrogenase (EC 1. EC 4. This enzyme. 5 ¼ ribose-5-phosphate isomerase.1. The characteristic enzyme of sugar metabolism by the genus Bifidobacterium is fructose-6-phosphate phosphoketolase (F6PPK. Figure 2 C.1. 10 ¼ L (þ) lactate dehydrogenase. 11 ¼ phosphoroclastic enzyme. 13 ¼ alcohol dehydrogenase (EC 1. which act on glyceraldehyde-3-phosphate.2. All Rights Reserved.2. 8 ¼ acetate kinase. Inc.

[32] Biavati et al. folic acid (B9). whereas B. The Vitamins Produced Deguchi et al. Lactobacillus. or human. B.1. that is. Desjardins and Roy[41] used API ZYM systems to determine 22 strains of human origin that possess a. . The results are shown in Table 2. and nicotinic acid (PP). choerinum.2) and 29 isoenzymes of 6-phosphogluconate dehydrogenase (6PGD) (EC 1. Arthrobacter. longum. longum is exceptional. folic acid.2. Galactokinase (EC 2. be used in a preliminary study.[43] were interested in the synthesis of six vitamins by Bifidobacterium of human origin: thiamine (B1). In contrast. however.[36] The F6PPKs of B.6) (galactose þ ATP ! galactose-1-phosphate þ ADP) of B. breve and B. respectively. and a large proportion of each (B6. suis.Hoang-Dung TRAN and friends confused with the bifidobacteria. which are generally deficient in detectable glucose6-phosphate dehydrogenase.44) have been identified. using electrophoresis in starch gel (zymogram) followed by comparison of electrophoretic metabolism.[39] Tochikura et al.[44] However. and N-acetylglucosaminidase activities have also been demonstrated. Five of these vitamins (with the exception of riboflavin) are synthesized by most of the strains examined. 3. bifidum and B.1. Propionibacterium. The production of vitamins B2 and B6 by B. and B. 14 isoenzymes of transaldolase (EC 2. riboflavin (B2).7. and nicotinic acid. infantis are good producers.[37] Transaldolase is an apparently essential enzyme characteristic of the fructose-6-phosphate shunt. B9 and B12) is excreted.and b-galactosidases and a-glucosidase activities. and Actinomycetaceae. three different types of this enzyme depending on the ecological source of the strain: mammalian. longum release small quantities and B.[35] have demonstrated. globosum (animal type) and B.[38] determined the following activities by HPLC: Arylsulfatase (R-sulfate þ H2O ! R – OH þ sulfate). B.[37] Using the same method.1. Inc. This method can. at least in cells cultured on glucose. All Rights Reserved. bee. adolescentis does not synthesize any of these vitamins. B. longum (lactose þ H2O ! galactose þ glucose). 2.1. pyridoxine (B6). These studies were confirmed in the same year by Chevalier et al. These authors also note that with regard to thiamine.[32] Some research has been carried out on other less characteristic enzymes: 1. breve and B. but 6PGD is apparently nonfunctional in the bifidobacteria. Corynebacterium. Nutrient Requirements 1.[40] purified b-D -galactosidase from B.or alkyl-b-glycosides). E. cuniculi develop only Copyright © 2004 by Marcel Dekker. b-glucuronidase. Nitrogenous Matter Most strains of Bifidobacterium are able to use ammonium salts as their only source of nitrogen. dentium (human type) have been purified.[42] b-Glucosidase. bifidum or B. bifidum purified and characterized after growth on galactose. cyanocobalamine (B12). b-glucuronidase (R-glucuronide þ H2O ! R – OH þ glucuronic acid) and b-glucosidase (hydrolysis of the aryl. B. D. Miura et al. b-glucosidase has not been detected in either B. magnum. B. infantis are characterized by a high level of production of vitamins PP and H.

Hoang-Dung TRAN and friends Table 2 Vitamin Production by Bifidobacterium B. In vitro and in the absence of any organic source of nitrogen. adolescentis and B. longum were able to develop in the unsupplemented medium. 1984). Only B. breve B. cyanocobalamine (B12). longum þ þþþ þþþ þ þþþ þþþ þ þþ B. In fact. manganese. for example. folic acid (B9). Through the intermediary of ferroenzymes. and nicotinic acid (PP) for their growth (Teraguchi et al. Transport depends on a membrane ATPase. ¨ Bifidigenic Factors. and above all iron.bifidum can be divided into two variants: the “A” variant or B. and aspartic acid and up to 150 mg/L of threonine. bifidum Tissier required protein factors and not N-acetylated sugars for its development.[46.. adolescentis þ þ þþ þ þ þ þ þþ Thiamine (B1) Riboflavin (B2) Pyridoxine (B6) Folic acid (B9) Cobalamine (B12) Ascorbic acid (C) Nicotinic acid (PP) Biotin (H) þ þ þþ þ þ þþ þþþ þþ in the presence of organic nitrogen. and its incorporation may be competitively inhibited by zinc. the species B. and the “B” variant or B. bifidum. Gyorgy[53] discovered a strain of B. Strains of human origin seem to need thiamine (B1). pyridoxine (B6). bifidum (then known as L. B. valine. Growth Factors Poch and Bezkorovainy[52] supplemented an entirely synthetic minimum base medium with growth factors in order to identify those essential to the development of the various species of Bifidobacterium. produces alanine. bifidum Copyright © 2004 by Marcel Dekker. bifidus) which was to develop only in the presence of human milk and more specifically in the presence of derivatives of N-acetylglucosamine[54 – 56] and showed soon afterwards that the strain B. Vitamins It is impossible to draw up any rule for the genus Bifidobacterium with regard to vitamin requirements. which Tissier found in adult human beings. In 1953. bifidum grows only in the presence of magnesium.[48] Fe3þ ferric iron is used only at neutral pH. bifidum in both oxidation forms depending on the acidity of the medium. infantis þþþ þ þþ þþþ þþ þþ þþþ þþþ B.47] the glutamine synthetase and glutamate dehydrogenase of the Bifidobacterium may be involved in the assimilation of nitrogenous compounds by these microorganisms.[48 – 51] Fe2þ ferrous iron is used at pH 5. 2. bifidum. bifidum. All Rights Reserved. All the other species required the presence of growth factors of various types. iron is involved in the production of acetic acid by B.[43] 4. Inc.[45] According to Hatanaka et al. Iron may be assimilated by B. 3. the bifidobacteria may synthesize large quantities of amino acids. . Trace Elements B. bifidum þþþ þþ þ þþ þ þþ þþþ þþ B.

bifidum requires the sugar factors from human milk in varying quantities depending on the strains. or yeast extract. B. porcine gastric mucin. human milk and rat milk. The activity of the meconium in vitro is 1. The trypsin or chymotrypsin hydrolysis of native human k-casein gives rise to fractions containing 60– 70% of sugars such as galactose.[57 – 59] Most species of the genus Bifidobacterium are unable to develop in a totally synthetic medium and require complex biological substances such as bovine casein digestate.[62] and porcine mucin[23] contain BBb factors (Table 3).[41] Native human casein[66] or its trypsin hydrolysate[63] consisting of glycoproteins may be effective versus B.71] Copyright © 2004 by Marcel Dekker. and colon. respectively. glucosamine. bifidum var.65] human casein hydrolysates. lyophilized milks.[64. The mucins (glycoproteins of mucus) are produced and secreted by the mucus cells of the salivary glands.[53] Mild hydrolysis of the mucins give rise to oligosaccharides similar to those in human milk. Three groups of natural BB factors can be distinguished. the esophagus.62. lactoserum of bovine milk. bifidum have differing nutritive requirements. The factors BBa and BBb are characterized as the elements in human milk that do not lose their stimulant activity for B. BF2. Inc.[67] The mucins. bovine casein hydrolysate. and sialic acid. which may be the following: galactose. and galactosamine.Hoang-Dung TRAN and friends ¨ var. infantis. and glycoproteins) and the BI and BL factors (Table 3). which is active particularly on variant b. 1. All Rights Reserved. N-acetylgalactosamine. which are themselves active. We can now distinguish three main groups of bifidigenic factors which differ depending on the species with which we are concerned:[61] the BB factors (BFl. which Gyorgy isolated from infants. bifidum var. bifidum var. longum and B. the stomach. bifidum var. . Porcine gastrointestinal mucins and the meconium are an abundant source of BB factors.[52. BB FACTORS .60] These growth factors required for the development of Bifidobacterium are known as bifidigenic factors.[70. This is factor BF1 found in milk and colostrum and in the form of gynolactose. N-acetylglucosamine.[23. The BBa factors are found mainly in yeast extracts.2– 2 times greater than that of human milk. b. appears to be insensitive to N-acetylglucosamine derivatives and to require protein factors in the same way as B. liver extracts. b.63] whereas colostrums. fucose. It would seem that the presence of an N-acetylglucosamine structure in the oligosaccharide structure is essential but not sufficient to the expression of bifidigenic activity. ¨ Gyorgy’s bifidus factor I or BF1. pennsylvanicus has N-acetyl-D glucosaminidase activity. which is considerably greater than that found for other bifidobacteria. These oligosaccharide chains are linked to peptide segments accounting for 20% of the weight of the molecule and consisting of more than 70% proline. pennsylvanicus.[66] In addition. whereas the “B” variant of B. These observations suggest that the various strains of the same species B. serine. a and B. and porcine mucin. the small intestine. heating or irradiation.[69] The oligosaccharide chains contain between 2 and 20 monosaccharide residues. bifidum var. and threonine. Their molecular weight exceeds one million daltons. B. which are the major constituents of mucus[68] consist of 70– 80% sugar.

Lyoph. a B. Characteristics of the Main Bifidigen Factors Bifidigen factor Species concerned Active structure N-Acetylglucosamine glycoproteins BB BF1 B. bifidum var. a B. longum Plant extracts Liver extracts Milk Plant extracts Liver extracts Milk 2 2 Proteic different from BB factors Hoang-Dung TRAN and friends . b B. infantis Milk and colostrum Human casein hydrolysate Mucins Casein hydrolysate Human milk and colostrum þ + Nonglycosylated peptides Glucidic part 2 Proteic part BL B.Table 3 Resistance Source þ þ Heat Ray. bifidum var. bifidum var. All Rights Reserved. bifidum var. b Copyright © 2004 by Marcel Dekker. BF2 Glycoproteins þ + BI B. Inc.

however.2. Lactulose.[74] Lactulose and Lactitol. isolated lactulose from human milk. The BI factor. longum. The other growth factors.and radiostable BI. is sensitive to heating and irradiation.[66] BI AND BL FACTORS . It would be important to explore the specificity of these factors with regard to the species of Bifidobacterium that colonize the intestine. Zn) have a promoting effect on the growth of eight species of Bifidobacterium. Lactoferrin and its three metal complexes (Fe. are active with regard to certain species only.[73] These early studies should. infantis. longum. Lactulose (4-O-b-D -galactopyranosyl-D -fructose) is not metabolized by human or animal species. this factor is not active in vitro and is not present in the free state in mother’s milk. since the degree of difference between the collection strains and strains encountered in nature is doubtless considerable. All Rights Reserved. 3. Furthermore. They appear to consist of nonglycosylated peptides obtained by the action of a protease on casein. which appear to be highly complex. the studies conducted were carried out in vitro or in vivo on axenic or monoxenic animals. five of human origin and three of animal origin. and this type of activity appears to be related to their sugar fraction.[72] ROLE OF THE BIFIDIGENIC FACTORS AND CONCLUSION . coli and Staphylococcus aureus. be repeated on the basis of the recent understanding of the taxonomy of Bifidobacterium and the biochemistry of the bifidigenic factors. In vivo. Cu. These factors are proteins. these lactoferrin-metal complexes demonstrate an anti-bacterial activity versus E. and bovine albumin serum digestate. The growth-promoting activity resides in the k-casein portion and not in the carbohydrate portion after trypsin digestion. and these require extrapolation to humans. bifidum and B. Manciaux[4] reported that Petuely. which stimulates the growth of B. as are the BL factors of human milk. porcine gastric mucin. in the 1930s. Hoang-Dung TRAN and friends BF2 factors. . whereas the BL factor.[52] In fact. porcine gastric mucin. Their nature has been described essentially by Raynaud[56] from a strain of B. human or bovine milk lactosera. a. which is not detected in raw milk. which activates the growth of B. In vivo. Glycoproteins. the disulfide/sulfhydryl residues of k-casein are important biologically active compounds responsible for this phenomenon in B. and yeast extract.[75] its action is Copyright © 2004 by Marcel Dekker. is present in dairy products subjected to heat treatment. The factors with general activity are hydrolysates of bovine casein and yeast extracts rather than human milk lactoserum. However. at the beginning of the logarithmic growth phase. Furthermore. the administration of a dairy-based food supplemented with BF1 or BF2 factor to infants restores the bifidum flora partially. It appears that the combination of disulfide/sulfhydryl residues with something else is the basis of the microbial growth-promoting activity in hydrolysates of casein. Inc. The correlation between a given factor and a given species appears to be an important aspect of the study of the bifidigenic factors. lactulose can increase the development of B. bifidum var.[59] ACTIVE CONSTITUENTS OF THE BIFIDIGENIC FACTORS.and radiolabile BI and thermo. is destroyed by lyophilization. According to Petuely. What is the influence of the “bifidigenic” factors on the other bacterial genera of the intestinal flora and particularly of the human flora? Lactoferrin. bifidum. The glycoproteins isolated from human colostrum and milk lactoserum appear to be effective versus both variants. The BI factors from human milk are of two types: thermo. These factors are abundant in many plant extracts as well as liver and milk extracts.

infantis and not by other intestinal bacteria such as E. and insulin (polyfructose) with molecular weights of less than 4500 are used only by B. The sensitivity of bifidobacteria has been the subject of little research. and vancomycin strongly inhibit most species.to pentasaccharides of dextran are also metabolized specifically by B. penicillin G. infantis. acidophilus. 73). kanamycin. Lactulose is not used specifically by the bifidobacteria and may be metabolized by other intestinal bacteria. bifidum. stachyose. coli. clindamycin.Hoang-Dung TRAN and friends due to the fact that it resists better than lactose degradation by lactases in the digestive tract and can therefore be used massively by the bifidobacteria.79] than to purify fructooligosaccharides from natural sources. faecalis. RESISTANCE TO ANTIBIOTICS Knowing which antibiotics the bifidobacteria are resistant to is of interest for two reasons: 1. semisynthetic. 2. However.82] In contrast. It makes it possible to incorporate antibiotics as selective agents in culture media for the isolation of bifidobacteria from complex flora derived from medical or dietary samples. and streptomycin—but the sensitivity of the species varies from 10 to 500 or more mg antibiotic/mL[73] in B. polymyxin B. CULTURE MEDIA AND CULTURE PARAMETERS Three types of medium have been designed for the isolation. nitrofurantoin. Inc.[77] Oligoholosides and Polyholosides. we can accept the following points: most bifidobacteria are resistant to numerous antibiotics—notably to nalidixic acid. and characterization of bifidobacteria: complex. as must be the case for the bifidigenic factors. Raffinose. bacitracin.[76] Fructooligosaccharides. Copyright © 2004 by Marcel Dekker. metronidazole. particularly during antibiotic treatment. infantis and B. All Rights Reserved. . gentamicin. These polymers of fructose with a degree of polymerization of between 2 and 35 have a stimulant effect on the growth of bifidobacteria. It offers the possibility of maintaining the bifidobacteria in the digestive tract without aggression. culture.[80] V. The most important source of fructooligosaccharides is the Jerusalem artichoke tuber. Ref.[76] Lactitol is considered to be a bifidigenic factor with a less marked effect. VI. In contrast. unpublished. L. and the works done before the publication of an international standard are difficult to compare because experimental conditions vary. and synthetic media. ampicillin. lincomycin.[20] Sensitivity to tetracycline varies from one species to another and even from one strain to another (Matteuzi.[77] Today it is easier to prepare a similar substance by an enzymatic route from sugar[78. the oligosaccharides of amylose and cellulose are not specific to B. and S. breve.[78] They are metabolized by bifidobacteria and also by other types of bacteria and are not degraded by human digestive enzymes nor generally by undesirable microorganisms within the digestive tract. chloramphenicol. Oligosaccharides higher than the trisaccharide of inulin and the tri. oleandomycin.[81. erythromycin. neomycin.

We will note particularly the following: Tomarelli’s medium[90] for the culture of B.[5. described by Ochi et al. or human milk and permit the growth of as many strains of Bifidobacterium as possible.[51] developed a synthetic medium for the culture of the ES5 strain of B. We will consider the following: BL agar medium.[83] and then by Mitsuoka et al.21] can be used for the culture and isolation of bifidobacteria but also of other lactic bacteria from all habitats.[52] D. a wide range of peptones.[85] is considered to be the optimum culture medium for the detection of bifidobacteria.[91] Tanaka and Mutai medium.[92] Ueda et al.[20] Since the recent enthusiasm for incorporating bifidobacteria in fermented dairy products.[31] which is a modification of the Tomarelli medium ¨ Gyorgy’s medium. it is difficult to define a selective medium appropriate for all species. horse blood.. Scardovi’s tryptone phytone yeast medium (TPY)[20.84] and finally slightly modified by Teraguchi et al. Mention should also be made of the YN-6 medium[86] and YN-17 medium. tomato juice. Entirely Synthetic Culture Media Petuely[75] was the first to propose a synthetic culture medium. yeast extract.[65] which is also a modified Tomarelli medium. Semisynthetic Culture Media Complex constituents of known composition are included in these media.Hoang-Dung TRAN and friends A. Since the physiological requirements of bifidobacteria are extremely varied.[88] whereas YN-17 medium inhibits some of the bifidum population. Poch and Bezkorovainy medium. All Rights Reserved. Copyright © 2004 by Marcel Dekker. cystine. Hassinen medium.[44] Gyllenberg modified the Petuely medium. The media listed above are efficient for the maintenance of pure strains but are less effective for isolating them from complex flora since they often permit the growth of other genera. they are supplemented with substances with a low redox potential: cysteine. Inc. or sodium sulfite.[89] B. Selective Culture Media All the constituents of these media are chemically defined. C. several selective media have been proposed in order to differentiate between Bifidobacterium and other lactic bacteria and to isolate bifidobacteria from the intestinal flora. ascorbic acid. Complex Culture Media These richly varied media are prepared from liver or meat extracts. bifidum. In addition. bifidum Norris’s medium. .[15] A wide range of complex culture media have been proposed in recent years.[87] These media are not very efficient since YN-6 medium inhibits some species of Bifidobacterium but allows other genera to develop.

.[96] took into account the fact that bifidobacteria are able to metabolize carbohydrates such as fructo. convex. ascorbic acid and sodium azide were used as selective substances. E. TTC makes it possible to differentiate between Bifidobacterium and other species since the bifidobacteria develop in large white colonies. This medium is a reinforced clostridial medium (RCM) containing added antibiotics (nalidixic acid. the new medium containing cysteine. polymyxin B. This is macromolecule consisting of linear Copyright © 2004 by Marcel Dekker. Matteuzzi et al. This medium is relatively commonly used. white. In general. Iodoacetate. the colonies formed are round. or murein. but Scardovi[20] and Boventer[99] distinguished two differing types of colony for B. VII. Culture Parameters The appearance of the colonies of Bifidobacterium cultured on agar medium under anaerobic conditions may vary in function of the medium and the species used. Sonoike et al. and 2.[95] were able to selectively isolate B. with uneven edges and map. Twenty-two species of Bifidobacterium are able to develop on a medium containing transgalactosylated oligosaccharides as a carbon source. peptidoglycan. However. propionate. COMPOSITION OF THE WALL The main constituent of the bacteria wall of the genus Bifidobacterium (gram-positive) is mucopeptide. considerably reduces the growth of nonbifidum colonies. azide. Beck[93] isolated Bifidobacterium on a medium containing added bifidigenic growth factors (N-acetyl-D -glucosamine). and China ink in order to isolate numerous species of Bifidobacterium.[97] Mitsuoka used propionate as one of the selective agents added to BL agar medium for the selective counting of intestinal bifidobacteria. The base medium was similar to Norris’s medium.5-triphenyltetrazolium chloride (TTC).8) and anaerobiotic culture makes it possible to eliminate most enterobacteria. All Rights Reserved.5. kanamycin). iodoacetic acid. Inc. Ushiima et al.[82] suggested the addition of 80 mg of kanamycin/mL to the medium. Poch et al. which inhibits glyceraldehyde-3-phosphate dehydrogenase.Hoang-Dung TRAN and friends Initially. intraspecific variations of resistance are so great that the isolation of unknown strains with a medium of this type would be unreliable. Munoa and Pares[89] attempted to quantify Bifidobacterium from water on a new selective medium: Bifidobacterium iodoacetate medium 25 (BIM-25). Beerens[98] proposed a selective and elective medium by adding 5 g/L of propionic acid to Colombia agar and adjusting the pH to 5. whereas other colonies were rough. but also within a given species.3.[94] modified the MRS agar medium. Some colonies were smooth. Chang et al. adolescentis from a complex gastric flora by using selective agents: polymyxin.and galactosyl-oligosaccharides. The BS agar medium thus obtained is not entirely satisfactory for the detection of bifidum in the stools. dull or glossy and of variable diameter.[52] supplemented a synthetic base medium with various substrates in order to identify the growth factors necessary for each species of Bifidobacterium. and linoleate. and shiny. The relatively low pH of this medium (5. bifidum.

101] The amino acids may be the same or may differ from one strain to another. which are polymers of glycerol phosphate. longum. . and rhamnose comprising the polysaccharide portion of the wall. This macropeptide is covalently linked to other macromolecules.20] Figure 3 Peptidoglycan structure of Bifidobacterium bifidum. A. aspartic acid. B.) Copyright © 2004 by Marcel Dekker. The amino acids constituting the various peptides are alanine. or even a tripeptide.2 and 64. VIII. All Rights Reserved. These teichoic acids are attached to the NAG-NAM skeleton of the peptidoglycan. or threonine. The polysaccharide chain consists of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). ornithine or lysine associated with one or two of the following: glycine. such as (a) polyosides: glucose. The tetrapeptides are linked to the NAM residues and to each other through the intermediary peptides (Fig. glutamic acid.[100. (From Ballongue. 1989. CHARACTERISTICS OF THE GENOME DNA Base Composition The G þ C% of the bacteria of the genus Bifidobacterium is between 57.5%. and (b) teichoic acids.Hoang-Dung TRAN and friends polysaccharide chains which are linked with each other by tetrapeptide bridges associated with peptides.[17. may consist of a simple amino acid. their sequence within the tetrapeptide and their types of cross-linkage may vary.. for example. galactose. 3).e. a dipeptide. but even if they are the same. serine. possesses an ornithine-type tetrapeptide and the link peptide is [15] L -Ser – L -Ala – L -Thr – L -Ala.[102] i. Inc.

No phenotypic characteristic has so far correlated with the presence of plasmids. 5. enterococci) decline sharply by about 1000-fold. longum. asteroides has 14 types of plasmids (1. Copyright © 2004 by Marcel Dekker. After birth. B. B. B. Inc. longum contains 13 model plasmids (1. 2. 24.5. whereas the levels of other bacteria (E. (B.2 – 22 MDa). the colon contains 109 –1010 bacteria per gram of stools consisting mainly of enterobacteria. Do the bacteria invade from the digestive tract from the mouth or from the rectum? Several studies tend to disprove the hypothesis that the digestive tract is colonized by the rectal route[116. Plasmids B. bifidobacteria enter the body of the neonate by an oral route: Kleinschmidt[118.[105] Very curiously. adolescentis. breve. Boventer[99. staphylococci. B. coli.25 – 9.5. pseudocatenulatum. B. as we shall see below. 9 are isolated essentially from humans: B.[112 – 115] Anaerobes such as Bacterioides and Clostridium and other putrefying bacteria are enormously reduced and may disappear. Sgorbati.120] clearly showed that the implantation of Bifidobacterium in the digestive tract occurs by descending route. and B.[104.107] has no plasmid. which are structurally very varied.119] detected B. A. catenulatum. breve. angulatum. All Rights Reserved.[103] had not found any plasmid in B. unpublished). and 46 MDa). Origin of Colonization This rapid invasion of the sterile digestive tract at birth raises the question of the origin of the bacteria and more particularly of anaerobes such as Bifidobacterium. In some strains these plasmids may be temperate phages. since the rectum remains sterile until colonization is complete. B. . on the contrary. dentium. breve: Iwata et al. 3. B. but this appears to be unusual. bifidum in the upper segment of the digestive tract of a child operated due to the absence of an anal perforation.117] and demonstrate that.[57.105] B. They reach a level of 99% of the fecal flora.5 MDa). infantis. only 5 species have plasmids:[103.104] 1. BIFIDOBACTERIUM ECOLOGY Of the 24 species of Bifidobacterium so far recognized. longum. B. B. even though Sgorbati et al.[109 – 111] The bifidobacteria appear only between day 2 and day 5[109] and become dominant 10 (10 –1011 per gram of stools) barely one week after birth.[108 – 110] Forty-eight hours after birth. globosum contains a plasmid belonging to each of the three molecular weight categories (13. bifidum. Of the 24 species of Bifidobacterium. lactobacilli. which survive only precariously in the atmosphere. Implantation in the Neonate It is generally admitted that until the time of birth the fetus is surrounded by a completely sterile environment.Hoang-Dung TRAN and friends B. 4. 1. the digestive tract is rapidly colonized by bacteria. B.[106] have demonstrated plasmids in 40% of the strains of this species. indicum: 60% of the strains isolated contain a single 22 MDa plasmid.[103] IX. and streptococci. which constitutes a continuum with B. B. infantis.

125 – 131] The stools of a breast-fed child are also characterized by a granular appearance. in artificial feeding the fundamental difference lies in the maintenance of high levels of optional aerobic species (E. INFLUENCE OF THE TYPE OF FEEDING ON THE COMPOSITION OF THE INTESTINAL The effects of breast-feeding and bottle-feeding have been compared in numerous studies. Propionibacterium. From subsequent research it has emerged that there is no difference in the qualitative distribution of species between these two types of feeding.[122] Mitsuoka.123. the predominant popu- FLORA .124] Method of Feeding. . whereas following cesarian section only Propionibacterium and Enterococcus were isolated in 8 out of 9 neonates investigated. Tissier wrote that the flora of a neonate raised on the breast consisted entirely of Bifidobacterium.[57] Some authors. which we have just seen directly affects the speed of invasion by bifidobacteria. Bacteroides. Peptostreptococcus. At the beginning of the twentieth century. Tests for bifidobacteria in mother’s milk have always been negative. Enterobacteriaceae.132 – 134] In contrast. Prematurity. Factors Influencing Colonization In addition to the method of delivery. Clostridia. Eubacteria. Fusobacterium. whereas enterobacteria and Bacteroides readily to colonize the colon. et al. several other factors also influence colonization.[110] with the exception of the findings of Mayer and Moser.[135. Copyright © 2004 by Marcel Dekker.2).[137. and marked acidity (pH 48– 5. Inc.[112 – 114.[132] which is probably due to the abundance of Bifidobacterium and therefore to considerable acetate production. whereas Bezirtzoglou[110] found them in 21% of infants aged 4 days and born by cesarian section (41% after 15 days).[109] isolated bifidobacteria in 41% of vaginal births.Hoang-Dung TRAN and friends Mutai and Tanaka[121] isolated and observed in the mouth of 23 neonates Bifidobacterium. Thus. The difference lies in the quantitative level in the proportion of Bifidobacterium and other species. All Rights Reserved.[22] who isolated B.138] on the contrary. Peptostreptococcus. the stools of children fed artificially are more similar to those of adults (pH 6. coli and streptococci) which initially colonized the digestive tract and the development of anaerobes (Bacteroides. Enterococcus. note that breast-feeding does not increase the level of Bifidobacterium in the first few days after breast-feeding. Streptococcus). bifidum from the colostrum and milk of a woman before breast-feeding commenced.136] Bifidobacterium appears fairly late and is found in lower proportions in the stools. Clostridium. Another weighty argument in favor of the hypothesis of colonization by the vaginal or fecal flora of the mother is the observation by many authors that invasion of the digestive tract of the neonate occurs much more rapidly after vaginal birth than after birth by cesarian section. with clearly higher levels of Bifidobacterium for children breastfed who show high levels of Bifidobacterium. 2. Lactobacillus. whereas lactobacilli were predominant in the flora bottle-fed infants.[5. which are generally higher than those of enterobacteria and Bacteroides within the first week of life (see Table 4 and Fig. This is a cause of difficult implantation of Bifidobacterium due to the lack of receptors and/or endogenous substrates. 4).0)[58] which indicates the presence of putrefying organisms.4 – 7. slightly vinegary smell.111. and Enterobacteriaceae 10 minutes after birth via the genital tract.[109.

3 9.9 5.9 Enterobacteriaceae Streptococcus Staphylococcus Lactobacillus Bifidobacterium Eubacteria Bacteroidaceae Peptococcaceae Cl.9 10. Figure 4 Comparison of fecal flora between breast-fed and bottle-fed infants.5 9. Copyright © 2004 by Marcel Dekker.1 2.8 Bottle-fed infants 9.9 6.3 5.1 6.9 7.6 7.0 7. All Rights Reserved.Hoang-Dung TRAN and friends Table 4 Bacteria Comparison Between Fecal Floraa of Breast-Fed and Bottle-Fed Infants Breast-fed infants 8.0 1. perfringens Clostridium Veillonellae a log cfu.4 0. .4 1.9 5. Inc.7 3.8 5.0 10.8 7.5 5.

There is a remarkable proliferation of Bacteroides. Inc. Eubacterium. (From Mitsuoka. . CONSTITUENTS OF HUMAN MILK AFFECTING THE EQUILIBRIUM OF THE INFANT’S Humanized milks have an organoleptic composition similar to that of human milk: high concentration of lactose. no industrial dairy formula has made it possible to maintain the same equilibrium as that found during breast-feeding.[140] did not observe any change in the distribution of the species in breast. 1984. bifidum appears to be the dominant species during breast-feeding. in Italy. B. FLORA . To date. B. a sudden change occurs in the fecal flora following the first bottlefed[139] or solid food.[129] B.[141] They are still incapable of providing the conditions favorable to bifidobacteria in breast-fed infants. and a low buffering potential. Bifidobacterium constitutes the predominant genus (Fig. B. In some children the bifidum flora falls sharply. Figure 5 Changes of intestinal flora from birth to old age. 5). and Clostridium.) Copyright © 2004 by Marcel Dekker. bifidum longum infantis breve 3% 8% 12% 11% The discrepancies between these findings are probably a result of the differing techniques used to identify the strains. in others. Peptostreptococcus. low concentrations of the proteins used by putrefaction organisms.[131] note that in both types of feeding. however.or bottle-fed infants: B. Biavati et al. At weaning.[136] In all cases. INFLUENCE OF THE TYPE OF FEEDING ON BIFIDOBACTERIUM SPECIES .[59. Human milk provides factors essential to the intestinal development of Bifidobacterium. whereas the Italian authors identified species using DNA-DNA hybridization. the ratio of Bifidobacterium “putrefying flora” falls and is reversed. some lactoferrin and lactulose.127. The proportions of the various species of Bifidobacterium also vary with the type of feeding. it remains stable. the French and Japanese workers used carbohydrate fermentation. All Rights Reserved.130] In contrast.Hoang-Dung TRAN and friends lation consisting of enterobacteria and not of strictly anaerobic species. Benno et al.

whereas the numbers of Bacteroides. hospital. B. longum biovar. They are produced by the host and may be used by bacteria.[109] and Biavati [154] isolated B. and each age group has its characteristic species (see Table 5). breve was recognized as the dominant species. catenulatum. breve. infantis from the stools of Japanese infants. Numerous observations suggest that the environment and. They then observed the appearance of this species in the stools and its disappearance if the administration was stopped. The species present and the biotypes found also vary with time. These species. adolescentis. bifidum var.[145.149] B. and even the unit within which the delivery takes place influence the rapidity of colonization by Bifidobacterium.[143] administered relatively large quantities of N-acetylglucosamine to infants with low levels of B. b. but 10 years later B. longum biovar. and b-glycosidases.[138. which they named gynolactose and allolactose [b-D -galactopyranosyl(1. a. obstetric and therapeutic customs (increasingly frequent use of antibiotics) play a role in the colonization of neonates by bifidobacteria. in particular. In children fed milk containing added porcine mucin. Enterobacteriaceae. B. b. Clostridium.[147] Environment. Mitsuoka et al. and B. Streptococcus. Inc. The fall-off in Bifidobacterium is accelerated in the elderly. To date. Polonowski and Lespagnol[142] isolated oligosaccharides other than lactose from human milk. pseudocatenulatum) are well Copyright © 2004 by Marcel Dekker. These bacteria include species of the genus Bifidobacterium: B. bifidum and B. sialidases. in children under 7 months of age. Peptostreptococcus. infantis. the pH of the stools fell and the bifidum population rose. Endogenous Substrates. Endogenous substrates exist within the digestive tract without a dietary source. The country. are not present in young children and disappear in the flora of older children and adults. longum biovar. where there is an increase in Clostridium and optional aero-anaerobic species. Some strictly anaerobic bacteria produce enzymes able to degrade blood group antigens and mucin oligosaccharides.[109] isolated mainly B.[114.146] They are able to remove the N-acetyl-D -galactosamine residues from the blood group A factors and also secrete a-L -fucosidases. Mitsuoka et al. Figure 5 shows the change in the intestinal flora throughout life. and Lactobacillus increase. .[144] Numerous substrates therefore appear to be involved in maintaining the equilibrium of the infant’s intestinal flora. with the exception of B. It would even appear that very strict conditions of hygiene delay the implantation of Bifidobacterium. no active molecule has been identified. B. The proportions of the various Bifidobacterium species also vary with age. b. Levesque et al. Eubacterium. infantis. infantis appears to be specific to neonatal infants[35] and to show a higher incidence in the stools of breast-fed infants. 1. In children and adults. B.148.6)-D -glucopyranose].Hoang-Dung TRAN and friends In 1930. and species with similar fermentation characteristics (B.150 – 153] This change is usually due to a reduction in the gastric secretion in this age group. Change from Weaning to Adulthood Influence of Age Many authors observe that the number of bifidobacteria falls significantly in adult stools and particularly in those of the elderly. B. bifidum in the flora. B. All Rights Reserved. Thus.

A. bifidum biovar. longum B.154] However.[109. breve B. the only identification criteria were phenotype characteristics. adolescentis biovars. infantis B. and this species may be confused with four other species. breve are not present in this age group. It would seem that foodstuffs have little impact on the constitution of the dominant intestinal flora. adolescentis B. Identification of the Genus Bifidobacterium Until the 1960s. adolescentis biovars.109. . longum B. dentium (formerly Actinomyces eriksonii) is isolated from dental caries or abscesses. b B. bifidum biovar. bifidum biovar. IDENTIFICATION BY PHENOTYPE INVESTIGATION When the bifidobacteria were discovered by Tissier at the beginning of the twentieth century. Mitsuoka[153] noted a reduction in the level of Bifidobacterium after the administration of a western diet to individuals used to Japanese food. X. longum B. b Adults Older adults B. B. adolescentis type b increases considerably in the elderly. a represented.[123. Copyright © 2004 by Marcel Dekker. a and b B. and the proportion of B. All Rights Reserved. Inc. Influence of Diet The high degree of variability of the intestinal flora in infants depending on diet contrasts with the apparent stability of the flora in adults despite differences in diet. This lack of differentiation criteria explains the numerous debates which preceded the creation of the genus Bifidobacterium.140] B.Hoang-Dung TRAN and friends Table 5 Population Distribution of Bifidobacterium Species in Human Colon Predominating species B. adolescentis (see Section XIII and Table 13). breve B. Pathogenicity B.[35. infantis and B. adolescentis is the species characteristic of adult flora.153] 2. taxonomy was based entirely on morphological observations. longum Minor species Breast-fed infants Bottle-fed infants Children B. In contrast. infantis B. 3. notably B. Taxonomy subsequently was based on increasingly numerous phenotype characteristics and today can make use of progress in genotyping. b B.

. although the peptoglycan structure of Bifidobacterium is closer to that of the Lactobacillaceae than the Actinomycetaceae. or glycerol Copyright © 2004 by Marcel Dekker. it cannot be considered a specific characteristic but only an indicative criterion. C16:0 (palmitic acid). In addition. It should be noted that the growth temperature and the composition of the culture medium have a marked influence on the distribution of lipids and phospholipids. Inc. The detection of F6PPK can be completed by a test for a-galactosidase. Enzyme Tests The association of a branched shape with the presence of fructose-6-phosphate phosphoketolase (F6PPK) in a strain indicates that it belongs to the genus Bifidobacterium. C16:1 (palmitoleic acid).[21] 3. accompanied by marked acidification No acid production from rhamnose. In addition.102] 7. for which this band is located at a slightly greater distance from the anode. Metabolites The determination by gas chromatography of organic acids produced at the end of fermentation and notably an acetic acid/lactic acid ratio of about 3/2 provides excellent identification criteria for the genus Bifidobacterium.[159. though the genera Bifidobacterium and Lactobacillus both contain diphosphatidylglycerol and phosphatidylglycerol. alanylphosphatidylglycerol. Other Identification Criteria Other tests can be used to identify the genus Bifidobacterium. dulcitol. fructose. it is important to note that bifidobacteria produce the L þ isomer of lactic acid. Actinomyces). Study of Electrophoretic Patterns All soluble cell protein electrophoretic patterns show a band that migrates over the same distance. 6.[42] 5.[156] This test can therefore be used as an identification indicator. boum. and C18:1 (oleic acid). with the exception of B. Lipids and Constituents of the Cell Wall Membrane Bifidobacteria have the following fatty acids: C14:0 (myristic acid). Culture Conditions Bifidobacteria develop under anaerobic conditions at 378C in species of human origin or 428C and higher for species of animal origin and require an incubation time of 48 hours. adonitol.Hoang-Dung TRAN and friends 1.160. notably:[15. All Rights Reserved.[32] 2. levulose. and galactose. sorbose. erythritol. 4. Propionibacterium. C18:0 (stearic acid). lactose. The API ZYM system indicates a-galactosidase activity in bifidobacteria[155] but not in lactobacilli. Corynebacterium. and the lyso derivatives of diphosphatidylglycerol. Morphology Since a branched appearance is seen in other bacterial genera (Arthrobacter.[158] Analysis of the cell composition in terms of lipids and phospholipids therefore provides a good criterion for distinguishing between the genus Bifidobacterium and the Lactobacillaceae.[157] The presence of this band therefore appears to provide an appreciable criterion for the identification of the genus.161] Rapid and complete coagulation of milk without the formation of gas Fermentation of glucose. only Bifidobacterium possesses polyglycerolphospholipids and their lyso derivatives.

pseusolongum var. rat. breve ferments ribose. longum ferments arabinose and melezitose. most researchers classed all bifidobacteria together as a single species: Bifidobacterium difidum. asteroides.Hoang-Dung TRAN and friends The development of these microorganisms in peptone water Negative catalase No reduction of nitrates No indole formation No liquefaction of gelatin No fermentation of glycerol No attack of coagulated proteins B. animalis a and b. All Rights Reserved. and d. Roy et al. guinea pig. Sugar Fermentation This criterion has been used most frequently to identify and define new species. A mixture of these seven sugars is monitored by gas chromatography and should make it possible to identify six to eight typical strains of Bifidobacterium in less than 24 hours. longum subsp. b. B. In addition. . In 1957. chicken. thermophilum) and B. He proposed two new species. Copyright © 2004 by Marcel Dekker. b. B. whereas B. and d. globosum and then B. B. bee). c. and the results obtained have been compared with the identification tables produced by Mitsuoka[5. Using fermentation profiles and the ability to grow at 46. B. 1. mannose.[155] developed a rapid method for identifying bifidobacteria species based on the fermentation of seven sugars: arabinose. c. Until 1957. Dehnert[162] was the first to demonstrate the presence of several Bifidobacterium biotypes and used 24 sugar fermentation processes to classify the various species into five groups. whereas B. melezitose.167] The ability of a strain to ferment certain sugars is the test first used to identify species. B. whereas B. thermophilum var. esculine.164] associated serological properties to sugar fermentation to identify new human-derived species isolated from the stools of adults and children and their various biotypes. a. mouse. infantis does not ferment arabinose. and a new variant. ribose. longum ferments melezitose. This method presents no major operating problems but does have several drawbacks: it is lengthy and tedious because a panel of 30 sugars must be studied for 10 days. B. A few years later. sheep. cellobiose.[166. for example: B. the interpretation of the results using identification tables remains controversial and can at best give an indication of an identification based on the fundamental characteristics not open to doubt. a. and salicin. thermophilum does not ferment pentoses but does ferment starch. indicum. ruminale (synonym of B. calf. B. B. lactose. pseudolongum ferments pentoses and starch. and amygdaline but does not ferment arabinose or xylose. Species Identification It is obvious from the multiple taxonomic revisions that have taken place in a few decades how difficult species identification is within the genus Bifidobacterium. coryneforme were isolated in the same year. mannitol. animalis is unable to ferment this sugar.58C enabled Mitsuoka[165] to separate human strains from animal strains (pig.153] and Scardovi[20] (Table 6). and B. Numerous sugars have been tested. Inc. Reuter[163.

pseudocatenulatum B. bifidum B. minimum B. angulatum B.Hoang-Dung TRAN and friends Copyright © 2004 by Marcel Dekker. thermophilum B. pullorum B. catenulatum B. asteroides B. All Rights Reserved. animalis B. v ¼ variable. Salicin Starch Inulin . cuniculi B. globosum B. subtile B. dentium B. infantis B. adolescentis B. choerinum B. boum B. L-Arabinose Melezitose Cellobiose Gluconate D-Ribose Melibiose Trehalose Galactose Raffinose Mannitol Mannose Fructose Sorbitol Maltose Sucrose Lactate Xylose B. indicum 2 þ þ þ þ þ þ þ þ þ þ 2 2 þ 2 2 þ þ 2 2 þ þ þ þ 2 þ 2 2 þ þ þ þ þ v þ þ 2 þ 2 2 þ þ þ 2 2 þ þ 2 þ þ þ þ þ þ þ þ þ þ v 2 þ þ v v þ 2 þ 2 2 2 2 2 2 2 2 v þ 2 þ v þ 2 v 2 2 v v 2 2 2 2 2 2 þ þ þ 2 þ 2 v þ 2 2 2 þ 2 v 2 2 v v 2 2 2 2 2 þ 2 2 2 2 þ þ þ þ þ þ þ þ þ þ 2 þ þ þ þ þ þ þ 2 þ þ þ þ 2 2 2 v v v þ v 2 2 2 2 2 2 2 2 2 2 2 2 þ 2 2 2 2 2 2 2 þ þ 2 þ þ þ þ þ þ þ þ þ 2 2 2 þ þ 2 2 2 2 2 2 2 þ 2 v v þ 2 2 2 2 2 2 2 2 2 2 2 þ þ v þ 2 v v 2 þ þ þ þ þ v þ 2 þ 2 þ þ þ þ 2 2 þ þ 2 2 v v þ v þ 2 þ þ 2 þ 2 2 v 2 2 2 þ v 2 2 2 2 v þ þ þ þ þ þ þ þ þ þ þ 2 2 þ þ 2 þ þ v þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ 2 þ þ þ 2 þ v v v þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ 2 þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ v v 2 2 2 v v 2 v v þ 2 2 2 2 v v 2 þ 2 2 v 2 2 2 v þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ 2 þ þ þ þ 2 2 2 v v 2 v 2 þ 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 v v v þ v 2 2 2 2 2 2 2 v þ 2 þ 2 2 v 2 2 2 2 2 þ þ þ þ þ þ 2 2 2 2 þ v 2 2 þ 2 2 v þ þ þ þ ¼ positive. 2 ¼ negative. breve B. coryneforme B. longum B. Table 6 Sugar Fermentation by Bifidobacterium sp. pseudolongum B. Inc. magnum B. suis B.

[16] Two types of study can be envisaged for the comparison of Bifidobacterium species with each other. choerinum B. indicum Migration (cm) n. Electrophoresis in a starch gel (zymogram) of the 14 enzymes of transaldolase and the 19 isoenzyme of 6-phosphogluconate dehydrogenase (6PGD) can be used to compare the electrophoretic mobility of these enzymes in the original strain (Table 8). cuniculi Note: n.d. heat inactivation. The sequence of amino acids. All Rights Reserved. and the net electrical charge of each protein are determined by the sequence of nucleotides in the DNA. infantis B. but many studies have been carried out using only the soluble fraction of disintegrated cells. molecular weight. dentium (human origin) demonstrate activities which vary with regard to optimum pH. The protein profile of each strain is therefore a fingerprint of the genome. Study of F6PPK Isoenzymes A test using a colorimetric reaction following starch gel electrophoresis for three isoenzymes of F6PPK can give an indication of species identity.Hoang-Dung TRAN and friends 2. bifidum B. The cell proteins are dissolved using detergents such as SDS. suis B. The migration distances are linked to the ecological origin of the species: human (15 cm).d. thermophilum B. Species B. longum B. Copyright © 2004 by Marcel Dekker. globosum (mammalian origin) and B. and affinity toward the substrate. asteroides B. The distribution of the protein bands is Table 7 Species Migration of F6PPK in Bifidobacterium sp. purified preparations of F6PPK from B. not determined. 10 10 10 10 10 – 15 16 16 16 B. breve B.. A colorimetric method applied to 3-phosphoglyceraldehyde dehydrogenase is able to identify other strains. Study of Protein Profiles A bacterial strain cultured under standard conditions always gives the same protein profiles. 6-phosphogluconate dehydrogenase. the molecular weight.[169] Electrophoresis in a polyacrylamide gel of the lysate of a strain provides electrophoretic profiles of the soluble cell proteins. pullorum B. animalis B.d. pseudolongum B. dentium B. 15 10 10 n.d. Inc. 10 10 n.[35] These isoenzymes catalyze the same reaction but are distinguished by differing electrophoretic patterns. but the same is not true for the other glucose metabolism enzymes of Bifidobacterium—transaldolase. boum B. the identity of the metal inducing maximum activity.d. 1986. and aldolase. or bee[20] (Table 7).[168] The electrophoretic migration distances for F6PPK appear to be linked to the ecological origin of the species. globosum B. angulatum B. coryneforme B. pseudocatenulatum B. transketolase. In addition.[104] 3. Migration (cm) 15 15 15 15 15 15 15 n. catenulatum B. mammalian (10 cm). subtile B. minimum B. Source: Scardovi. adolescentis B. . magnum B.

since it is able to distinguish between strains with DNA-DNA homology levels of up to 80%. animalis B. asteroides B. All Rights Reserved. catenulatum B. infantis B. globosum. B. dentium. pseudolongum. .[157] but it is an onerous method. indicum a Transaldolase 7 (5)-6-8a 5-(6)-(8a) 6 8 5 5 4a-(5) 4 2 2 1 3 5 (7)-8a 6 5 2 6 10 3 6 (6)-(7)-8-(9) (6)-7-8-9a 6PGD 7-(8) 5-(6a) (3)-4a-(5) (5)-6-7 5 5 6a-8 1a-3 (2) (3)-(4)-(5)-6-(7) 7 4 4 8-9a 7-8-9a 8a-9 7 Absent 5-8 6 2 6 (9)-10a-(11)-(12)-(13) 6-(7)-8-(9a) Number of isoenzymes for type strains. Electrophoresis in polyacrylamide gel enabled Biavati et al. which is now distinguished from B. suis B. choerinum B. adolescentis and B. magnum B. dentium (the only species thought to be pathogenic) and B. breve B. subtile. cuniculi B. requiring reference strains. angulantum B. Source: Scardovi.Hoang-Dung TRAN and friends Table 8 Migration of Transaldolase and 6PGD in Bifidobacterium sp. Inc. pseudolongum B. B. subtile B. pullorum B. can also be differentiated by their zymograms (starch gel electrophoresis). bifidum B. boum B. The homology between B. 1986. which differ. Electrophoretic pattern Species B. globosum B. then compared with those for a reference strain. eriksonii (formerly Actinomyces eriksonii) was established by comparing their electrophoretic patterns (zymograms). Copyright © 2004 by Marcel Dekker. pseudocatenulatum B.157. adolescentis B. minimum.[35. dentium B. B.170] This method is doubtless the most discriminating and is both reliable and sensitive. ( )Number of isoenzymes in less than 10% of strains. Use of these two types of electrophoresis has given the following results: 1. minimum B.[169] This identity complies with high DNA-DNA homology (80 –100%). 3. and is difficult to interpret.[157] to recognize four new species: B.[157] 2. thermophilum B. longum B.[169] Polyacrylamide gel electrophoresis also confirms these findings. which have identical phenotype profiles. cornyeforme. and B. coryneforme B.

to 8. C. When the preparation conditions for the extracts and their electrophoresis are standardized. B. Transaldolase Serology An immunological approach investigating the serology of the transaldolases can also be used to differentiate between species within the genus Bifidobacterium. present similar profiles. three different isoenzymes. thus defining a “continuum. . Archiving of a large number of models in a reference data bank. expressed as taxonomic distance. infantis. highlighting what is doubtless a very close degree of relatedness between these species. infantis. and G) detected by this model can be split into four distinct groups closely linked at the ecological origin of the species. In this case. This diagram illustrates the interrelationships existing within the genus and shows that the seven groups defined by Sgorbati and London[107] (A. III. Figure 6 shows the results of the studies performed. Hoang-Dung TRAN and friends The comparison of zymograms and protein electrophoretic patterns in polyacrylamide gel of B. thermophilum of bovine origin are similar. longum. and that which goes furthest. 6. II. and B. cuniculi. B. The attribution of unknown bacteria to their group and their possible identification. B. and the numerical analysis of the patterns of a large number of strains makes it possible to achieve: Rapid grouping of the strains. longum and in many strains of B. In contrast. Determining the homogeneity or heterogeneity of the taxa. B. D. B. Study of the protein patterns provides valuable information about a given strain. A quick method of determining whether two colony types in a culture are due to variation or contamination. Inc. E. are shown in the dendogram (Fig.171] The zymogram of B. that is. infantis. B. adolescentis with electrophoretic mobilities of 8 and 5.174] This method consists of preparing immunosera against the highly purified transaldolases of eight species of Bifidobacterium. 6). which migrate to a distance of 5.[36. is found most frequently in B. These four antigenic groups (I. respectively.[107. asteroides and testing them against 21 bacterial species of the genus in order to determine their immunological distances. B. suis. although they belong to different species. angulatum. are found in 50% of B. and IV) coincide with the arrangement Copyright © 2004 by Marcel Dekker. These results. confirming that they are indeed three separate species. All Rights Reserved.” Table 8 shows that the transaldolases and 6PGD of B.4. the electrophoresis patterns of the cell proteins for these three strains differ.157. infantis and B. The only difference is the incidence within the strain: the isoenzyme migrating to a distance of 5 occurs more frequently in B. longum is interesting. Only a few strains of B.157] We are therefore faced by a very unusual phenomenon: these strains.[172] 4. minimum. or 8 units. F.173. globosum. thermophilum. bifidum is in contrast highly specific. B. the differentiation is based on the mobility of the 3-phosphoglyceraldehyde dehydrogenase.[35.[169] The bands obtained on the electrophoretic diagrams of these two species show an identical distribution. These two species have the same isoenzymes of transaldolases. a high degree of reproducibility (in excess of 96%) can be obtained. Only the concentrations of the proteins differ. This species is the only one to show a transaldolase and F6PPK migration distance of 7 units.

All Rights Reserved. B. bifidum has a poly-(1.[102] In addition. subtile.2)-glycerophosphate skeleton in the lipoteichoic acids. .174. D. which is substituted in the end position by a polysaccharide. bifidum. such as B. making it possible to separate species from which are relatively close to each other. A and B.175] and are also confirmed by DNA-DNA hybridization studies. In addition.Hoang-Dung TRAN and friends Figure 6 details. and B. The most widely separated species (IV) (group F) were isolated from waste water. catenulatum. boum from B. infantis. this dendogram is able to distinguish two subgroups.[176] In addition. angulatum. Copyright © 2004 by Marcel Dekker. breve is one of the few species to produce b-glucuronidase activity. B. and G. B. longum. thermophilum or B. and (2) B. minimum from B.) Immunological relationships among transaldolases in Bifidobacterium. E. adolescentis. The two species found only in the bee (group H) are antigenetically very distant from the other members of the genus (III).[41] It is suspected that this enzyme may convert procarcinogens into carcinogens. dentium. Composition of the Wall The sugar composition of the wall varies with strain. Inc. and B. among the strains of human origin (I): (1) B. 6. B. The sequence of amino acids in the peptidoglycan may vary from one species to another.[174] The species associated with mammalian animal habitats (II) belong to groups C. Enzymes B. B. Bezirtzoglou[110] noted that only the species B. breve. longum is the only species to have neither b-glucosidase nor N-acetylglucosaminidase activity. B. particularly with regard to the percentage of rhamnose and glucose[15] (Table 9). (See text for of the species of Bifidobacterium based on electrophoretic mobility[36. pseudocatenulatum. 5.

Strain selection Number of characteristics examined (greater than 50) Rigorous standardization of the methods of analysis Weight attributed to each characteristic in the evaluation Classification of the reactions as positive. B.[177] prepared antibodies to the lipoteichoic acids of B. thermophilum adolescentis indicum pseudolongum Op Den Camp et al. 2. Copyright © 2004 by Marcel Dekker. 4. They were specific towards the polyglycerol phosphate core (essentially poly 1. Crossed reaction tests with phenolic extracts of lipoteichoic acids of Bifidobacterium and Lactobacillus have shown that only the former react. . bifidum infantis breve liberorum parvulorum asteroides suis Origin Adult stool Infant stool Infant stool Infant stool Infant stool Bee Pig Adult stool Pig Adult stool Bee Pig B. The usefulness of numerical taxonomy depends on several factors:[167] 1. bifidum by coupling with an immunogenic protein. Most of the problems involved in processing data tables resulting from the examination of the physiological and biochemical properties of the bacteria are solved by the use of computer-assisted numerical taxonomy. B.Hoang-Dung TRAN and friends Table 9 Cell Wall Composition of Bifidobacterium sp. B. Kind of peptidoglycan cross linkage Orn-Ser-Asp-Ala Lys-Gly Lys-Gly Lys-Gly Lys-Gly Lys-Gly Orn or (Lys)-Ser-AlaThr-Ala Orn or (Lys)-Ser-AlaThr-Ala Orn-(Lys)-Glu Lys or (Orn)-Asp Lys-Asp Orn or (Lys)-Ala Polysaccharide Glucose Galactose þ þ þ þ þ þ þ þ þ þ 2 þ þ þ þ þ þ þ þ þ þ þ þ þ Rhamnose þ þ þ þ þ 2 (þ) þ þ 2 þ þ Species B. IDENTIFICATION BY STUDY OF THE GENOME Identification of the Genus Bifidobacterium The DNA base composition of the chromosome of Bifidobacterium differs from that of Lactobacillus [17] and other lactic bacteria. A. B. Processing of the Results All these identification criteria give responses that must be classified and interpreted. longum B.2) and to a small extent to the polysaccharide portion. negative. making it possible to envisage a serogroup with lipoteichoic acids as group antigens. All Rights Reserved. B. 7. B. 5. Inc. 3. B. B. 6. B. or noncomparable Type of computer software used XI.

[157]—B. and B. breve pseudolongum and B. indicum. bifidum and B. ruminale. In addition. . A few years later. whereas that of B. longum belonged to two different species has been confirmed. lactentis. infantis under the name of B. magnum. globosum. minimum and B. animalis and proposed raising these subspecies to rank of species: B. longum subsp. animalis and B. longum. pseudolongum is 60. dentium. The more DNA base pairing there is between two strains. coryneform. choerinum. animalis. parvulorum under the name of B.7– 50. The results of DNA-DNA hybridizations between the various species of Bifidobacterium are shown in Table 10. B. B. cuniculi. B. B. All Rights Reserved. thermophilum breve and B. B. infantis and B. longum is distinguished from B. The GC% cannot therefore be considered as an exclusion characteristic in bacterial taxonomy.[167. Species Identification DNA Base Composition B.Hoang-Dung TRAN and friends Genus Lactobacillus Streptococcus Leuconostoc Bifidobacterium GC% 34. animalis. 1. B. B. 2. in 1982.[175. and B. A fragment of denatured DNA from a reference strain is labeled and then used as a probe to hybridize with a single strand of DNA from the strain to be identified. adolescentis into four groups. longum (50 –76% Copyright © 2004 by Marcel Dekker. B. B. two organisms with similar G þ C (GC%) analysis are not necessarily closely related. infantis into three subspecies and B.2– 64. B. From this table it can be seen that two continua can be defined from the human-defined strains B. animalis and B. the closer they are genetically. 11 different species can be described: B.5 However. B. but the following species have been combined to form a single species: B. catenulatum. and B.178] On the basis of the DNA-DNA homology percentages. pullorum.8 33– 44 39– 42 57. globosum [175] In the following year and using the same methods. The GC% of B.179 – 182] The fact that B. infantis ruminale and B. B. liberorum. Holdeman and Moore[183] divided B. B. subtile. longum is 58. some new species were described by Scardovi[21]—B. longum subsp. B. and B. B. The use of DNA-DNA hybridization methods has advanced the taxonomy of the bifidobacteria. B. angulatum. B. cornutum and B. eriksonii. DNA-DNA Hybridization DNA-DNA homology is a mean measurement of similarity in which the entire genome of one organism is compared with that of another. boum—and then. by Biavati et al. Inc. pseudocatenulatum. asteroides. In 1974. pseudolongum by its GC%. longum and B. suis. they described two new species: B. Scardovi and Trovatelli[184] detected genetic differences between B.

magnum B. catenulatum B. thermophilum B. adolescentis B. coryneforme B. suis B. angulantum B. indicum à 100 / / à à à à à à à à à 88 65 / à à 63 88 / à à à 0 0 à à à à à à à à à à à à à 100 à à à 0 0 0 0 à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à B: suis 0 à à 0 0 0 0 à à à à à à à à à B: subtile à à à à à à à 86 à à à à 88 à à à à à à 0 à / à 90 67 à 65 97 à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à à 90 à à à à à 0 à à à à 0 à à à à à à 0 à à 100 67 / / à à 75 100 à / à / / à / à à 98 à à à à à à à à à à à à à à / à à 98 à à à à à à à à à à à à à à à à à à à 86 à à à à à à à 0 à à à à à à à 98 / à / 82 à à 0 0 90 à à à à à à à à à à à à à à à à à à à à 102 à à à à 0 à à à à à à à à à à 100 à à à à à à à à à à à à à à à à à à à à 0 0 à à à 0 0 0 102 à à 0 0 0 10 0 à à 85 0 à à 0 0 / à 0 0 0 100 à 0 100 Value between 40% and 60%. Inc. bifidum B. subtile B. pseudolongum B. B: indicum 0 0 0 0 à / à . cuniculi B. minimum B. dentium B. All Rights Reserved. animalis B. breve B. globosum B.Hoang-Dung TRAN and friends Copyright © 2004 by Marcel Dekker. pullorum B. asteroides B. boum B. pseudocatenulatum B. Table 10 DNA-DNA Homology in the Genus Bifidobacterium B: pseudocatenulatum B: pseudolongum B: adolescentis B: catenulatum B: angulatum B: choerinum B: thermophilum B: globosum B: animalis B: magnum B: pullorum B: minimum B: asteroides 0 0 0 0 à à B: bifidum B: longum B: infantis B: dentium B: cuniculi B: breve B: boum B. infantis B. longum B. choerinum B.

adolescentis appears to be distant from these four species (less than 20% homology) but close to the species in the other subgroup. electrophoresis of soluble proteins. Grimont and Grimont[187] suggest that the parameters used to describe species and strains should include the size of the DNAr restriction fragments following agar medium electrophoresis. This method can demonstrate that two strains are different. . Studies have been carried out in order to identify restriction enzymes able to cut DNA at infrequent restriction sites. hope is based on the use of new methods. dentium). angulatum. adolescentis. infantis. The whole bacterial DNA can be processed using restriction enzymes. New Methods Today. and B. B. However. 2.[175] The other continuum is that of B. pseudocatenulatum. All Rights Reserved. The number of plasmids detected in a strain of Bifidobacterium may suggest the identification of certain species because only some possess plasmids. Comparison of these sequences with those of reference strains should give information extremely useful for identification. Inc. although the DNA-DNA hybridizations between these strains confirm the relationship between B. the results obtained by electrophoresis of soluble proteins and the zymograms appear to distance B.[185] But even if the genome is relatively small. which are very well-conserved molecules throughout the animal kingdom. bifidum (and not to the subgroup containing B. on the contrary. longum and B. and B. dentium. and B. infantis. adolescentis. Pulsed field electrophoresis requires large fragments of DNA. which should lead to further changes. B. B. longum (0% hybridization). but can in no case be used to confirm the similarity of two bacteria. Plasmid Tests 1. breve. infantis. dentium (some strains showing up to 49 –57% homology). The hybridizations challenge this finding since. catenulatum and B. pseudocatenulatum (60 – 80% homology). longum and B. longum. infantis. B. and B. and growth factor requirements[57] and suspected by Scardovi et al. there are too many restriction fragments for the electrophoretic pattern to be easily read. in particular to B. B. 3. B. catenulatum. adolescentis appears to belong to the subgroup as B.[174] These genotypic findings nonetheless raise some questions: The electrophoretic mobility of the transaldolases and 6PGD had suggested proximity of B. The serology of the transaldolases makes it possible to separate the strains of human origin into two subgroups. In contrast. longum. which had already been detected by the zymogram.[104. Copyright © 2004 by Marcel Dekker.[186] The successive use of several restriction enzymes should make it possible to pinpoint identification. The DNA fragments carrying genes (DNAr) coding for ribosomal RNA are then localized on filters by hybridizing with either a DNAr 16 þ 23S from Escherichia coli or with a DNA probe that codes for the well-conserved portions of ribosomal RNA 5S or 16S. B. Many more studies are required before a new level of identification can be defined that could be achieved by sequencing the RNAr 5S and 16S. 4.105] This technique is particularly useful for the separation of B.Hoang-Dung TRAN and friends homology). genetically. adolescentis and B.[187] DNA-DNAr hybridization remains to be tested regarding its value in improving the classification of bifidobacteria. appears to be genetically very distant from B.

and their use does not provide a sufficiently reliable answer. and d B. thermophilum Genotype B. including those parts that do not code for proteins. adolescentis a. animalis B. gallinarum Note: DNA-DNA homology higher than 50%. infantis B. animalis B. dentium B. The new genome methods now under investigation hold the promise of a more accurate and quicker identification method. catenulatum B. Inc. thermophilum B. Table 11 Relationship Between DNA-DNA Homology and Phenotypy Phenotype B. cuniculi B.Hoang-Dung TRAN and friends C. . the identification is not definite. Conclusion The identification of species within the genus Bifidobacterium is difficult. pseudolongum B. bifidum B. b. B. breve a and b B. minimum B. infantis ss. globosum B. choerinum B. infantis a and b B. liberorum B. indicum B. magnum B. The kits and commercial tests that provide phenotype identification information are not appropriate for these bacteria. infantis ss. Genotyping today provides a more accurate and reliable approach to bacterial taxonomy. All Rights Reserved. boum B. c. cuniculi B. gallinarum Copyright © 2004 by Marcel Dekker. indicum B. suis B. breve ss. asteroides B. pseudolongum B. Table 11 shows the accuracy and precision of identification obtained using genotyping. coryneforme B. lactentis B. minimum B. breve B. adolescentis B. DNA-DNA hybridization methods take into account the entire genome. longum a and b B. DNA-DNA hybridization is widely used and is a particularly appropriate method since it makes it possible to study the entire bacterial genome. Several species that cannot be differentiated by phenotyping can be distinguished by their genetic characteristics. bifidum a and b B. breve ss. infantis ss. Even with 100 phenotype characters. longum B. subtile B. pullorum B. whereas phenotype analysis involves only the segments of the genome in which the phenotype expression can be measured[188]—about 10%. asteroides B. angulatum B. parvulorum a and b B. pseudocatenulatum B.

A. depending on the presence of a substrate and a redox potential appropriate for its growth requirements.XII. These are the polysaccharides of the capsule or slime. Fimbriosomes are rounded structures closely associated with the fimbriae. there is a uniform interstice between the bacterium and the host cell measuring less than 40 nm. Inc.[192] Elements known as “fimbriosomes” have also been detected in hyperadhesive mutant strains.. in which case they belong to the subdominant flora. All Rights Reserved. coli. The polysaccharide fraction of B. The Adhesins These structures.[189] B. 2. They have been described in the superficial adhesion process of enteropathogenic E. Colonization Conditions To obtain a probiotic or pathogenic effect depending on the invasive bacterial species concerned. Mechanism of Adhesion There are numerous conflicting hypotheses concerning the possible implantation of ingested microorganisms.[193] It has not yet been demonstrated whether these structures potentiate the effects of the fimbriae or whether they act separately. Protein Type. Hoang-Dung TRAN and friends RELATIONSHIP BETWEEN INTESTINAL FLORA AND INTESTINAL CELLS: ADHESION AND COLONIZATION METHODS The study of the mechanisms of adhesion has been facilitated by the development of investigation methods and can be carried out today in vivo by taking a human or animal colon biopsy sample. These are either external membrane proteins or individualized structures such as the pili or fimbriae of gram-negative bacteria. It must be able to multiply. This adhesiveness depends on three factors: adhesins. the bacteria must adhere to the cell surfaces of the digestive tract. . It must be able to reside in situ. adhere to the cells or mucus and avoid expulsion.[190] The bacterium may be completely encircled by the membrane of the apical pole of the cell or only partially surrounded. adhesin receptors.[189. and mucus. In most cases.[191] The pili may be bound to the glycoproteins and glycolipids of the cell membrane of many vertebrate species. infantis is involved in the adhesion of this bacterium to Copyright © 2004 by Marcel Dekker. and in some cases the association takes place without penetration. Nonprotein Type. which are implied in the attachment process and are present in some bacteria. 1. These bacteria may either simply pass through or colonize the intestine by adhering to the cell wall.e. Two conditions must be present to allow the implantation of a bacterium: 1. may be of two types. which is then examined under electron microscopy or in vivo by the culture of intestinal cells or tissues under survival conditions and exposed to bacteria before being examined under the electron microscope. which is filled with fibrillar material.190] These non-protein structures may also be fibrillar structures other than fimbriae. i.

[177] An important phenomenon that should be highlighted is “phase variation. This results from differences of structure and composition of the enterocytes of the host. Analysis of the Adherent Flora Compared to the diversity of the intraluminal flora. their relationship with the epithelium of the colon or rectum has not been studied. coli to uroepithelial cells and isolated human colon cells appears to involve the intermediary of glycolipids (glycolipids account for about 20% of the membrane lipids of the enterocyte) containing Gala 1 –4 Galb residues. Using electron microscopy. This phenomenon is familiar in E. Adhesin Receptors The most probable receptor on the membrane of the intestinal epithelial cells is a protein or glycoprotein with fatty acid –binding sites.[196 – 199] Binding occurs spontaneously through the intermediary of the lipid fraction of the LTAs. Inc.195] Many studies have demonstrated that the LTA of gram-positive bacteria have a high binding affinity to the membranes of the epithelial cells of mammals. If biopsies are taken from various points of the duodenum and jejunum. coli both in vitro and in vivo.[199] The binding of the LIAs of the bifidobacteria to human epithelial cells in culture is dependent on cell concentration and time and appears to be reversible.[194. which are mature in adults and immature in young subjects. LTA is bound through the fatty acids. .[206] have confirmed the close association of bacteria with the mucus layer.” which allows a bacterium to modify its surface and thus its adhesive potential.203] and the portion of the digestive tract involved. The bacterial concentrations are generally lower by one or two log factors than the intraluminal populations. Croucher et al. Only the adhesive capacities of the optional anaerobes (such as enterobacteria) have Copyright © 2004 by Marcel Dekker. the adhesion of E.[200] It appears that the Gal a 1 –4 Gal b receptors are indeed irregularly distributed among intestinal cells.Hoang-Dung TRAN and friends the epithelial cells of the ileum or the lipotechoic acids (LTAs) of gram-positive bacteria.[201] The surface receptors trap the bacteria in mucus secretions. depending on its phase. The type and quantity of specific glycolipid receptors is also determined genetically.[177] However.[207] Despite the marked predominance of anaerobic bacteria in the lower portion of the gastrointestinal tract. and it consists of a sort of protective elastic and viscous gel consisting of glycoproteins.[202] the age of the host[193. as well as through the intermediary of receptors which are sensitive to mannose and which could consist of one or several glycoproteins. and adults may be related to this change of receptors in function of age. but its presence is also essential in the mechanisms of bacterial adhesion.[204] The differences found in the composition of the intestinal flora in infants. Its most obvious function is to provide specific protection against bacterial penetration. C. The membrane receptivity of the host cells is strongly influenced by cell maturity. children. the flora adhering to the epithelium is generally limited to a few species of bacteria. All Rights Reserved. which are themselves bound to the esters. Mucus Mucus covers the entire mucosa of the digestive tract from the stomach to the colon. the bacterial population appears to be equally distributed along a given segment of the gastrointestinal tract.[205] 3.[190] 2.

have made it possible to carry out detailed exploration of all portions of the digestive tract. such as biopsy of the intestinal mucosa and collection of luminal aspirate fluid using a Camus probe or weighted tube. More elaborate sampling methods. The dominant flora consists of highly anaerobic microorganisms. and few studies have been devoted to this topic.[210] The adhesion of Bifidobacterium and more particularly the hydrophobic interactions are promoted by a high level of fatty acids in the LTAs. Methods of Evaluation Conventional stool collection allows only investigation of the terminal flora of the digestive tract.209] The density of bacteria ranges from 106 to 107 cfu/g of fresh tissue.[189] E. The characteristics of the epithelial cell of the human colon/lipoteichoic acids of Bifidobacterium complex have been investigated by determining the radioactivity of carbon-14 – labeled LTAs after interaction with suspended colonocytes.[208. resulting in an inhibition of the adhesion of the microorganisms which use them.[208] to demonstrate that the same strain of E. THE INTESTINAL FLORA Knowledge of the intestinal flora has developed simultaneously with the methods of investigation with regard to both sampling technique and analysis of the flora. These new sampling methods and the design of more appropriate culture media for the various species now make it possible to use a good direct Copyright © 2004 by Marcel Dekker. D.Hoang-Dung TRAN and friends been studied. A. The bonds that permit this adhesion are probably strong. Modifications of Adhesion Several studies have shown that some bacterial species may modify the receptor sites of epithelial cells.190. resu1ting in a high level of hydrophobicity of the bacterium. The strong electrostatic charge of the polysaccharides of gram-positive bacteria also favors adhesion. . Examination of colonic biopsies allowed Hartley et al. The adhesion of E.[189. This phenomenon reflects an increase in the membrane permeability of these bacteria. Adhesion of the Bifidobacteria Sato et al. Thus. may degrade the receptors within the cell or mucus and also eliminate any bacteria bound to them. All Rights Reserved.[194] have demonstrated the role played by the polysaccharides in the adhesion of the bifidobacteria by inhibiting this phenomenon using antibodies targeted against polysaccharides. coli and other enterobacteria has been demonstrated by electronmicroscopy of biopsies.211] These enzymes. Gram staining is negative in cell-bound Bifidobacterium. The most probable receptor on the colonocyte membrane is a protein or glycoprotein with fatty acid– binding sites.[202] XIII. which are therefore difficult to isolate and keep alive. some of which are glycosidases. Inc. coli is closely associated with the intestinal wall throughout the length of the colon. extracellular enzymes of Bifidobacterium may degrade specific sites of pathogenic organisms or their toxins.

Counting of a given species of Bacteroides from a mixture of bacterial DNA isolated directly from the stools and exposed to a species-specific labeled DNA probe has been suggested. the effects of which on the host were the first to be understood. Generally. chemical. Of the aero-anaerobes. which means there are more living entities in the flora than there are cells in a normal body and the bacteria consist of 500 species. aerobic Lactobacillus. All Rights Reserved. The subdominant population. The dominant population. The acidity of the stomach maintains a low concentration of bacteria in the upper part of the digestive tract and destroys some pathogens. and biological regulatory mechanisms. which can easily be explained from the choice of culture methods. which accounts for less than 1% of the total bacterial population but which. shown in Table 12.2) followed by Streptococcus. Composition The intestinal flora of a human weighs between 1 and 2 kg. B. come Eubacterium.213] However. This flora can be divided into two categories. The main results. in decreasing order. The importance and role of the microbial flora within the host can also be assessed indirectly by determining the bacterial metabolites produced using the following methods: The breath test or measurement of the hydrogen expired by a subject after ingesting lactulose. Measurement of the activities of various bacterial enzymes in intestinal samples. Numerous studies have been carried out to define the composition of the intestinal flora.Hoang-Dung TRAN and friends approach to understanding the intestinal bacterial flora by identifying and counting the species samples. the most numerous population in adults is Bacteroides (about 1010. 2. that is.4). this method would not make it possible to differentiate between viable and nonviable bacteria. 1. The interactions that exist between various bacterial species are also important in maintaining the equilibrium of the intestinal microflora. Inc. roughly the same weight as organs such as liver. Intestinal peristaltism results in the elimination of many microorganisms.3/g of feces). there are the enterobacteria (108. 1. or lungs. Thereafter. Bifidobacterium.[5] C. and finally Staphylococcus (104.[146] may play a nonnegligible role in the equilibrium of the intestinal ecosystem. Factors Affecting the Flora Factors Ensuring the Equilibrium of the Intestinal Flora The diversity of bacterial species and their quantity at various levels within the digestive tract can be preserved only by means of physical.[212. show some discrepancies. The digestive tract houses about 1014 bacteria. Tests for fatty acids excreted in the stools by gas chromatography reflecting bacterial metabolism. and then the Peptococcaceae. isolation media. . brain. and a confirmation of the percentage of live bacteria would still have to be carried out using conventional dish culture methods. and counting methods. It is possible to observe Copyright © 2004 by Marcel Dekker. according to studies.

9 9. two parts should be distinguished within the colon: (a) the ascending colon.8 4.0 4. creating the redox conditions for the implantation of anaerobic species in more distal portions. which contains mainly gram-positive bacteria.1 8. Source: Ref.3 8.6 0 9.2 8.[214] Thus.3 0 5.0 6.3 8. The various factors active along the digestive tract result in qualitative and quantitative differences in the digestive flora.1 7.2 3. coli and anaerobes such as Clostridium.5 8.9 9.5 6.8 7.5 9.8 4. 1 – 4 days 10. . Copyright © 2004 by Marcel Dekker. by a flora consisting of E. a given well-defined resident flora corresponds to each portion of the tract.4 9.5 Infant. [153].6 10. Fusobacterium.8 7. All Rights Reserved.9 4. 20 –64 Adult.4 3.2 10.0 7. Thus. within the ileum.7 4.4 7. This aero-anaerobic flora is subsequently replaced. the flora present in the proximal small intestine (duodenum and jejunum) consists of aerobic gram-positive microorganisms (streptococci and staphylococci) and a few yeasts.2 5.8 8.6 6. and (b) the descending colon. known as “putrefaction flora.4 6. Finally.[5] This switch from a dominant aerobic population within the stomach to a strictly anaerobic population within the colon can be explained if we accept that the aero-anaerobic bacteria use any oxygen present.Hoang-Dung TRAN and friends Table 12 Fecal Flora of Different Human Groups Infant.7 5. Bacteroides).8 8.2 8.9 5.2 7.7 6.0 9. in which the flora. 65 –86 years years 10.8 10.9 10.0 4. symbioses between species as a result of the production of vitamins or amino acids or other metabolites which can be assimilated by other species and also of antagonisms due to the release of antibiotics. 4 – 6 years 10.3 4. 2. Inc.5 Bacterian group Total bacteriaa Aerobic or facultative anaerobic Enterobacteria Streptococcus Lactobacillus Staphylococcus Yeast Anaerobic Bacteroides Eubacteria Bifidobacterium Peptococcus Clostridium perfringens Veillonella a 9.1 log cfu.7 6.3 9. bacteriocins.8 Adult.1 Infant. or factors such as the volatile fatty acids.3 6.2 9. and Bacteroides (106 total bacteria per mL).7 9.0 4.” consists mainly of gram-negative bacteria but also some gram-positive bacteria (Clostridium.9 6.7 10. which have the primary role of sugar fermentation. as shown in Table 13.0 8. 5 – 90 days 10.6 8.8 8. Location and Physiology The composition of the intestinal flora varies depending on the rate of transit and luminal secretions as well as the intestinal segment.

It also follows enterocyte maturation and development Copyright © 2004 by Marcel Dekker. . Inc. once more highlighting the importance of the reciprocal host-bacteria relationship. whereas others may be pathogens. Age and Diet This change in the flora is closely linked with the maturation of the digestive system.Hoang-Dung TRAN and friends Table 13 Human Gastrointestinal Flora Stomach Jejunum 0– 105 Ileum 103 – 107 Colon 1011 – 1012 Total microbial concentration Strict aerobic or facultative anaerobic bacteria Enterobacteria Streptococcus Staphylococcus Lactobacillus Fongy Anaerobic bacteria Bacteroides Bifidobacterium Peptococcus Clostridium Fusobacterium Eubacteria Veillonellae a 0 – 103a 0 – 102 0 – 103 0 – 102 0 – 103 0 – 102 Rare Rare Rare Rare Rare Rare Rare 0– 103 0– 104 0– 103 0– 104 0– 102 0– 102 0– 103 0– 103 Rare Rare Rare 0– 102 102 – 105 102 – 106 102 – 105 102 – 105 102 – 103 103 – 106 103 – 107 103 – 104 103 – 104 Rare 103 – 105 103 – 104 104 –1010 105 –1010 104 –107 106 –1010 102 –106 1010 – 1012 108 –1012 108 –1012 106 –1011 109 –1010 109 –1012 103 –104 Number per gram of intestinal contents. This tool has been found to be most useful in demonstrating the effects of a given species or small group of species on the host. associating a region of the colon with a bacterial function and consequently with particularly dominant species[214] has been challenged by the work of Croucher et al. This conventional theory. D. Role and Effect of the Intestinal Flora “The gastrointestinal tract is a complex ecosystem with characteristics which depend at each moment on a dynamic equilibrium between the host and the native bacteria. Some may be probiotic and others simply commensal.”[214] Exogenous bacteria also influence all the bacteria within the intestinal flora.[206] Their studies of human colon biopsies tend to demonstrate that there is no specific location for the various species within the colon. All Rights Reserved. The overall effect of the microbial flora on the host is generally evaluated by comparing an axenic animal with a holoxenic animal in which the flora has developed normally.[215] E. Bacteriological examination of the feces shows that diet has little or no effect on the constitution of the dominant intestinal flora. 1. Effect on Physiology of the Intestinal Wall and the Immune Defense System The intestinal flora modifies the morphology of the mucosa and the rate of turnover and differentiation of epithelial cells.

2. It would appear that 90% of human cancers are due to the environment and could therefore be avoided. tyramine. and bacterial toxins. This is the case of lactobacilli ingested with yogurt.217] In the axenic animal.[218] Rowland and Grasso[222] have investigated the degradation of the N-nitrosamines by the intestinal flora.[221] Thus. ammonium. which can produce the lactase activity missing in lactose-intolerant subjects. vitamin B12. The intestinal bacteria ensure the maintenance of the immune status by providing repeated antigen stimulation throughout the human life span.[214. These bacteria are able to compensate for enzymatic. disaccharidase. pantothenic acid. notably histamine. Major differences have been observed between cancer risks.218. and proteins. folic acid. but no directly active carcinogen has been isolated. facilitating their detoxification and elimination. riboflavin. phenols. Another important action of the intestinal bacteria is their involvement in the enterohepatic cycle and the detoxification of numerous substances and drugs. Enzymatic Action. but it would seem that neither the place where the population live nor their race is responsible. Production of Harmful Substances. which are partially absorbed and used by the host.[220] Detoxification. short-chain fatty acids. An important role played by the flora is its action in cell maturation observed in the normal development of Peyer’s patches. All the aspects of the intestinal metabolism of the host are influenced by the enzymatic activity of the bacteria it shelters and more particularly the anaerobic bacteria. Simon and Gorbach[215] observed an increase in the activity of enterocytic enzymes. Bacteria as Nutrient Sources The bacterial mass of the intestine is itself an important source of nutrients: thiamine.[43. 4. .Hoang-Dung TRAN and friends of the velocities in the neonate. and b-glucosidase.219] 3. Inc. cholesterol is converted to form coprostanol and the bile salts to form bile acids and then lipocholic acid and other derivatives conjugated with amino acids such as glycine and taurine. A diet containing low fiber and high quantities of animal fats appears to promote the onset of cancer of the colon. Tumoral Action Cancer of the colon is the second greatest cause of death in Great Britain and in the United States. since it determines the uptake of nutrients and permits the formation of the ecological site for other bacteria. These observations. In contrast.[223] Considerable research has been carried out in an attempt to identify carcinogens. Metabolic Effects The bacterial flora produces a very large and varied quantity of enzymes. in particular alkaline phosphatase. All Rights Reserved. some microorganisms produce substances toxic to the host. amino acids. cadaverine. Aries Copyright © 2004 by Marcel Dekker. rather that the etiology of the disease is related to diet. which are used by the flora itself but also by the host.216. N-nitrosamines. All these studies have demonstrated the importance of the role of the flora. We will list here some examples of the effects of bacterial metabolism on the host. first made as a result of histological investigation of the intestinal wall. agmatine. have subsequently been confirmed by numerous studies demonstrating that resistance to various pathogens is conditioned by the presence of a flora. deficiencies of the host if they are introduced in a sufficiently large number into the digestive tract.

These properties have subsequently been clearly defined: the ingestion of fermented milk results in stimulation of the immune system. Numerous studies have shown that deficient subjects do not show any intolerance toward yogurt.[230] Two explanations can be suggested for this: the activity of bacterial b-galactosidase may persist in the intestine.[220] A large portion of the world’s adult population shows a deficit in galactosidase. nutritive. . In fact. BIFIDUM-INTESTINAL RELATIONSHIPS: PROBIOTIC ROLE OF BIFIDOBACTERIUM Tissier[228] observed a close relationship between the immunity of the breast-fed child and his or her specific flora. Inc.[229. azoreductase. It is reasonable to think that the intestinal flora could produce or potentiate carcinogens or procarcinogens. and producing bacteriocin and volatile acids. probably as a result of the enzymatic activity of the bacteria of the digestive flora on a harmless procarcinogen substrate derived from the diet.[225] This recently discovered role challenges the theory of the anti-infectious barrier effect suggested by Ducluzeau et al. and other growth factors are.[220] which holds that the barrier effect can be observed only in bacteria belonging to the dominant flora.[227. a probiotic effect can only occur if the bifidobacteria survive their transit through the stomach. particularly lactic and acetic acids. We can now attribute the functions described below to the bifidobacteria. deconjugating bile acids. of bacteriocins. Bifidobacteria had long been recognized as bacteria with probiotic. 5. which stimulate peristaltism. the subdominant flora uses the endogenous substrates for its own metabolism. and this resistance is increased by the food bolus. In the case of the bifidobacteria. preventing the proliferation of pathogenic bacteria and the adhesion of other organisms. the determining factors in a probiotic action. and therapeutic properties. Some strains of Bifidobacterium are able to resist gastric acidity.[231] which has also been shown to be active in the degradation of nitrosamines. which inhibit pathogenic bacteria in their conjugated forms. together with the stimulation of the immune system and accumulation of specific metabolites. Effect of the Anti-infectious Barrier Toward Pathogens The microbial population of the gastrointestinal tract forms a barrier against proliferation of exogenous pathogens.[224] One explanation may be that the colonization of the endogenous flora maintains the pathogens at a subclinical level by preventing the colonization of the undesirable flora by competition for the substrate or epithelial receptors. and nitroreductase formed by the flora are reduced by the ingestion of Lactobacillus acidophilus. All Rights Reserved.[224] therefore believe that carcinogens may be produced in situ. but also for the dominant population.228] XIV.230] The quantities of b-glucuronidase. Copyright © 2004 by Marcel Dekker.[232] The bacterial production of organic acids. particularly volatile fatty acids. and that pathogenicity can be effective only above a certain colonization threshold of the invasive bacteria. where a fraction of the lactose present may be metabolized or the ingestion of yogurt may stimulate any intestinal lactase that is still active. The intestinal microorganisms inhibit the growth of the invasive pathogens by producing organic acids. and even antibiotics as well as the secretion of enzymes. vitamins.Hoang-Dung TRAN and friends et al.

.5 g/mL. and threonine). Bacteria-producing lactic acid also produces hydroxy-methylglutaryl – CoA reductase. bulgaricus and Streptococcus salivarius ssp.146] They may also have an action on the immune system appended to the digestive tract. and the fact that they produce only L þ lactic acid. Action on the Morphology and Physiology of the Digestive Tract Wall Bifidobacteria influence the maturation and turnover cycle of the enterocyte. together with the development of the intestinal velocities.Hoang-Dung TRAN and friends A. B. B6. thus enabling them to produce a genuine effect. Jaspers et al.241] The consumption of fermented dairy products could lead to a reduction in serum levels of cholesterol.[233 – 237. bifidobacteria apparently affect the activity of HMG – CoA reductase. unlike Lactobacillus delbruekii ssp. maintains the mechanism of in situ production of various bacterial metabolites. Metabolic Effects Suppression of Lactose Intolerance Bifidobacteria.215] They are also involved in the degradation and replacement of intestinal mucins. and studies are in progress intended to demonstrate the involvement of bifidobacteria in the reduction of cholesterol levels.[240] It is difficult to determine the role that should be attributed to the bifidobacteria. 2. Inc. The bacterial biofilm.[214. valine. thermophilus. The administration to hyporcholesterolemic human subjects of fermented milks containing very large quantities of Bifidobacterium (109 bacteria per gram) results in a fall in the total cholesterol from 3 to 1. and PP). are resistant to biosalts[239] and as a result can have an in situ effect on the metabolism of lactose. which themselves have an effect on other bacterial genera and even on the host. In vitro. amino acids (alanine.[242] Rao et al. Nutritional Effects The production of vitamins (B1.219] D. In addition. Hypocholesterolemic Effect Several studies have tended to demonstrate a relationship between the presence of a lactic microflora and a reduction in plasma cholesterol. 1. which is involved in the synthesis of cholesterol. B9.[244] have shown that both orotic and hydroxymethylglutaric acids reduce serum cholesterol.145. which is completely metabolized by humans enhances the nutritional characteristics of the bifidobacteria. bound to the epithelial walls.218. B12. Adhesion to the Intestinal Epithelium The adhesion of the bifidobacteria to the epithelial cells permits the formation of ecological niches within which the growth of bacteria is maintained regardless of changes in the habitat.[231] C.[43. it has a defense function against pathogenic bacteria.[243] have shown that the metabolites produced from orotic acid during fermentation of fermented products could be responsible for this hyporcholesterolemic effect. aspartic acid.[240. whereas uric acid inhibits the synthesis of cholesterol. All Rights Reserved. Copyright © 2004 by Marcel Dekker.

Acetic and lactic acids are produced in the bifidobacteria in a ratio of 3/2.227.[248] In infants suffering from rotavirus-induced diarrhea.250] The production of lactic and acetic organic acids during the fermentation of carbohydrates by bifidobacteria. Other Metabolic Effects The ingestion of milk fermented with Lactobacillus acidophilus and B. which results in a reduction in the pH of the intestine and consequently inhibits the growth of the undesirable bacteria. and Inhibition of the Reduction of Nitrates Bile acids are secreted into the duodenum in their form of conjugates with glycine or taurine. coli and gram-negative bacteria. and these four parameters are good indicators of the fermentation capacity of the colonic flora. The pKa of acetic acid is 4. 2.220. The acidity Copyright © 2004 by Marcel Dekker. All Rights Reserved. Acetic acid has a stronger antagonistic effect against gram-negative bacteria than lactic acid. coli. Inc. the concomitant administration of B.[141] Acidification has a bactericidal potential. bifidum for a period of 3 weeks has no effect on the production of hydrogen or methane or on fetal b-galactosidase activity. Acetic acid 8.4% and lactic acid 1. The permanent acidity of the intestinal contents that results from the development of bifidobacteria has a bacteriostatic effect against E. especially against gram-negative bacteria. whereas that of lactic acid is 3.1% are present in an undissociated form at an intestinal pH of 5. longum.[218] The implantation of bifidobacteria is promoted in infants by breast-feeding. Reduction of Nitrosamines.8.76.[246] E.[249] Two hypotheses have been advanced to explain this probiotic effect: 1. which results in about 11 times more undissociated acetic acid than undissociated lactic acid.[15] 4. . but few studies have been carried out in human subjects: B. Most strains of the genus Bifidobacterium are able to hydrolyze sodium taurocholate and glycocholate in the colon. This disequilibrium of the intestinal microflora is countered by oral administration of B.86.[218] This difference appears to be due more to the quantity of undissociated acid than to the type of acid. Prevention of the colonization of the intestine by pathogens by competing for nutrients and for binding sites on the epithelial surfaces. and it is produced in greater quantities by Bifidobacterium. longum has a barrier effect against Escherichia coli in the axenic rat.3. Hoang-Dung TRAN and friends Deconjugation of Bile Acids.[227] Axenic mice monoassociated with B.[247] The intestinal flora of leukemia patients is modified by chemotherapy. longum –fermented milk with the antibiotic treatment results in a reduction in the number of stools and the number of Bacteroides and a more rapid regain of weight compared with treatment with antibiotic alone.[245] The hydrolases involved are constitutive and extracellular. longum live longer than truly axenic mice after the intravenous or intragastric administration of high doses of viable E. Effect of the Anti-Infectious Barrier to Pathogenic Bacteria It has long been possible to demonstrate close relationships between the probiotic and therapeutic effects of bifidobacteria. and bacteria that are usually rare in healthy subjects multiply considerably. but does increase the activity of fecal b-glucosidase.[218.

The most probable explanation for this phenomenon would be the reinforcement of the immune barriers.[267] The intestinal disorders in 34 Soviet cosmonauts were successfully treated by the ingestion of bifidobacteria.[260] 2. which facilitates elimination of any pathogens present.[268] Copyright © 2004 by Marcel Dekker.[264] Seki et al. previous implantation of B.[266] The ingestion of milk fermented with B. All Rights Reserved. longum is successful in regularizing digestive transit in pregnant women (reduced abdominal ballooning. heat-stable and active at pH values ranging from 2 to 10 versus other gram-positive species including some strains of Clostridia.[256] The antitumor action may be obtained as a result of (a) direct suppression of the procarcinogens. In contrast. Similarly.[7] The intestinal flora and health status of children suffering from diarrhea were restored more rapidly after the ingestion of milk fermented with B. kidneys.[252] have investigated the in vitro interactions between B. longum may be capable of affecting both humoral immunity and cellular immunity. 1. The antimicrobial substance detected was of a protein type. spleen. Prevention and Treatment of Other Diseases In l966 Bamberg[261] successfully treated digestive disorders induced by antibiotic treat¨ ment using a lyophilized culture of B.[226. but also a therapeutic role in patients suffering from diarrhea. . breve. longum can play not only a preventive role in healthy subject. diarrhea-type phenomena. weeks later. Inc.[6] have investigated the translocation capacity of E. longum has a direct inhibitory effect on liver tumors in the mouse. Enterococcus faecalis. Haller and Krauberg[262] and Neumeister and Schmidt[263] obtained similar results in radiation-treated subjects. coli from all the organisms invaded within one week. longum. Intragastric inoculation of E. Bifidobacteria were successfully administered to premature infants with an intestinal flora that had been disturbed by the taking of antibiotics.[251] and (d) reduction of the intestinal pH.[257] The number of tumors developed by mice with an intestinal flora including E. thus preventing diarrhea and constipation. infantis has an undeniable antitumor effect. lactobacilli. and Clostridium paraputrificum is considerably reduced if B. B. although they remain present at high levels within the colon.[258] (b) the reduction of indirect suppression of procarcinogens or bacterial enzymes which result in their formation.[227] The production of bacteriocins by bifidobacteria was studied by Meghrous et al. and lungs of monoassociated animals. F. coli in the axenic mouse. B. longum is present. adolescentis and Bacteroides ovatus.255] B. (c) activation of the host’s immune system.[256] In the mouse.[265] developed a treatment for constipation in the elderly based on Bifidobacterium. longum in the axenic mouse allows the animal to survive and results in the disappearance of E. In a continuously renewed complex medium they observed that the inhibition of Bacteroides by the bifidobacteria appears to be due to the production of certain metabolites by the latter. Therapeutic Effects Antitumor Effect The research so far carried out has been essentially concerned with the direct or indirect antitumor action of streptococci.251] Knocke et al. in the appearance of this strain in the liver.Hoang-Dung TRAN and friends stimulates the peristaltic movements of the intestine. coli at sublethal doses results. or constipation).[254. and bifidobacteria. by maintaining high levels of bifidobacteria in the flora. The ingestion of milk fermented with B.[253] in 13 strains. Yamazaki et al. coli.

and B. B. 1958. The Prolongation of Life. B. Universite de Nancy. Recent trends in research on intestinal flora. microflore intestinale et nutrition. In Vitro and In Vivo Verification of the Probiotic Potential of the Strain This potential can only be demonstrated through clinical and nutritional studies. Int. longum constitute no danger. 1908. but other bifidobacteria in this group could be confused with B. Mitsuoka. the tolerance of the strain of acidity and above all of oxygen. 3.. infantis. CONCLUSION The current use in fermented milks of probiotic bacteria and of bifidobacteria in particular presumes that the user will take all necessary precautions with regard to the following aspects. A.P. eight species could theoretically be used. M. Since B. Recherche sur la flore intestinale des nourrissons (etat normal et pathologique). Laiterie 1984. Essais d’implantation chez ´ ` le nourrisson.M. G. Technological Potential The choice in this field must take into account several factors such as the acetate/lactate ratio. . H. Bifidobacterium bifidum: ses facteurs de crossance. 3 –24. Fuller. Metchnikoff. the choice must still be based on the probiotic activities desired. longum that had been used. E. breve. Marshall. Furthermore.. C. ` ´ ´ These de medecine de l’Universite de Paris. B. XV.Hoang-Dung TRAN and friends This nonexhaustive review of the beneficial effects of bifidobacteria demonstrates that in all cases it was bifidobacteria of human origin and more particularly B. Copyright © 2004 by Marcel Dekker. 54– 59. the relationships of the bifidobacteria with other species in the product. bifidum. REFERENCES 1.E. 4. Fed. and no strain has all the qualities demonstrated for the genus Bifidobacterium as a whole. 1990. Laits fermentes. Putnam. 4 pp. Choice of Species This is the most important factor if it is intended that the product should have genuine probiotic qualities. ´ 2. dentium if genetic methods are not used to identify the strain (Table 11). T. These. Gurr. Manciaux. Inc. the distribution conditions for these products must be such as to ensure the survival of the bifidobacteria under good physiological conditions. M. 5. Bifidobacteria Microflora 1982. D. V. In this field. dentium is recognized as being pathogenic. All Rights Reserved.I. Bull. Tissier. 1 (1). Strain Identification B. R.

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Yanai.D. G.. North Eur. S. 1987. Ed. 1982. C. A. 1980.R. 1975. 221. M.Lj.. 21. H.. Alterations in fecal microflora enzymes related to diet age. T. A. Recherche 1988. Beauchop E.. 1989. Dig. Environ. Eur. 529–533. Schellenberg. 1984. P. Actualite de la Recherche. 225 – 230. 257–260.A. Leedle. Salyers. Shaughnessy. Lemonnier.. enjeu scientifique et industrial.T. J. Gnotobiologic evidence for functions of the microflora.N. 577. . Lee. 40. acidophilus. All Rights Reserved. Antonie van Leeuwenhoek 1989. De Macias. S. A. 232...E.. Kuritza. Breakdown of mucin and plant polysaccharides in the human colon. G.. ¨ Luckey.and acidophilus bacteria in nutritional and health. Pasteur 1923. 235.. 52 (2). A. Dairy J. Interaction between the host and its microbes. 192– 250. J. 63 (1). ´ Abrams. 1475– 1625. Bexter. Ed. B. Salyers.A. 1983.R. 1977. Inc.. ´ Berrada.. Raibaud.L. Bacteria and the aetiology of cancer in the large bowel. Masson: Paris. 215. 29.. In Human Intestinal Microflora in Health and Disesae. J. 226. Appl. Biochem. 10 (2 – 3). 625. 225. 228.. 10. Symp. 543 – 545. Clin. 1986. 33 –38. Les anaerobes. 65– 75.D.P. J. A. 229. Cancer 1977. G. 361. D. Environ. W. Microbiol. Microbiol. In Clarke R.D. Ernahrungs-forschung 1965. J.S. J. 219. 51. Drug metabolism by the intestinal flora and its implications. R.S. Clostridium difficile and gut disease. 218.A.. Goodwin. Perdigon. 1977. 224.O. Bifidobacteria Microflora. 217. Rosenblatt.J. J.J. 1986. T.P. Simon. A. 19.T.D. Grasso.E. M.. 15. 55. 222. Actualites Scientifiques et Agronomiques Ecologie Microbienne du Tube Digestif. H. Academic Press: London. 7 –12. D. Appl. 1983. Gorbach. 277 – 310. Ther. Kawashima.P.R. H. M. ´ Ducluzeau. Survie de bifidobacteries dans l’estomac ´ ´ de l’homme. Salyers. A.. casei and L. Masson. S. Microbiol. eds. Hill.H. Borriello.. Vercellotti.P. Okamura. New York: Academic Press.. Bullard. R. Inst.Z.A. Rowland. 1985. Int. Savage. Barrier effect of Bifidobacterium longum on Escherichia coli in the germ-free rat. Aries. 6 (3). 55. G.S.. B. Gut 1969. Enumeration of polysaccharide degrading Bacteriodes species in human feces using species – specific DNA probes. J. 1979.. 1986. De Ruioz Holgado. INRA.. La putrefaction intestinale. Dis. D. 216.. Wilhelm. B. 49 (4). T. 213. Lactobacillus supplements and dimethylhydrazine. Degradation of N-nitrosamines by intestinal bacteria. Salyers. Crowther. 266– 270. J. N. In Les laits Fermentes. The human intestinal microflora.Hoang-Dung TRAN and friends 212... 34. Vercelloti. G.C. 2421– 2426.J.. R... Interaction of shigella with bifidobacteria. S. In Microbes and Infections of the Gut. 231. A. Le yaourt. H.P. Hentges. 230. 17 – 23..E. P.A. Wurzner.C. 1977.E. Wilkins. Laroche. Drasar. 80 – 88.. D. Carbohydrate utilization in the human colon.. T. 236. Nakaya. Microbial ecology of the gut.C. Res. Coates and Fuller (1977) Rasic. 334. Gorbach. 227. Alvarez. The role of dairy foods containing bifido. D. S. J. Williams. J. 51 – 55. Sci. Nutr.. P. Vit.. Microbiol. 129– 146. West. ´ Tissier. Olivier. P.. Fermentation of mucins and plant polysaccharides by anaerobic bacteria from the human colon. 220.. 233. Copyright © 2004 by Marcel Dekker. Goldman. N. Yokota. 1190– 1196. 385– 390. Can. Molecular and biochemical approaches to determining what bacteria are doing in vivo. 214. Immunology 1988.. Tonetti. Microecol. V.. Bull. Teraguchi 1984. Appl.F. Morphological aspect of gastro-intestinal tract colonization. Goldin. Systematic augmentation of the immune response in mice by feeding fermented milks with L. N. John Libbey Eurotext. 31 (9). 409.A.D. Faure.. 223. Lemeland.. 234. 259– 260. 5 (1). Bacterial. Salyers. Wilkins.

Cancer Inst. L. Food Sci. B. J. Microecol. 81. Rehabil. 658–688. J. H. 1365– 1370. Bifidobacteria Microflora 1984. Swenson. J. S. 54 (7).L. 240. T. 1980. Cancer Inst.. J. Antifidus cocci. 241. Santos. Chem. 1974. 64 (2). 255. 242. 239.V. Doctorat en medecine: Lille. Survival of microencapsulated Bifidobacterium pseudolongum in simulated gastric and intestinal juices..V. A note on bile acids transformations by strains of Bifidobacterium.C. G.B. L. Mizutani. A. Inc. 1990. J. Nishihara. Wardojo. Maharaj. 252. M. Pacini.S. R. 250. Raibaud. Technol. 163– 172. Dev. Effect of diet and Lactobacillus acidophilus supplements on human faecal bacterial enzymes.. Marteau.. J. Bifidobactera Microflora 1982. longum dans le traitement des diarrhees a rota´ virus du nourrisson.... B. Food Sci. E.. Y. K. J.. Phys. Kohwi. 1984. K. Momose.. Clin. Kinderheilk 1967. J. Z.R.. 193 – 197. their biological properties and clinical significance. Petitdemange. Nutr. 29 – 33. Stimulation of intestinal biocenosis in continuous flow culture. 1 (1). C.Hoang-Dung TRAN and friends 237.. Chawan. S. Gorbach. Hoskins.C.. Role des bacteries anaerobies stricyes ` ´ ´ dans les effects de barriere exerces par la flor du tube digestif. R. Tamura.. L. Screening of Bifidobacterium strains for bacteriocin production. 1989. K. Kamimura. Jaspers. Yamazaki. Flourie. Hoskins.. 86– 95. Otani.K. P.F. 55– 59. 243. 49. Y. T. I. 1990. Nicolas. Miller... S.R. Mann. 246.O. Effect of chronic ingestion of a fermented dairy product containing Lacto-bacillus acidophilus and Bifidobacterium bifidum on metabolic activities of the colonic flora in humans. Mschr. Inhibitory effects of lactic acid bacteria from fermented milk on the mutagenicities of volatile nitrosamines. T. Copyright © 2004 by Marcel Dekker. 67. N. Rao. 1980. 12 (8). Am. A. Interrelationships between diet. Nakano.T. 251. 238. Ueda.. Kageyama. S. 3 (1).. Hannelore. A. 27.C.. 1178. R. Clin. Junelles. Hashimoto. Y. A.. 254.. Food Sci. H. Protective effect of Bifidobacterium-monoassociation against lethal activity of Escherichia coli. Natl. L... 127– 135. 345–351. Can. Nutr. 244. Studies of a surfactant and cholesteremia in the Maasai. 759– 765. Ther. 25. Pochart. 16– 23.. J. Ballongue. J. 1988.. 1990. Paris. Clin. P. 245. Influence of milk and thermophilus milk on plasma cholesterol levels and hepatic cholesterogenesis in rats. Dwyer. Biotechnol. ´` ´ ` Romond. Desjeux.M. Pellier. Mucin degradation in human colon ecosystems. 115 (4). Knocke. A. 253.R. 257.. N. P. 1969. 296. J. L. 14. Invest. Bifidobacteria Microflora 1988. dans Les anaerobies. Rao. Rambaud. J. Kawashima. S. Hudault. 1639...F. P. Gann 1978. 46.. 1339. 1980. H.. Ferrari. 49. 1979. 255–261. 22 (4). Goldin. 247. 575– 580. The effect of oral administration of Lactobacillus and antibiotics on intestinal bacteria activity and chemical induction of large bowel tumor.L. 1981. All Rights Reserved. 249. Homma. Appl.. 10 (1). Bifidobacteria as a resistance factor in human beings.. N. P. 464. Homma. Imai. N. L. 7 (1). Effect of consuming yogurts prepared with three culture strains on human serum lipoproteins. Mayer. J.. Isoda.. Goldin. Meghrous. E.B. 256. 63. 69 (5). ˆ ´ ´ Ducluzeau. Sexton. Massey. Leudecke. Biol. Symp. 1986. A. M. Lett. 248.. 258. Gorbach. Tanoda.R.. Pulusani. Mitsuoka.. Med. D. H.. Boulding. Agric. Antitumor effect of Bifidobacterium infantis in mice. 613– 618. Indust. Gastroenterology 1981. Spoerry. Euloge. Am. J. 35– 43.L.A. 1981. 52 (4). B.. Bacteriol. Effect of intestinal bacteria on incidence of liver tumors in gnotobiotic C3H/HE male mice. Apport d’un lait fermente a B. intestinal flora and viruses. D. Canzi. Masson. B. Hosono. ... Shiwnarain. 1984. Natl. The effect of Bifidobacterium administration in patients with leukemia.. Mucin degradation in human colon systems. K. Inst. T. T. Microbiol.. 139–144. J.

Role en sante humaine.. Med... Ono. 113 (2). Zentralbl. 1980. Pasteur 1959. Materin. S. 53.. Igarashi.. 1990. Y. Inst. J. Fukuwatari. 1990.. 379– 387. Y. Klin. K.. Food Sci. Kawashima. M. Japan 1982..... H.. 261. Sekine. 50 – 53. Bifidobacterium et facteurs bifi´ ˆ ´ digenes. S. Takayama.. E. Ogasa. Can. Voprosy Okhr. Lizko. Kozlova. Oncol. Vergleichende Untersuchungen uber die Laktobazillen aus den Faeces von ¨ Menschen. D. All Rights Reserved. Med. 89 – 95. T.. 29– 35. 270. Sunakawa. 157–169. 58 (20). E. R. Strahlentherapie 1960. Food Hyg. Copyright © 2004 by Marcel Dekker. Kawashima. 83 – 95. Yajima.. 39. The effect of Bifidobacterium cultured milk on the “regularity” among on aged group.. 1986. 210.. 1969a.. 275. Inhibitory effect of some intestinal bacteria on liver tumorigenesis in gnotobiotic C3H/He male mice. J. Uncoupling of growth acids production in Bifidobacterium ssp.. T. Sakurai. 273. ´ ` La Vergne. 32 (3). M. 274. J. T. 104 – 107. S. J. H.. Y. 97 (1). I. 342– 348. Y. Teraguchi. ¨ Bamberg. Orig. Simamura.S. 1978. 268. 31 (4). 263. 276. .. bifidum a onze antibiotiques. Sato.. 33 – 45. 704. 271. Abt. Det. Fernandes. Kiyosawa. 2086. 267.. 265. Okonogi.. Soc. M.. J. ¨ Mitsuoka. C. J. Fukuda. Antibiot... Mitsuoka. J.. 184– 190. The influence upon radiation of the intestines by the oral use of combination of living acidophilus – bifidus and colibacteria. ´ Ballongue. Cancer Lett. ´ Asselin. T. Dairy Sci. N. Toupin. 116. Neumeister. Schmidt. Uber die Sanierung von Typhus dauerausscheidern mit Ampicillin. M. M.. Effets de l’ingestion de laits fermentes au B. Arai. M. Clin. Tatsuki. Araki. T.. Manciaux. 1989. 262. Med. 842– 844. 264.. Biotekhnol. Food Pro. 1987. II. 1478– 1484. Toida. K. longum sur la flore intestinale ` ´ humaine. 260.. Intestinal microbial flora in premature infants with septicemia and its correction with Bifidobacterium. 272. T. T. 1988.. Sensibilite de B. Y. K. ¨ Haller. Biochemical characterization and antitumor activity of a new cell wall preparation. Iwata. C. Teraguchi. Shahani. Seki. S. D. Soc. 1963. Hashimoto. 1990.N. Inc.. 1974.Hoang-Dung TRAN and friends 259. Saito. Treatment of intestinal radiation reactions. J. Nutr. K. M. K. 272.P. Sakurai. Desjardins. Kalnitsky. M. J. Kraubig. T. J. G.. Keio J. S. 1976. T. ARBBA. M.M. 269. Evolution de la taxonomie des bifidobacteries. Anticarcinogenic and immunological properties of dietary lactobacilli. E. The effect of fluoroacetate on the metabolism of yeast and bacteria.. Jpn. Test tube method for counting bifidobacteria in commercial dairy and pharmaceutical bacteria products. 266. Mutai. Oikawa. Sekiguchi.C. 32– 51.L. Res. Schweinen und Huhnern. I. Schmitt. These de l’Universite de Caen en Sciences Pharmaceutiques. Ann. Biol. Clinical effects of Bifidobacterium preparations on pediatric intractable diarrhea. Bakteriol. 23. 21.. J. Hotta. 1966. Med. 1987. S. J.F. T.. 32 (2). Chem. Kuboyama. Wellt.. H. J.. 298– 314. Watanabe. 73 (6). W. Mizutoni. N. Dysbacteriosis under extreme conditions. Guzman Barron. Goulet. Tanaka.. 170. whole peptidoglycan (WPG) from Bifidobacterium infantis. K. Yamashita. Burdin. S.. Roy. Kawashima. Vitamin production by bifidobacteria originated from human intestine. Parasitenk.

the question is often raised as to why bifidobacteria have joined lactobacilli as a target for probiotic 125 Copyright © 2004 by Marcel Dekker. . In 1990 there were approximately 30 publications in the international literature focusing on bifidobacteria. our understanding of the ecology of bifidobacteria in the human intestinal tract. WHY BIFIDOBACTERIA? Lactic acid bacteria have a long history of use in fermented foods. II. New therapeutic applications for bifidobacteria have emerged.Hoang-Dung TRAN and friends 3 An Update on Probiotic Bifidobacteria ROSS CRITTENDEN Food Science Australia. It focuses on recent advances in food technology. there have been numerous changes within Bifidobacterium taxonomy and the development of new applications and food technologies. INTRODUCTION Research and commercial interest in the genus Bifidobacterium has flourished in recent years. However. In 2002 there were more than 10 times that number. and emerging evidence for clinical benefits and mechanisms of probiotic action. This chapter provides an update on our current knowledge of bifidobacteria as probiotics. phenotypic examination of microbial diversity to the application of cultureindependent molecular techniques has provided new insights into the ecology of bifidobacteria within the intestinal tract. and an overview of the beginnings of a new wave of interest in these organisms as probiotics. Australia I. Since Ballongue’s chapter was published in 1998. Exciting advances have been made in demonstrating the efficacy of probiotic bifidobacteria in human health. The previous chapter by Jean Ballongue provides an historical perspective on the discovery of bifidobacteria. their basic physiology. Inc. Victoria. including roles in the treatment of allergy and the maintenance of remission in inflammatory bowel disease. A fundamental switch from culture-based. Werribee. All Rights Reserved. such as their use to deliver targeted gene therapy to hypoxic tumors. driven almost entirely by their potential health benefits in probiotic functional foods.

with a saccharolytic and acidogenic physiology. probiotic bifidobacteria now appear alongside lactobacilli in a large proportion of probiotic functional foods. longum are recognized as heterotypic synonyms.3] Additionally. Like lactobacilli. for most strains. animalis Bif.[5] while Bif. With the theory that two is better than one. denticolans Bif. Bif. infantis Bif. III. lactis has been absorbed into Bif. inopinatum have been reclassified into new genera. inopinatum Bif. longum Scardovia inoponata Bif. Table 1 Changes in the Taxonomy of Bifidobacteria New designation Parascardovia denticolens Bif.[2. scardovii Bif. lactis Bif. globosum Bif. bifidobacteria were considered desirable. Inc. whereas infants who were fed using cow’s milk –based formulas developed a mixed microbiota. gallinarum Bif. and Bif. .[4] Bif. without gas formation. and so they can be used in fermented dairy products. the consumption of live bifidobacteria to maintain or restore a population of “healthy” intestinal bacteria was popularized in a similar way to lactobacilli before them. Hence. the traditional food vehicle used for probiotic lactobacilli. Technological considerations also facilitated the adoption of bifidobacteria as probiotics. saeculare Bif. and. and were replaced by putrefactive bacteria. suis. pseudolongum subsp. suis Copyright © 2004 by Marcel Dekker. and without involvement in putrefying or toxigenic reactions or pathogenicity. without compromising the organoleptic qualities of fermented dairy foods.[6] The names Bif. globosum Bif. denticolans and Bif. there were some sound scientific and technological reasons for the initial development of interest in this genus. ruminantium Bif. merycicum Bif. Bifidobacteria produce lactate and acetate during sugar fermentations. The science to evaluate the theoretical benefits of the consumption of bifidobacteria has largely lagged behind their commercial application. animalis. Many species grow well using lactose as a carbon source. coryneforme Bif. gallicum Bif. the application of molecular techniques to determine relatedness of bacteria at the genome level has led to some recent reclassifications of species within the genus Bifidobacterium (Table 1). longum Recent additions to the genus Bif. Notably. Bif. From an historical perspective. healthpromoting bacteria. suis have been amalgamated into Bif. the levels of bifidobacteria were shown to decline in the elderly. including higher levels of potentially deleterious organisms. TAXONOMY As for most bacterial genera. thermacidophilum Old designation Bif. infantis. longum.[1] Interest in reinforcing the numbers of these bacteria in the intestinal tract stemmed from studies showing that bifidobacteria constituted the numerically dominant microbial population in the intestinal tract of infants who were fed breast milk. All Rights Reserved. especially clostridia and enterobacteria (see Chapter 2). First among these was that they were known to constitute a major population group within the human colonic microbiota.Hoang-Dung TRAN and friends research and commercial application. infantis and Bif.

safety. which is associated with dental caries. Bif. Taxonomic classification should be included as an early step in the development of any new probiotic strain. Inc. On the basis of distinct clusters from PCR methods targeting the 16S– 23S internally transcribed spacer region of Bif. longum/infantis have GRAS (generally regarded as safe) status. Bif. and identifying a new probiotic strain at the species level is important in assessing its relative risk. There is still considerable room for improvement. thermacidophilum[7] stands out from other Bifidobacterium species. . Bif.0).[11] if only by limiting designations to the genus and strain level. Taxonomy is important not only from scientific. lactis. a strain designation is the primary identification promoted for many probiotics. dentium. lactis is rather more palatable to marketers of probiotics for human consumption than Bif. there are almost certainly new species awaiting discovery. lactis/animalis group are considerably more acid-. animalis and Bif. Strains formerly classified as Bif. The reclassification of Bif. Bif.[16] After more than 20 years of use in probiotic foods and investigation in a large number of animal studies and in human feeding trials.14] The constant flux in species designations can cause headaches for commercial suppliers. animalis/Bif. IV. Demonstrating a safe history of use in foods facilitates passage of probiotic ingredients through food regulatory frameworks. but also from regulatory. As its name suggests. adolescentis. SAFETY Except for Bif. lactis. animalis. evolutionary. dentium (dental caries). deleterious effects have yet to be reported for bifidobacteria. All Rights Reserved. species other than Bif. oxygen-.[15] The application of molecular techniques to characterize currently uncultivable bacteria will undoubtedly lead to the discovery of many new species and genera of bacteria in a range of habitats.[8] In the past there has been substantial discrepancy between the Bifidobacterium species designated on a product label and the species found in the product itself. 2) and technologically suited to fermented dairy products. lactis are the most commonly used bifidobacteria in fermented dairy probiotic products.[11] and the name Bif. and ecological points of view.10] Manufacturers of probiotic dairy products appear to have improved their attention to correct taxonomy in recent years. Strains within the Bif. and Bif. lactis to Bif. and thermotolerant than most human intestinal Bifidobacterium species (Figs. it can grow at elevated temperatures (49. animalis at the subspecies level has been proposed.[8] A number of bifidobacteria now have a long history of safe use as dietary adjuncts. animalis is probably the most contentious of the new changes in taxonomy.18] to the elderly[19 – 22] and in healthy individuals and those with intestinal disease. bifidum.Hoang-Dung TRAN and friends A number of new species of bifidobacteria have also been isolated and characterized (Table 1). and the taxonomic map of the bifidobacteria can be expected to undergo further changes in the coming years. 1. bifidobacteria are regarded as safe.[12] and the problem appears to still be particularly widespread in freeze-fried probiotic preparations[11] and animal probiotic products. Human feeding studies have been performed without reports of adverse health effects in age groups ranging from infants[17.[13. Among these. but since probiotic attributes vary within species and must be defined at the strain level.[9. Bif. and marketing standpoints.58C) and at relatively low pH (4. are generally regarded as safe. the separation of Bif. In the case of bifidobacteria. lactis from Bif. breve. In the complex milieu of the intestinal tract of humans and animals.[23 – 26] Copyright © 2004 by Marcel Dekker.

O Bif. In the rare cases of bacteremia where bifidobacteria have been found. This figure represents viable counts of bacteria before (A) and (A) after incubation in HCl/KCl at pH 2.) Figure 2 Copyright © 2004 by Marcel Dekker. the subject has been predisposed to infection by surgical trauma or underlying health disorders.[27] The rarity of reported infections and inadequate taxonomic characterization Bifidobacterium lactis is able to grow at higher temperatures than Bifidobacterium species indigenous to the human gastrointestinal tract. B Bif. W Bif. All Rights Reserved. 39.) Figure 1 Reports of human bacteremias involving bifidobacteria are rare. 39. breve. lactis. (Modified from Ref. . longum. Inc.0 for 105 min at 378C. and the bifidobacteria have formed part of a mixed culture bacteremia involving other usually benign commensal bacteria. adolescentis.Hoang-Dung TRAN and friends Bifidobacterium lactis is considerably more tolerant to acidic conditions than species indigenous to the human gastrointestinal tract. (Modified from Ref. A Bif.

TECHNOLOGY Probiotics are believed to elicit their beneficial health effects when viable.[8. the number of species with a safe history of use in foods may be lower than the history of product labeling would suggest.11] Therefore. Not only viability. animalis/lactis compared to human intestinal isolates remains to be investigated and may provide important clues to improving the survival of pH-sensitive strains. All Rights Reserved. A number of parameters.Hoang-Dung TRAN and friends of offending isolates has precluded any associations being made between particular species and human bacteremias. salts. storage. and intestinal delivery. formulation. Currently. and technological approaches such as immobilization.[32 – 36] However. animalis. and flavoring and coloring compounds.[39] This is sometimes achieved by limiting fermentation by traditional yogurt starter cultures or by omitting starter species.[37] The relative oxygen sensitivity of a range of Bifidobacterium strains has been reported by Beerens et al. lactis can survive well in fermented dairy products throughout their shelf life if the pH is prevented from dropping below 4. Fermented dairy products are still the major food vehicles in which probiotic bifidobacteria are delivered. The physiological mechanisms behind the acid tolerance of Bif..[9. The low incidence and predisposing circumstances of bacteremias involving bifidobacteria. Oxygen sensitivity can be addressed by strain selection and with appropriate packaging techniques and materials. and induction of stress proteins have been employed to improve stability. their use in functional foods provides greater technological challenges. Since bifidobacteria are strict anaerobes and generally less acid tolerant than lactobacilli.27. when combined with the long and safe history of use of some strains in foods.) V. although the inclusion of Lactobacillus delbrueckii subsp. Most human intestinal Bifidobacterium isolates are more challenging to grow and maintain higher viability during product storage than Bif. Surveying of the probiotic bifidobacteria used in yogurts and lyophilized products has demonstrated regular discrepancies between species labeled and those in the product. microencapsulation.[38] and in general species of animal origin are more oxygen tolerant than those isolated from humans. supports the conclusion that bifidobacteria are generally safe for probiotic use. although this dogma is being increasingly challenged. including interactions with other starter and probiotic strains. Copyright © 2004 by Marcel Dekker.31] Whether viability is an essential requirement for all health effects elicited by probiotics is an intriguing question that demands further investigation. this does not mitigate the need for adequate safety testing of new probiotic Bifidobacterium strains before they are used commercially. however.1. but also functionality must remain stable during manufacture. manufacturers aim to maintain a high level of probiotic culture viability throughout the shelf life of their products. . The outcomes of some of these strategies are discussed later in this section. with yogurt being dominated by Bif. Bif. However. lactis/animalis. can influence the survival of bifidobacteria in these products.[30. Inc. sugars.29] (See Chapter 19 for further details on the safety assessment of probiotics. One example is a potentially novel Bifidobacterium species identified in deep caries that may play a role in disease. oxygen and pH exert perhaps the greatest influence on Bifidobacterium survival during storage. bulgaricus is mandatory in yogurts in many countries. The application of culture-independent methods of investigating microbial ´ diversity in niches within the host will lead to the identification of new species that require characterization from a safety viewpoint.[28] A number of articles discuss safety tests that should be applied to new probiotic isolates.

[58 – 65] carrageenan. but it is questionable if the level of improved viability could justify the increased production costs in commercial food applications. Those reported have included imparting a grainy texture[67] and stimulating production of off-flavors. bifidobacteria and bifidogenic oligosaccharides are now included in some powdered infant formulas.[69] and cellulose acetate phthalate[70] have all been trailed. Alginate.A. and modified starch have generally failed to substantially improve the survival of acid-sensitive bifidobacteria. Another impediment to the application of some microencapsulation techniques in certain food matrices is the imposition of deleterious effects on sensory properties. and with suitable sensory properties. encapsulation with alginate. The technical hurdles in the application of microencapsulation for probiotic bifidobacteria are not insignificant.[54] Attempts to maintain high viability of bifidobacteria in coleslaw[55] and a table spread[56] have proved less successful. Microparticles manufactured using alginate and poly-l-lysine and particles produced using cellulose acetate phthalate in spray drying have been reported to significantly improve survival of bifidobacteria in acidic environments.[66] Microencapsulation may yet prove to be a valuable approach to maintain the viability of environmentally sensitive bifidobacteria in long shelf-life food products.[61. Hoang-Dung TRAN and friends Food Applications Other Than Fermented Dairy Products Bifidobacteria have now been successfully applied to a range of food matrices beyond dairy yogurt and fermented milk. Inc.48] products.[66. presumed to be due to mass transfer –induced nutrient limitation effects on metabolism. In laboratory models simulating the acidic conditions in the stomach. and camel’s milk[43] and in fermented soybean[44 – 46] and oat-based[47. and all with an encapsulating material that is inexpensive. carrageenan. C.[42] sheep’s. A Bif. More encouraging results have been reported for improved survival in food products. the story of microencapsulation of bifidobacteria is not all doom and gloom.[40. efficient release of the bacteria within the gastrointestinal tract. while at the same time improving viability. One interesting application of these bacteria has been as a biocontrol agent in fermented meat.67] modified starch. foodgrade.[57] B. protection against low pH. stable. . lactis strain incorporated into a Hungarian salami was reported to inhibit the growth of Listeria monocytogenes and Escherichia coli strain 0111. To simulate the bifidogenic effect of human breast milk. Copyright © 2004 by Marcel Dekker. Most have been only partially successful. longum.[68] gellan-xanthan. The demands include protection against oxygen and other environmental stresses during product formulation and storage. and bile during gastric intestinal transit. Bifidobacteria have been reported to grow and survive in a variety of cheeses[49 – 52] and remain viable in ice cream[53] and in frozen yogurt. Microencapsulation A number of microencapsulation technologies to protect bifidobacteria against acidic conditions have been developed. proteases. Induction of Stress-Response Proteins Perhaps the most judicious approach to improving the tolerance of bacteria to environmental stresses is by preconditioning cultures through induction of shock proteins. The presence of the heat shock protein gene dnaK has been demonstrated in Bif.[65] However. All Rights Reserved.70] Microencapsulation has also been shown to provide a beneficial impact on sensory parameters in yogurt by preventing excessive acidification.41] They have been used in fermentations of goat’s.

Bioconversions The metabolic activities of bifidobacteria have been exploited to produce bioactive compounds during fermentations that potentially increase the health functionality of the food products. Knowledge of the interactions between the microbiota and the health and disease of the host is an essential prerequisite for a mechanistic understanding of probiotic efficacy. storage.75] Occasionally intestinal isolates prove quite fastidious with respect to their growth environment. Until the mid-1990s most investigations of intestinal microecology had relied on culture techniques to isolate the bacteria. longum strain resulted in increased tolerance to both freeze-thawing and lethal heat stress. and gastrointestinal transit. Freeze-drying remains the most commonly employed method of drying bifidobacteria for commercial use. Inc. One example is the apparent requirement of some strains for high cell density for growth.[45] synthesis of conjugated linoleic acid from free linoleic acid. inhibits the growth of such strains.[51] VI. spray-drying has been used successfully for some strains when carriers such as gelatine gum arabic. Preconditioning with bile salts was shown to provide improved protection against lethal concentrations of bile for Bif. . The complexity of the intestinal microbiota was still recognized.[61. Companion cultures.[78] transformation of isoflavone phytoestrogens in soy milk. including lactobacilli and propionibacteria. including whey-based media supplemented with yeast or beef extract. Fermentation and Drying Bifidobacteria generally grow well in industrial media that support the growth of lactobacilli. Recent years have seen a surge in the development of the molecular tools needed to investigate bacterial population dynamics in complex ecosystems.[79] and production of bioactive peptides with potential antihypertensive effects. adolescentis.Hoang-Dung TRAN and friends Bif. ECOLOGY OF BIFIDOBACTERIA WITHIN THE HUMAN GASTROINTESTINAL TRACT The application of bifidobacteria as probiotics has provided fresh impetus for research aiming to provide an understanding of the ecology of these bacteria within the human intestinal tract.76] The suitability of spray drying for bifidobacteria is highly dependent on the robustness of individual strains. adolescentis.68. Examples include the production of folic acid[77] and oligosaccharides in fermented milk and yogurt. breve. As in other bacteria. A too dilute inoculum or even gentle stirring of the cultures. even under anaerobic conditions. Inducing stress proteins during fermentation and maintaining their presence downstream may contribute to improved survival of environmentally sensitive bifidobacteria during manufacture. D. longum and Bif. and Bif.[73] and consideration of the carbon substrate is important since not all strains can utilize lactose.[72] Prehydrolysis of media with proteases often improves fermentation yields. However. however. Copyright © 2004 by Marcel Dekker. E. or starch were added and relatively low air outlet temperatures (508C) were used. All Rights Reserved.[74. have also been shown to increase Bifidobacterium yields in fermentations in a strain-dependent manner.[71] Such stress-response proteins in bifidobacteria might be exploited to promote survival of strains during intestinal transit. cross-protection was afforded by the stress-response proteins since salt pre-treatment of a Bif.

also supply sites for bacterial colonization. particulate matter. bifidobacteria are generally found in the order of 108 – 10 10 bacteria/g. and the surface of mucosal epithelial cells.85. However. Bacterial numbers and diversity increase through the intestine. there have been few reports of Bifidobacterium ecology at different sites in the human intestinal tract.[87] Many bifidobacteria adhere to human intestinal mucus in vitro. A. including dietary fibers and other undigested food particles.[81] Tannock.[25] Within the lumen. made more onerous by the difficulties in sampling within the intestinal tract. the bifidobacterial population in feces (8 Â 108 bacteria/mL) was found to be 100-fold larger than in the cecum (5 Â 106 bacteria/mL).[87] The stomach and proximal small intestine contain relatively low numbers of bacteria (103 –105 bacteria/ mL) because of the low pH and rapid flow in this region.[80] The emergence of culture-independent.89] Due to the difficulties involved in obtaining samples. and the number of bacterial species identified within the intestinal microbiota may eventually exceed 1000. Using culture methods. in terms of the total bacterial population. distinct microhabitats also exist within each gut compartment.2% in feces (a figure in agreement with other estimates of the fecal bifidobacterial population using culture-independent methods[91 – 94]). reaching 108 bacteria/mL in the ileum and 1010 –1011 bacteria/g in the colon.[81. the relative proportion of bifidobacteria declined from 5.88..[84] and McCartney. . genetic methods to examine bacterial diversity has revealed that a significant fraction of the microbiota [up to 60%][80] remains uncultivable and that the intestinal ecosystem is far richer than was previously believed. These include the intestinal lumen.[90] investigated the differences in total Bifidobacterium numbers (and other bacteria) in samples collected from the cecum of healthy volunteers via an intestinal tube and compared the cecal population to that found in feces. Inc.[100] and it is likely that they colonize particulate substrates in the colon.[99] Many bifidobacteria adhere well to resistant starch. We are only at the very beginning of developing this knowledge. enabling investigation of microbial diversity at both the genus and species level.[82 – 85] Genetic techniques to study intestinal microbial ecology have been eloquently reviewed by Vaughan et al.[86] Understanding the role of bifidobacteria within the intestinal microbiota and their interactions with diet and with the host is an enormous task. but can expect rapid advances in coming years with the application of high-throughput genomic technologies to this task.[81] New taxa continue to emerge. the deep mucus layer within the intestinal crypts.[81] The discriminatory power of genetic fingerprinting techniques has allowed even deeper examination of bacterial diversity at the strain level. In addition to the different anatomical sections of the intestinal tract.[87] In the feces of healthy adults. The site of intestinal Copyright © 2004 by Marcel Dekker. All Rights Reserved. Marteau et al. Where Are Bifidobacteria Within the Intestinal Tract? Indigenous bacteria are not distributed evenly throughout the gastrointestinal tract but are found at population levels and in species distributions that are dictated by the environmental conditions that are characteristic of specific regions of the gut.Hoang-Dung TRAN and friends and it was estimated to contain more than 400 species dominated by perhaps 30 –40. the unstirred mucus layer of the epithelium. investigated using 16S RNA probes.[81] The 16S ribosomal RNA (rRNA) gene has become an important tool in classification.[95 – 98] and introduced probiotic strains have been found both associated with the mucosa throughout the colon and in the lumen.8% of total RNA in the cecum to 3.

A study from the United Kingdom[105. Bif.5% in adults (25 – 55 years) and actually increased to 8. with considerable variation between individuals. breve. mainly oligosaccharides. but further work on the molecular bases for health mechanisms is required before such assessments can be made.108] or in a Dutch study by Harmsen et al. Hopkins et al. forming up to 90% of the total fecal bacteria.102 – 104] 3. Intestinal mucus isolated from the elderly supports in vitro adhesion of bifidobacteria relatively poorly compared to mucus isolated from infants and Copyright © 2004 by Marcel Dekker.[87. In adults. Bif.[41. such as colonization resistance and immunomodulation. longum are the most frequently reported species. and Bif. longum appear to be the most common intestinal species in the elderly. Bif. . In adults. Which Species Are Present and How Does the Population Change with Age? 1. All Rights Reserved. cultural. 16S rRNA analysis.Hoang-Dung TRAN and friends colonization may be important in the mechanism of health benefits imparted by probiotic bifidobacteria. In contrast.[101] The dominance of bifidobacteria is induced by bifidogenic components in breast milk.[93] comparing the fecal bifidobacterial levels from different age groups using fluorescent in situ hybridization (FISH). Elderly Early studies using culture methods reported that Bifidobacterium levels decreased as a proportion of the total fecal microbiota in elderly Japanese.108] with Bif.[106] One of potentially many mechanisms for this change in the size and composition of the intestinal bifidobacterial population in the elderly may be altered mucosal adhesiveness. and genetic variations on intestinal ecology are still poorly understood and may result in regional differences in intestinal microecology. bifidum. bifidum. adolescentis and Bif. longum/ infantis.[93] found that bifidobacteria declined from 68% of total bacteria in the feces of breast-fed infants to 5.[96. angulatum also reported.102] 2. The influence of diet. Inc. Although considerable variation exists between individuals. B.[107. bifidum [108] and Bif.90 – 94] The numerically dominant species in the intestinal tract also changes with age.[1] This finding has only recently been readdressed using modern bacteriological and molecular techniques.5% in elderly subjects (75 –95 years).catenulatum/pseudocatenulatum. they have been reported to account for 1 –5% of the total bacteria in feces in studies using molecular techniques that account for the uncultivable microbiota. adolescentis.[81. In addition to a drop in bifidobacterial numbers. Bif.[89. the reported fecal Bifidobacterium numbers were not abnormally low in two independent studies of elderly Italians.94. and Bif. and community cellular fatty acid profiles. Adults The proportion of bifidobacteria in the colonic microbiota drops following weaning and the introduction of solid food. Bif.101] The Bifidobacterium species most commonly isolated from breast-fed infants are Bif.[106] observed that the diversity of Bifidobacterium species in the intestinal tract decreases with age. Infants Bifidobacteria colonize the human intestinal tract during or soon after birth and in breastfed infants eventually dominate the microbiota.6% of total bacteria in young children (1 –10 years) and 2.106] confirmed the earlier Japanese observation[1] of a drop in Bifidobacterium numbers in the elderly using viable counting procedures. Harmsen et al.

bifidobacteria isolated from the elderly. bind poorly to intestinal mucus compared to bifidobacteria from adults. Consumption of a range of oligosaccharides can induce 10.[110] At the strain level.111] The bifidogenic effect of dietary NDOs can be replicated in adults.85] Despite these differences in taxonomy.Hoang-Dung TRAN and friends healthy adults. and species level.81. C.83.[109.109] In adults.89] It appears that we all harbor our own particular combination and proportion of Bifidobacterium species. Similar results were reported by Tannock.[80] including a study in which the fecal bifidobacterial population of an individual was monitored for 8 months using species-specific PCR-DGGE. All Rights Reserved.[95] Additionally. When differences between individuals’ intestinal bifidobacteria have been investigated at the strain level. the intestinal microbiota is unstable and developing and through childhood to adulthood develops increasing complexity and stability.to 100-fold increases in Bifidobacterium num- Copyright © 2004 by Marcel Dekker.[82.101] and can be simulated through the supplementation of cow’s milk – based formulas with nondigestible oligosaccharides (NDOs). E.109.[81. How Stable Is the Bifidobacterium Population Over Time? In infants. Inc. Can Diet Influence the Bifidobacterium Population Dynamic? The example of differences between the size and composition of the intestinal Bifidobacterium populations in breast milk-fed and formula-fed infants clearly demonstrates that diet can influence bacterial population dynamics during the early maturation of the intestinal microbiota. This effect is elicited largely by oligosaccharides within human breast milk[87. differences in Bifidobacterium population stability were observed between two adults monitored for 12 months.[80.[41. No substantial changes in the species composition or relative intensity of bands were observed.110] Satokari et al.[110] This uniqueness of species and strain combinations may have important implications for the introduction of “foreign” probiotic strains into the intestinal tract of adults with a developed and stable intestinal microbiota. each person has generally harbored unique multiple strains. especially strains of Bif.[89] used PCR-DGGE (PCR-denaturing gradient gel electrophoresis) to follow the Bifidobacterium species diversity in five healthy adults over a 4-week period.[96] perhaps reflecting a change in selective pressures for mucosal colonization. D.[80. although to a lesser extent. It opens debate into the relative merits of the prebiotic approach of stimulating proliferation of the native intestinal bifidobacteria versus probiotic effects within different age groups. Are My Bifidobacteria Different from Yours? The application of molecular techniques to profile the complex microbial communities has revealed that each person has a unique intestinal microbiota at the community.110] PCR-DGGE analysis of the composition of Bifidobacterium species in the feces of different individuals has shown that each person has a unique pattern. genus. the bacterial population of the colon at the genus and species level appears to be very stable over time. it is likely that the functionality and biochemistry of the intestinal bifidobacterial population as a whole differs little between individuals. adolescentis.[82] Further analyses of larger sample sizes over long periods at a strain and species level are required to provide a better picture of ecological stability at the strain level. indicating that the Bifidobacterium population remained stable at the species level. .

85.[118 – 124] Differences have been observed in the respective intestinal bacterial populations in allergic and healthy infants. but probiotic intervention may prove beneficial in the control of these conditions.[128] and in elderly people with Clostridium difficile –associated diarrhea. including the development of tolerance to dietary antigens. All Rights Reserved.119.[89.[118. differences in the microbial ecology of healthy and allergic infants have only been investigated for a narrow range of organisms.[98] However.[112] Bifidogenic oligosaccharides form part of a class of dietary supplements called “prebiotics. G. other organisms with similar activities occupy the same niche.89] it appears that a proportion of healthy adults have intestinal bifidobacterial populations several orders of magnitude lower. Feeding of 8 g/day of galacto-oligosaccharides to healthy adult volunteers did not result in marked changes in the composition of their intestinal bifidobacterial population. The total numbers of bifidobacteria are lower in feces of allergic infants. .131] It is yet to be determined how the total number of bifidobacteria within a stable microbiota influences the long-term health of the human host.81.130. probably due to the high initial level of bifidobacteria. a prominent bifidogenic effect was also not observed in this feeding trial.[114 – 116] However. Inc.122] and the Bifidobacterium species composition in allergic infants is more adult-like. the apparent stability of the intestinal bifidobacterial population dynamic in adults suggests that dayto-day fluctuations in diet have little impact on the species composition. The long-term effects on the composition of the bifidobacterial Copyright © 2004 by Marcel Dekker. and if in individuals with naturally low levels of bifidobacteria. such as arabinoxylans.[80.[126] The size of the intestinal Bifidobacterium population has also been shown to be relatively small in subjects afflicted with inflammatory bowel disease (IBD). Although the level of bifidobacteria in the feces of healthy adults is normally in the order of 109 bacteria/g. being dominated by Bif. has stimulated investigations of differences between the intestinal microbiota of atopic and healthy infants. adolescentis.Hoang-Dung TRAN and friends bers in the intestinal tract of adults. How Do Antibiotics Affect the Bifidobacterium Population in the Gut? Administration of certain antibiotics is well known to perturb the intestinal microbial population dynamics. Significant differences were not observed in the Bifidobacterium populations of milkhypersensitive and healthy adults.125] The Bifidobacterium isolates from allergic infants have also been shown to be less adherent to intestinal mucus than isolates from healthy infants. Although bifidobacterial numbers can be increased using prebiotics.[117] can alter the composition of Bifidobacterium species within the intestinal tract. and cause/effect links between these observed differences remain to be established. It remains to be seen if prolonged consumption of dietary carbohydrates that are selectively fermented by only a narrow range of Bifidobacterium species.[98.[129] Direct cause/effect links between these diseases and a diminished bifidobacterial population remain to be established. including within the bifidobacteria.”[113] which are discussed in more detail later in this chapter.[127] irritable bowel syndrome (IBS).102. Is the Intestinal Bifidobacterium Population Dynamic Associated with Health and Disease? The growing recognition of the importance of the intestinal microbiota to the healthy maturation of the host’s immune system. F.

lactis. infantis strain could be isolated from the mucosa throughout the length of the colon. different from before treatment.[132] Tannock[80] reported the results of a trial in which the composition of the intestinal microbiota of 10 children was examined before and after treatment with antibiotics. Fujiwara et al. One contentious question has been the importance of host specificity in probiotic function. The impact of introducing probiotic bifidobacteria on the already resident intestinal Bifidobacterium population dynamic and on the composition of the wider bacterial community is of considerable interest. Probiotic supplementation following antibiotic treatment may have a role in preserving correct immune development in infants in addition to maintaining colonization resistance.[18] Using genus-specific PCR-DGGE. PCR-DGGE analysis of the intestinal microbiota of children before and after consumption of Bif.114 – 116] and the use of antibiotic-resistant mutants[25. Through investigations of colonic biopsies obtained during colonoscopies.[18.114 – 116.114 – 116] Human intestinal species were shown to adhere better to human intestinal mucus than to bovine intestinal mucus.[133] recovered a strain of Bif. Molecular strain fingerprinting techniques[18.133] have enabled tracking of ingested strains within the complex intestinal microbiota. Copyright © 2004 by Marcel Dekker. it adheres well to human intestinal mucus in vitro[97] and transiently colonizes the human intestinal tract following consumption. Following 6 days of treatment with oral rifampicin and streptomycin.[25] determined that a probiotic Bif.[97] but the question of the importance of mucosal adhesion and strain origin in colonization and probiotic activity of bifidobacteria remains unresolved. Inc. It is clear that selected probiotic bifidobacteria do survive transit through the stomach and small intestine and can be recovered in feces. In most cases the strain persists only transiently in the intestine. lactis/animalis is the predominant Bifidobacterium species currently used in probiotic foods. All Rights Reserved. H.[134] However.Hoang-Dung TRAN and friends microbiota following administration of antibiotics to which they are sensitive differs with the antibiotic used. . the Bifidobacterium strains collected in the dominant fecal microbiota of five human subjects were.[18. Bif. as determined by genus-specific PCR-DGGE. 3 months after 8day treatment with oral amoxicillin-clavulanic acid (Augmentin). It is also taxonomically distant from human intestinal species. Prolonged antibiotic treatment in early childhood has been significantly associated with a subsequent history of asthma.25. In 7 out of 10 children.[85] The impact of antibiotics on the microbiota of children may be particularly critical if it impacts on immune development. von Wright et al.46.133] but the duration of persistence in the intestinal tract is dependent on the individual and almost certainly the host – bacterial strain compatibility. lactis for 12 weeks revealed no major disturbances of the dominant bacterial groups in the intestine.[85] In contrast. in most cases. What Happens When Probiotic Bifidobacteria Are Introduced into the Intestinal Tract? The theoretical bases for many of the anticipated probiotic effects of bifidobacteria rely on the bacteria being viable in the intestinal tract. antibiotic treatment resulted in a marked change in the level and species composition of the bifidobacterial population. Satokari et al. It would be undesirable for an introduced probiotic to perturb the resident microbiota.[115] also observed no effect on the qualitative composition of the indigenous Bifidobacterium population after consumption of Bif. but is not an autochthonous member of the human intestinal microbiota. the bifidobacterial strain composition remained largely unchanged from before treatment. longum in the feces of some individuals up to 30 days after cessation of probiotic ingestion.

lactis was present in the intestinal tract. it appears that ingestion of Bif.141. the most consistent evidence accumulated for prebiotic effects has been for fructo-oligosaccharides. Just as important as bacterium numbers are the biochemical and functional activities of probiotics within the intestinal tract and. Interest in bifidogenic factors developed from attempts to replicate the bifidogenic effect of human milk oligosaccharides in infant milk formulas. in cow’s milk –based infant formulas[111] and may be useful in reinforcing bifidobacterial numbers in the elderly. A number of nondigestible oligosaccharides have now been demonstrated to act as bifidogenic factors. longum numbers returned to normal following cessation of the probiotic feeding. Bifidobacteria have also been used to produce synbiotic yogurt through de novo synthesis of galacto-oligosaccharides during milk fermentation.[113] There is now little doubt from the volume of animal and human studies investigating prebiotic effects that some indigestible oligosaccharides can induce a significant proliferation of bifidobacteria in the intestinal tract of individuals with initially low bifidobacterial numbers. although galacto-. factors that specifically promote the growth or activity of probiotic organisms within the intestinal tract have been termed “prebiotics. . PREBIOTICS The consumption of live bacteria is not the only strategy to increase the size of the Bifidobacterium population in the intestinal tract. xylo-.”[113] There is an obvious synergy between probiotics and prebiotics. slowly fermented carbohydrates may have advantages over oligosaccharides by minimizing rapid gas formation in the gut and promoting fermentation more distally in the colon. bifidum substantially reduced hydrolytic activity.[140] To date. some dietary fibers have been reported to have prebiotic potential. All Rights Reserved. isomalto-.[137. breve and Bif. lactis transiently supplanted a portion of the indigenous Bif.[142] The resulting enzyme had highly efficient galactosyltransferase activity and was able to channel carbon to galactooligosaccharide synthesis even at relatively low lactose concentrations. Advances in functional genomics and investigation of in situ mRNA expression may provide insights into the activities of bifidobacteria within the intestinal tract.138] The unfolding genomics/proteomics revolution is set to dramatically accelerate our understanding of host-microbe interactions and provide new insights into probiotic mechanisms. often in synbiotic combinations with bifidobacteria. their interaction with the host. However. The genomes of Bif. An example is the induction of intestinal mucin gene expression in the cell lines HT-29 and Caco-2 by lactobacilli and the subsequent suppression of pathogen adhesion to the intestinal epithelial cells. lactis does not significantly alter the intestinal bifidobacterial population of healthy adults in the long term. Bif. longum numbers while Bif.[136] Host responses to probiotics and their role in disease prevention are also being uncovered. which could suggest that Bif.[116] examined the same samples using PCR-ELISA and observed a decrease in Bif.[112] Bifidobacteria have themselves been exploited as a source of enzymes to synthesize NDOs with prebiotic effects. VII. and products containing both have been called “synbiotics”. and soybean oligosaccharides have also been shown to increase colonic bifidobacterial numbers. longum are now being sequenced.Hoang-Dung TRAN and friends Malinen et al.139] They are used.117] These large.[112.[135] and the genomics approach is beginning to yield information increasing our understanding of the physiology of these organisms.142] Metabolic engineering of a b-galactosidase from Bif. Inc.[112] Since they promote the growth of probiotic bacteria.[78. Copyright © 2004 by Marcel Dekker.[78] In addition to oligosaccharides. longum population. gluco-.[112. most critically.

Inc. and/or the immune system of the host. This figure displays the growth yield of Bifidobacterium lactis following growth using various mono.and host-derived complex carbohydrates and glycoproteins provides a competitive advantage. 3). VIII.) Copyright © 2004 by Marcel Dekker. .[136] Another consequence of their evolution in an environment with limited availability of simple sugars is that many Bifidobacterium strains grow poorly using some monosaccharides. HEALTH EFFECTS OF PROBIOTIC BIFIDOBACTERIA The theoretical benefits of probiotic bifidobacteria include both intestinal and systemic effects. Genomic analysis of Bif. studies of the probiotic effects of bifidobacteria have been focused in four major areas: Modulation of the host immune system Resistance to infectious diseases Control of inflammatory bowel disease (IBD) Prevention of colorectal cancer Although suffering from many limitations in design and control.Hoang-Dung TRAN and friends The ability of bifidobacteria to use a wide variety of oligosaccharides and other complex carbohydrates reflects their evolution in the hindgut of humans and animals where the ability to use a wide range of food. but grow well when supplied with oligosaccharides composed of those same sugars[39.117. Both therapeutic and prophylactic roles have been proposed and trailed in animal models and in humans. longum revealed a large number of proteins specialised for the catabolism of carbohydrates. the gut barrier. mediated by modulating the functionality of the intestinal microbiota. FOS ¼ fructo-oligosaccharides. All Rights Reserved. early studies at least provided indications that the theoretical benefits of consumption of probiotic bifidobacteria Figure 3 Bifidobacteria are often able to use oligosaccharides as carbon and energy sources more efficiently than the monosaccharides from which the oligosaccharides are composed. This suggests that bifidobacteria lack transport mechanisms for these simple sugars and import oligosaccharides before hydrolysing and metabolising them. (Modified from Ref.143] (Fig. XOS ¼ xylo-oligosaccharides. 39.and oligosaccharides. In the past few years.

but had different influences on serum transforming growth factor (TGF)-b (an anti-inflammatory cytokine) concentrations.147] It is becoming increasingly apparent that modulation of the host immune system may contribute to.[17. but decreased in the Bif. crossover of treatments. Amelioration of Allergy Symptoms The intestinal mucosal immune system in healthy individuals is able to create a balance between protective immunity against pathogens and tolerance to commensal bacteria and dietary antigens. Inc. placebo controls. supplementations with Lactobacillus rhamnosus GG[147] or Bif.150 – 152] The discovery of the importance of the intestinal microbiota in the development of oral tolerance fueled speculation into a possible role for probiotics in the treatment and prevention of atopic disease.144] Importantly.[17] Bifidobacteria have been shown to elicit a type 1 T-helper cell (Th1) response. lactis Bb-12[17. rhamnosus –treated atopic group.[119. A few studies have provided measurable improvements in clinical endpoints and a demonstrable amelioration of disease symptoms in the case of atopic eczema in infants.[145. the balance leading to tolerance is impaired and the immune system responds to the food antigen with an inflammatory response driven by a lymphocyte balance that is skewed towards type 2 T-helper cells (Th2).[118 – 125] including a reduced number and adult-like species composition of bifidobacteria in allergic infants.149. including randomization of subjects. Differences have been observed between the composition of the intestinal microbiota in tolerant and atopic infants. In recent years.125] In perhaps the best demonstrations. stability. many of the health effects attributed to probiotics. Both treatments resulted in deceased markers of inflammation and T-cell activation. blinding.119.[98.Hoang-Dung TRAN and friends could be translated into observable health effects in the host. Immunomodulation It is now well documented that probiotic bacteria can modulate both the humoral and cellular immunity of the host.18] Importantly.146] Both upregulation of immune protection against pathogens and cancer cells (Tables 2 and 3) and downregulation of aberrant inflammatory responses in allergy and IBD have been observed. an increasing number of studies have incorporated appropriate attention to the dose. we are now beginning to gain insights into some of the potential mechanisms of probiotic action by bifidobacteria. It has now been conclusively demonstrated that some Bifidobacterium strains can survive intestinal transit and persist transiently within the colon.114 – 116. .[118.150] It is now clear that the intestinal microbiota is essential for the development of oral tolerance in the neonate and that the type of bacteria present can influence the immune response and the development of immune homeostasis. the strains used as probiotics to date appear to be safe.[148] In atopic food allergy.118. lactis group. All Rights Reserved. so far. Serum TGF-b increased it in the L.46. or even underpin.[152] Bifidobacterium isolates from allergic infants generally failed to induce high levels of Copyright © 2004 by Marcel Dekker.25. 1. and meaningful health endpoints. and functionality of the fed probiotic and good clinical design. A. and the observed effect may be due to restoration of a tolerogenic Th1/Th2 balance mediated by interleukin (IL)-12[153] and possibly through induction of IL-10 –mediated downregulation of inflammation.[18.[17.18] proved effective in relieving atopic eczema in infants. The mechanism of action remains speculative and may be different for each strain.133. of clinically significant ameliorations of disease symptoms by probiotics.119.

Humans—healthy adults and elderly. double blind. Synbiotic effect: the magnitude of the immune stimulation was larger in the group fed Bif. Milk placebo. " Mucosal and systemic immune responses. All Rights Reserved. Free cells and encapsulated. " NK cell cytotoxic activity. " Mucosal total IgA and IgA-secreting cells. Randomised. Bif. [58] . lactis in milk. lactis HN019 Bif. Effect of probiotic Transient " CD4þ. Milk placebo. [21. Bif. lactis HN019 Bif.22] [20] Bifidobacterium Bif.Hoang-Dung TRAN and friends Copyright © 2004 by Marcel Dekker. Transient " in phagocytic and tumor-cell killing activities. Milk placebo. Bif. Bif. Bif. bifidum antibody response. Ref. " Mitogeninduced T and B cell proliferation. CD25þ and NK cell proportions in blood. lactis HN019 Bif. Table 2 In Vivo Studies Demonstrating Stimulation of Innate and Adaptive Immunity by Probiotic Bifidobacteria Model system Humans—healthy elderly. Effect enhanced by encapsulation. " Phagocytic activity of PMBCs " Phagocytic activity of peritoneal macrophages and blood leucocytes. Mice. bifidum Bb-11 [19] [165] Mice. bifidum in PBS administered orogastrically. Immunomodulatory activity was cell-associated and not secreted. No induction of specific Bif. lactis þ galacto-oligosaccharides. lactis in milk. " Humoral response to oral and injected toxin antigen. Double blind. Transient " in phagocytic and NK cell activity in PBMCs. lactis in milk. " Total IgA and IgM synthesis by mesenteric lymph node and Peyer’s patch cells and " reactivity of these cells to TGF-b1 and IL-5. Inc. lactis in milk with or without oligosaccharides. " IFN-a from mitogen stimulated PMBCs. Humans—healthy elderly. Milk placebo. lactis HN019 Bif.

Bif. Daily oral probiotic supplementation vs. Pre-feeding of autolyzed and heat-treated probiotic prior to challenge. Control with no probiotic. placebo controlled. lactis HN019 Salmonella enterica serovar Typhimurium Bif. # Rotavirus and E. and Potential Links to Immunological Mechanisms Pathogen Influenza. " Pathogenspecific intestinal antibody levels.coli and rotavirus Bif. orally vaccinated mice without probiotic. " IgG to influenza. Twice daily dose for 12 weeks. " Intestinal and systemic rotavirus-specific IgA. Piglets. Indication of reduction in duration of diarrhea. coli and rotavirus shedding. # Pathogen translocation to liver and spleen. Probiotics with or without prebiotics. " Protection against E. # Rotavirus shedding. breve YIT 4064 Bif. Effect of probiotic " Protection against influenza infection. Naturally acquired infections. infantis Rotavirus Rotavirus Infants in an institution. Daily consumption or probiotic for 28 days. Probiotic supplementation for 7 days prior to and after oral E. Model Mice. coli 0157:H7 Bif. " Feed conversion efficiency. bifidum and Bif. Mice.and post-challenge administration of the probiotic. coli. Inc.Hoang-Dung TRAN and friends Copyright © 2004 by Marcel Dekker. lactis HN019 E. No " in effectiveness by prebiotics. Table 3 In Vivo Studies Demonstrating Protection Against Infections by Probiotic Bifidobacteria. coli translocation. " Protection against low and high pathogen dose. coli challenge. Mice. Control. Intranasal challenge following oral vaccination. Mice. Low and high pathogen dose. " Pathogen-specific intestinal and systemic antibody levels. Single and multiple oral pathogen challenge. breve YIT 4064 Bif.coli-specific sIgA. [167] Bifidobacterium Bif. Pre. # Diarrhea. lactis Bb-12 Diarrhea in children Healthy children. Monitoring of E. All Rights Reserved. Non-infected control. " Blood leucocyte phagocytic activity and T cell proliferative response. Ref. # Morbidity and symptoms. which was low in both treatment and control groups. " Intestinal rotavirus-specific IgA " Delay in onset. no probiotic control. coli shedding. Placebo. " Blood leucocyte phagocytic activity and T cell proliferative response. No impact on incidence. and # severity of symptoms. [162] [166] [170] [18] [164] [163] . lactis HN019 E. Double-blind. " Phagocytic activity and E. # E. # duration.

none of the Bif. The ability of probiotic bacteria to modulate humoral and cellular immunity at the mucosal and systemic level has been established in animal and human studies.[18] This may have also been due to adult milk hypersensitivity predominantly being driven by mechanisms other than IgE-mediated reactions with Th2-skewed immunity. The immune-modulation effects that have been observed for bifidobacteria include: Increased mucosal IgA production[58. Increasing Resistance to Infections Evidence that probiotics can increase resistance to intestinal pathogens continues to accumulate. Evidence of strain and species variability in immune effects is demonstrated by the strong differences in cytokine responses they elicit.[145. strains isolated from healthy infants did induce IL-10.161] Importantly. but rather transient modifications of immune responsiveness beneficial to the host.[154. Recent in vivo studies of immune stimulation by bifidobacteria are displayed in Table 2. Limited attempts have been undertaken to test probiotics in adults with milk allergy.[154. it appears that adhesion of bifidobacteria to intestinal epithelial cells does not induce undesirable inflammatory responses.21.22.165] Increased lymphocyte responsiveness to oral and systemic challenge antigen [145.[159] The role of the intestinal microbiota in immune development and the use of probiotics for the alleviation of allergy symptoms are now burgeoning areas of research. but without success. adolescentis strains. probiotic lactobacilli and bifidobacteria do not elicit inflammatory responses that could be harmful. . a species usually associated with adult intestinal microbiota but also found in atopic infants.145. The importance of probiotic adhesion to the intestinal mucosa in immune modulation is still poorly understood.166] Stimulation of natural killer (NK) cell activity[20 – 22.162 – 164] Stimulation of phagocytic activity of mononuclear cells[19.125] induced IL-10.162.[156 – 158] Indications of a clinical benefit in adults with allergic rhinitis have been observed in individuals fed probiotic yogurt containing a Bifidobacterium strain and Lactobacillus acidophilus.155] Interestingly. Inc.[154.155] Hence.160.[98. All Rights Reserved. In contrast. As mentioned previously. Importantly. This was linked to an increase in interferon (IFN)-g secretion by peripheral blood mononuclear cells in the yogurt group. the adult microbiota is relatively stable and probiotics may not be capable of significantly impacting the population dynamics to a degree necessary to correct immune deviation in a mature immune system. 2.Hoang-Dung TRAN and friends IL-10 when used to stimulate macrophages in vitro. Stimulation of Innate Immunity Stimulation of the immune system provides the host with increased ability to fight infections and cancer. providing indications of an increased Th1 response.165.155] The window of opportunity for intervening in atopy development though probiotic supplementation may be limited to very early childhood when the immune system and microbiota are still maturing.148.167] In vitro studies have provided further insights into immunomodulation by bifidobacteria. careful screening and selection of the most appropriate strains to elicit the desired type of immune response for the targeted health effect is essential.[168] B. with the best protective effects seen to date in human trials for children Copyright © 2004 by Marcel Dekker.

Hoang-Dung TRAN and friends with rotavirus diarrhea. The probiotic preparation showed a clear benefit. Maintaining Remission in Inflammatory Bowel Disease IBD is a group of disorders characterized by chronic. with only 15% of the probiotic group relapsing compared to 100% in the control group ( p .[186] A genetic predisposition is recognized.[127] The dominant theory.166. which contained three strains of bifidobacteria in addition to lactobacilli and Streptococcus thermophilus. is that the inflammation results from a breakdown in normal immune tolerance to components (so far unidentified) of the intestinal microbiota. However.183 – 185] Binding or inactivation of pathogen toxins[171] Recent in vivo studies attempting to link increased resistance to infections to probiotic mechanisms are shown for immune modulation in Table 3 and pathogen inhibition and colonization resistance in Table 4. a bifidobacteria-fermented milk has now been trailed individually in ulcerative colitis patients with positive effects in a randomized. and those with preliminary evidence from in vitro and animal studies include: Stimulation of the host immune system[163. supported by experimental and clinical evidence. and pouchitis.179 – 182] Suppression of pathogen translocation or cell invasion[176. controlled trial. It is impossible to isolate the role that the bifidobacteria played in this effect.[169] The majority of human probiotic studies conducted so far have tested the effects of probiotic lactobacilli. Human studies investigating the application of probiotic bifidobacteria against rotavirus diarrhea in young children have provided indications that some strains may be protective. and since probiotics can modulate both the intestinal immune system and the intestinal microbiota.[18. but the etiology remains unknown. a number of recent trials in animal pathogen challenge models and in humans indicate that probiotic bifidobacteria may also afford some protection against enteropathogens (Tables 3 and 4).001). Conclusive evidence of clinically significant protection by bifidobacteria to bacterial pathogens remains to be demonstrated in humans.167. Several potential mechanisms have been proposed for improved resistance to infections. Inc. was trailed in a randomized.[24] Patients in clinically demonstrated remission were treated with the probiotic or placebo for a period of 9 months and were regularly clinically assessed. It seems likely that selected Bifidobacterium strains will prove able to provide clinically significant benefits in the maintenance of remission. C. Bacteriological analysis demonstrated recovery of the probiotics in feces of the treatment group. relapsing intestinal inflammation that include Crohn’s disease.164] Results from rodent pathogen challenge models are encouraging. However.186] Current steroidal therapies often elicit significant side effects. their application in control of these diseases has been investigated. double-blind. A probiotic mixture (VSL#3). ulcerative colitis. though they are only loose models of human infections. Copyright © 2004 by Marcel Dekker. placebo-controlled trial of 40 patients with chronic pouchitis.[127.[26] No adverse effects were observed in these trials indicating that bifidobacteria can be safely used in IBD. 0.170] Induction of nonimmune host responses such as increasing intestinal mucus production[138] or reducing toxin receptor expression[171] Inhibition of pathogens by specific antimicrobials[172 – 176] Inhibition of pathogens by metabolic end products[176 – 178] Competitive exclusion and inhibition of pathogen mucosal adhesion[176. . All Rights Reserved.

Treatment group dosed intragastrically with Bif. DVS [183] . Table 4 Resistance In Vivo Studies Demonstrating Protection Against Infections by Probiotic Bifidobacteria. enterica in vitro by lipophilic extracellular molecule(s) .LaftiTM B22. All Rights Reserved. Mice—oral pathogen challenge post colonization with Bif. longum supernatant before and after challenge. " Survival. B74. spp.3500Da produced by the bifidobacteria. Correlation of protection to " organic acid synthesis and # intestinal pH. controls of E. infantis. (CA1 and F9) isolated from infant stools. NE rat model. breve. Bif. Inoculated with bifidobacteria 1 week before oral pathogen challenge. coli. # Morbidity and pathology in both models. coli 0157:H7 vero cyclotoxin Model Mice—oral challenge with E. [171] Bifidobacterium Bif. Salmonella enterica serovar Typhimurium Salmonella enterica serovar Typhimurium [174] [173] Bif. Preand post-challenge probiotic administration. Control—no probiotic. Mice—oral pathogen challenge and simultaneous and post challenge administration of probiotics. Control—no supernatant. Inc. infantis Necrotizing enterocolitis (NE) Salmonella enterica serovar Typhimurium [182] Bif. Control. Conventional and gnotobiotic mice orogastric pathogen challenge. B97. spp. animalis and Bif. Probiotic vs. # Pathogen colonization and complete block of translocation. longum HY8001 (extracellular toxin binding molecule) Bif. Antagonism of S. bifidum. No difference in intestinal colonization of pathogen between treatment and control groups. Germ-free mice. breve (Yakult) Salmonella enterica serovar Typhimurium [178] Bif. # Intestinal colonization by pathogen and # symptoms of infection. and Potential Links to Pathogen Inhibition or Colonization Pathogen E. Outcome of probiotic Inhibition of toxin binding to receptor by direct binding to toxin by a soluble factor produced by the probiotic and reduced expression of the toxin receptor in renal cells of the mice.Hoang-Dung TRAN and friends Copyright © 2004 by Marcel Dekker. Colonization by Bif. coli fed and saline fed animals. # NE incidence and symptom severity. # Morbidity. Effect " in synbiotic. Synbiotic galacto-oligosaccharide. Ref.

[20 – 22.[192.190] while others were neutral.Hoang-Dung TRAN and friends D. Possible mechanisms by which probiotics may suppress cancer development are discussed by Reddy[187] and Rafter.[195] Yazawa et al. animal. It has been observed that bifidobacteria injected intravenously in mice selectively germinate and grow in the hypoxic regions of the tumors and not in other tissues. When injected intravenously into mice with solid tumors.[196] demonstrated that a strain of Bif. the enzyme was expressed only within the tumor. and human studies that bifidobacteria may contribute to maintaining a colonic environment and host immune system oriented to reduced cancer risk. exploiting these organisms’ physiology. and inhibited tumor growth.[189. adolescentis strain engineered to produce an antiangiogenic drug. expressed the anticancer drug. E.[191] A number of studies have investigated the suppressive effects of bifidobacteria on tumorigenesis using carcinogen-induced rodent colon cancer models. Copyright © 2004 by Marcel Dekker. Some demonstrated strong suppressive effects. To date. All Rights Reserved. However. A follow-up study showed that when the strain was genetically modified to synthesize an enzyme able to activate an antitumor drug.165] However. Anticancer Effects Anticancer effects remain perhaps the most controversial potential health benefit of probiotic bifidobacteria. Reduced fecal mutagenicity has been demonstrated in rodent models and in humans fed bifidobacteria. Genetically Modified Bifidobacteria in Anticancer Gene Therapy Though not a probiotic effect. with varying results.[187.[198] This represents an exciting new application of bifidobacteria.[188] and include: Stimulation of the host’s innate immune system Limitation of genotoxic reactions by the intestinal microbiota Alteration of physicochemical conditions in the colon Adsorption or degradation of potential carcinogens Nourishment of the intestinal epithelium with macro and micronutrients Production of antitumorigenic or antimutagenic compounds Stimulation of tumoricidal activity has been observed in ex vivo examination of NK cells from humans fed with probiotic bifidobacteria.190] and mutagen binding to Bifidobacterium cells has been observed in vitro.194] Further research is required to link purported mechanisms of probiotic action to cancer-suppressing effects in vivo. longum injected intravenously into a rodent breast cancer model specifically localized in the tumors. . The oxygen partial pressure is lower in these tumors than in normal tissues.[192 – 194] These variations were possibly due to differences in the Bifidobacterium strain and/or the models used. Inc. Applying prebiotics in combination with bifidobacteria (synbiotics) appears to enhance anticancer effects in the rodent models.[197] A separate group demonstrated similar results with a Bif. an interesting new application of bifidobacteria is their potential use as targeted vectors to deliver gene therapy to solid hypoxic tumors. there is no direct experimental evidence for cancer suppression in humans as a result of probiotic consumption. and amenability to genetic transformation to provide vectors for targeted gene therapy. there are indications from in vitro. safety. direct links between Bifidobacterium-induced immune stimulation and cancer suppression in humans are yet to be demonstrated. the bifidobacteria selectively colonized in the tumors.

Balmer. Y. CONCLUSIONS It is now clear that strains of probiotic bifidobacteria can survive passage through the upper gastrointestinal tract and transiently colonize the colon. M. T. Microbiol. Mitsuoka. the pace of scientific research into the roles of bifidobacteria in human health will no doubt accelerate. It is becoming apparent that the age of the host. Benno. M. 52. 1984. 3 – 24. and potential applications. Y. Evol. The role of the microbiota in healthy immune development in infants and possible effects on allergy will be a major focus for research into probiotic bifidobacteria in the short term. .Hoang-Dung TRAN and friends IX. T. of bifidobacteria in human health... B. 44. Bifidobacterium lactis Meile et al. Cai. Copyright © 2004 by Marcel Dekker.E. Syst. 4. Int. Sawada. Wharton. K. Marked progress has been made in our understanding of the population dynamics of bifidobacteria.. 28. 815 – 820. 2002. M. Child. stability of the microbiota. Microbiol. However. Y. Microbiol. we are still only at the very beginning of developing an appreciation of the functional relationships between the microbiota and the host in health and disease.. Benno. Some advances have been made towards elucidating possible mechanisms of probiotic action. Perhaps lagging in comparison to the pace of advances in other areas of probiotic research are technological solutions to maintaining the stable functionality of environmentally sensitive strains. and with the burgeoning development of new molecular technologies. S. facilitated by the shift away from culture-based. Matsumoto. The best demonstrations of health benefits thus far reported are for treatment of eczema in allergic infants and for the maintenance of remission in IBD. Inc. Hayashi. M.. Diet and faecal flora in the newborn: breast milk and infant formula. Y. Arch. Evidence continues to accumulate that certain strains can provide measurable and clinically relevant benefits to human health. and modulation of the host immune system is emerging as a key element in many of the beneficial effects seen to date. 5. phenotypic methods towards culture-independent. 1. 1672– 1677.. 2000.. The intestinal microflora of infants: composition of fecal flora in breast-fed and bottle-fed infants. Bifidobacteria Microflora 1982. Benno. Immunol. 3. REFERENCES Mitsuoka. H. 1. including nonrefrigerated. Dis. 1990. Importantly. Immunol.. An understanding of these relationships is essential to elucidating probiotic mechanisms and is needed to allow scientifically based selection of appropriate probiotic stains.M. 2. The application of high-throughput functional genomics to the study of host-microbe interactions promises to provide dramatic advances in our understanding of the role. 64. Sakamoto. and maturity of the immune system may have a dramatic influence on some probiotic effects. Kitahara. All Rights Reserved. Fukuyama. Unification of Bifidobacterium infantis and Bifidobacterium suis as Bifidobacterium longum. 1945– 1951.. uncovering mechanisms of action will add scientific weight to the observed clinical effects. 975 – 986. Technologies are needed to broaden the application of these functional ingredients to a wider range of products. long shelf-life foods. S. molecular techniques..A. Recent trends in research on intestinal flora. The once theoretical positive impacts on health for probiotics are beginning to be backed by hard scientific data. J. 1997 is a subjective synonym of Bifidobacterium animalis (Mitsuoka 1969) Scardovi and Trovatelli 1974. The application of bifidobacteria in functional foods remains largely limited to fermented dairy products. Sakata.

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nonsporing. P. freudenreichii freudenreichii subsp. OUWEHAND University of Turku. Although some strains may be relatively aerotolerant. avidum P. 159 Copyright © 2004 by Marcel Dekker. Inc. shermanii jensenii thoenii microaerophilum The species formerly known as P. inoccuum has been reclassified as Propioniferax innocua. and CO2 as their main fermentation products. P. P. acetic acid. nonmotile pleomorphic rods. acnes P. All Rights Reserved. lymphophilum P. acidipropionici australiense cyclohexanicum freudenreichii subsp. Their optimal growth temperature is between 30 and 378C. PAB were first described by Freudenreich and Jensen in 1906 and given the name Propionibacterium by Jensen in 1909. they are basically anaerobes that produce propionic acid. The main habitat of the classical PAB are dairy Table 1 Two Groups of Propionibacterium Species Cutanous strains P. P. .[1] PAB are divided into two principal groups: the classical or dairy PAB and the cutaneous PAB (Table 1). P. P.Hoang-Dung TRAN and friends 4 The Probiotic Potential of Propionibacteria ARTHUR C. granulosum P. Turku. Finland I. propionicus Classical (dairy) strains P. P. INTRODUCTION Propionic acid bacteria (PAB) are characterized as gram-positive.

they have also been isolated from rumen contents.Hoang-Dung TRAN and friends products. it appears that the classical and dairy PAB are also phylogenetically distinct. The species commonly included in dairy products are P. III. PAB play an important role in the production of flavor compounds and the ripening of cheese. and pigs.[6] During the maturation of the cheese. However. thoenii. Freudenreich and Jensen[5] first described propionibacteria when studying propionic acid fermentaion in Emmental cheese. in particular Swiss-type cheeses. and both subspecies of P. chickens. PROPIONIC ACID BACTERIA AS DAIRY STARTERS PAB are commonly used as starter cultures in the dairy industry. This proteolytic activity leads to an increase in free proline. METABOLISM The major means of energy generation of PAB is the production of CO2.3] II. Copyright © 2004 by Marcel Dekker. where they are part of the normal microbiota. They have also been isolated from the feces of humans. acidipropionici. P. freudenreichii. propionic acid. Based on 16S rRNA studies. but also by oxidative phosphorylation. These can be synthesized by the PAB and explains why they are rich sources of these vitamins. All Rights Reserved. PAB also have the ability to grow on lactate under anaerobic conditions. and spoiled orange juice.[2. jensenii. biotin and vitamin B12 are required. spoiled olives. The ATP is generated not only through substrate phosphorylation. The main habitat of the cutaneous PAB is the human skin. which contributes to the flavour of PAB-containing cheese. a property that is often used for the selective cultivation of PAB: Classical pathway: 3 lactate À 2 propionate þ acetate þ CO2 þ H2 O þ ATP ! Formation of succinate by CO2 fixation: 3 lactate À (2Àx) propionate þ acetate þ (1 À x) CO2 ! þ x succinate þ 3 ATP Aspartate as electron acceptor[4]: 3 lactate þ 6 aspartate À 3 acetate þ 3 CO2 þ 6 succinate ! þ 6 NH3 þ 3 ATP (3) (2) (4) For the propionic acid fermentation. Cutaneaus PAB have occasionally also been isolated from pathological material. . Inc. P. Dairy PAB strains are autolytic under the conditions found in cheese and degrade peptides and amino acids present in the cheese. and acetic acid through fermentation: 3 glucose À 4 propionate þ 2 acetate þ 2CO2 ! (1) This yields 4 ATP per glucose molecule and is thereby more energy efficient than lactic or acetic acid fermentation. the PAB will utilize the lactate that is formed by other starter bacteria present in the cheese.

and P. one of the typical flavor components of Swiss-type cheeses. acidipropionici or P. Milk whey has been suggested as an inexpensive growth medium for the production of propionic acid by P.[8] PAB may also produce exopolysaccharides in milk. while only 6 out of 52 tested P. When a large panel of indicator strains was used. Leversee and Glatz[14] observed that only 4 out of 52 PAB strains tested produced bacteriocins. B. jensenii. the low concentrations of the acids in the broth and the long incubation time make production and recovery too expensive. P. Commercial production of propionic acid from the fermentation of sugars by species of PAB has received considerable attention over the years. e. Propionic acid inhibits the growth of microbes through its accumulation within the cell and subsequent inhibition of enzyme activity. Holo and coworkers[15] observed that the majority of P. it has been observed that mild proteolysis of the bacteriocin produced Copyright © 2004 by Marcel Dekker. However. few bacteriocins from this genus have been described in detail (Table 2).[7] The utilization of lactate will also lead to the formation of propionic acid. The prevalence of bacteriocin production may be underestimated when a small number of indicator strains is used. homofermentative Lactobacillus species. which forms the eyes in the cheese. Propionic acid bacteria have been added to food in order to function as a preservative. acidipropionici.[10] Propionic acid fermentations may be improved through the combination with lactate-producing organisms.[12] It is. plasmid encoded together with immunity factor.[11] However. or to strengthen the barrier function of the normal intestinal microbiota with bacteriocin-producing probiotics. almost all strains of PAB exhibited antimicrobial activity against at least one of the tested strains. which originate from amino acid catabolism. Bacteriocins Bacteriocins have been defined as proteinaceous substances with a narrow inhibitory spectrum. uncertain if this type of propionic acid production can compete with the petrochemical production through oxidation of liquid phase propane or propanal. Alternative extractants like quarternary amines may further improve the removal of propionic acid from broth. Inc. freudenreichii exhibited such activity. All Rights Reserved.[13] However. however. A. Reports on the prevalence of bacteriocin production by PAB are variable. thoenii produced bacteriocin-like activity. but the solvents are in general toxic to the producing cells.[9] IV. many bacteriocins exhibit a rather wide spectrum of inhibition. . Separating the solvent phase from the growth broth by a membrane may eliminate this problem. freudenreichii.Hoang-Dung TRAN and friends As outlined above. this will lead to the production of CO2. as natural preservatives in foods. Continuous extraction of the acids with solvents has been attempted. This makes them of particular interest for potential practical applications. This may have important practical implications for modifying the rheological properties of certain fermented dairy products containing PAB or for the production of exopolysaccharide-based thickeners produced by food-grade microbes.[16] Despite the apparent prevalence of bacteriocinogenic strains of PAB.g. propionic acid is inhibitory to certain bacteria and fungi at low pH. Other important flavor compounds include branched-chain fatty acids. ANTIMICROBIAL ACTIVITY OF PROPIONIC ACID BACTERIA Propionic Acid Like most other organic acids. Although bacteriocins by definition are proteinaceous and thus are sensitive to proteolytic degradation.

[17] This may provide a new type of antimicrobial activity.300 13. no further investigations have been reported on this subject. acnes P. jensenii B1264 800 – 1200 Molecular weight (Da) Activity against Cutaneous propionic acid bacteria Gram-negative bacteria and yeasts Propionic and lactic acid bacteria Cutaneous propionic acid bacteria. jensenii LMG 3032 P.. jensenii DF1 P. mold. Even after this long incubation period. lactic and propionic acid bacteria 89 16 by P. freudenreichii and suggested to be of proteinaceous nature[20] with a molecular weight of 1000 to 1500 Da. thoenii P127 6383 87 9328 14 Propionicin T1 Propionicin SM1 Propionicin SM2 P. some gramnegative. Inc. since these early reports. lactic acid bacteria Propionic acid bacteria Ref.[18] This may relate to the fact that production in liquid media is in general poor compared to solid media and culture conditions need to be very carefully optimized to obtain higher yields.Hoang-Dung TRAN and friends Table 2 Bacteriocin Acnecin Bacteriocins Produced by Propionic Acid Bacteria Producing strain P. gram-positive and -negative Propionibacteria. where activation takes place first in an environment with proteolytic activity. thoenii LMG 2792 P.[19] These difficulties in the production are likely to limit the possibilities for practical applications of the bacteriocins of PAB. However. jensenii DF1 7130 88 22. It may require 3 days for propionicin T1 or 7 days or more for jenseniin G and propionicin PLG-1.600 Yeast. To what extent bacteriocins are produced in situ by PAB in a food matrix or in the intestine is currently unknown.[21] However. in general the formation of bacteriocins by PAB is slow. 85 86 Jenseniin G Jenseniin P . jensenii B1264 increases its inhibitory activity and its inhibitory spectrum. Antiviral Activity PAB have also been observed to produce antiviral substances.g. .000 18 29 Protease-activated antimicrobial peptide (PAMP) Propionicin PLG-1 P. Copyright © 2004 by Marcel Dekker. mold. lactobacilli Yeast. shermanii ATCC 9616 P. propionins. e.000 3000– 10.12. freudenreichii subs. The potential application of bacteriocins or bacteriocinogenic strains is great. the concentration of bacteriocins may be very low and antimicrobial activity needs to be concentrated in order to be detectable. These substances were found in the cell-free extracts of P. thoenii 419 P. the gastrointestinal tract. All Rights Reserved. jensenii P126 P. C.

Whether this actually will work in vivo remains. probably through antagonistic acitivities. However. freudenreichii subsp. however. molds. among others.g.[26] PAB have also been suggested for the preservation of meat products.[23] but may be added to aid in the inhibition of spoilage organisms. It is used to preserve dairy products such as cottage cheese and inhibits the growth of some molds and gram-negative bacteria. which may benefit animal husbandry. combinations with lactobacilli. shermanii. In addition to the use of pure cultures of PAB. The silage also has a better aerobic stability after opening.[27] PAB have been successfully applied in silage to prevent the growth of aerobic bacteria or yeasts and molds. are currently not known. have been observed to produce good quality silages. jensenii B1264 produces a bacteriocin (Jenseniin P) (see Table 2) that is active against. to the presence of propionic acid and acetic acid. E283. An interesting potential application of PAB bacteriocins has been suggested by. combinations of lactobacilli and PAB have also been observed to perform less then either strain alone.[24] The antimicrobial activity of Microgard is attributed to a 700 Da heat-stable peptide and. and calcium propionate (E281. It is effective in suppressing the outgrowth of spoilage organisms in dairy products and sourdough bread. Copyright © 2004 by Marcel Dekker. to be determined. Al-Zoreky and coworkers claimed that the inhibitory activity can be increased by varying the fermentation conditions under which Microgard is produced. The other commercial product using PAB for preservation is BioprofitTM.[28] This clearly indicates that strain combinations should be carefully assessed. The active components in Bioprofit. Inc. MicrogardTM is a skim milk product pasteurized after fermentation with P. but did not provide any details. and propionic acids.[25] Microgard is mainly marketed in the United States and Canada. V.[22] Propionic acid (E280) and sodium. which is mainly used in Europe. lactic. shermanii JS and Lactobacillus rhamnosus LC-705. acnes and could thus be used in the treatment of acne vulgaris. solely on the production of propionic acid to replace chemically manufactured propionate. E282. The combined effect of the two strains against spoilage yeast. Applications of PAB as Preservatives Propionate is commonly used in the manufacture of bread to inhibit spoilage organisms. Bioprofit consists of a combination of P. Such applications relied. was stronger than the effect of either culture alone.. freudenreichii subsp. This results in a better quality silage with a higher nutritional value. All Rights Reserved. The addition of PAB prolongs the storage life of the silage through the production of propionic acid and by lowering the pH. but not gram-positive bacteria. It is a much more powerful inhibitor of yeasts and molds than. PROPIONIC ACID BACTERIA AS PROBIOTICS The literature concerning the potential probiotic properties of PAB is very limited compared to that about lactobacilli and bifidobacteria. in recent years an increasing number of reports on the potential health benefits of PAB have been published. respectively) are therefore allowed as preservatives in most countries in the world. e. other than acetic. acetate or lactate. to a lesser extent. and Bacillus ssp. . potassium. Two products on the market exploit PAB and/or their metabolites for preservation purposes. This was suggested to be due to a synergistic effect of the different metabolites of the culture combination.[29] P. Propionic acid bacteria are not normally found in sourdough breads. The use of propionates will retard mold growth by several days. such as Bioprofit. P.Hoang-Dung TRAN and friends D.[26] However. thereby extending the shelf life.

Bile-sensitive strains of PAB were also permeabilized. the results need to be confirmed in vivo. this can be counteracted through the presence of milk or cheese. Copyright © 2004 by Marcel Dekker. Strains of PAB have also been observed to resist exposure to bile salts. which was subsequently hydrolyzed. Inc. such as L. pH 5). as these usually belong to the group of cutaneous PAB. Adhesion of four PAB to human intestinal mucus was observed to be low and mainly due to nonspecific interactions with the mucus. Classical PAB are nonpathogenic and would be well suited to compete with the cutaneous PAB and other skin microbes. Several PAB strains have been observed to survive exposure to low pH.[43] Although in vitro adhesion assays are useful screening tools.Hoang-Dung TRAN and friends It is still uncertain if PAB represent an important fraction of the intestinal microflora. in the case of PAB it may be advisable not to use intestinal isolates. This can be expected to contribute to a relief of lactose intolerance symptoms.[39] but were not observed to persist in the human intestine after feeding was stopped. acidipropionici CRL1198 were found to excrete PAB at levels of 108 colony-forming units (CFU)/g one week after cessation of PAB feeding. This may allow a probiotic organism to persist longer in the gastrointestinal tract to contribute to the competitive exclusion of pathogens and to modulate the immune response.35] Although incubation at low pH can affect the viability and the enzyme activity of PAB to a varying extent. rhamnosus GG or B.[42] This adhesion was reduced by 24% through prior adhesion of L.. the main protein in skin. Even exposure to other nonlethal stresses may provide protection against low pH.[44] Strain P. . which earlier had been observed to persist for at least a week. All Rights Reserved.[38] and their contribution to lactose digestion may therefore be more limited. freudenreichii ssp. rhamnosus GG.[40] Another important selection criterion for probiotics is their ability to adhere to the intestinal mucosa. some strains were apparently permeabilized by it.g. It was observed that P. lactis Bb12. The skin has been suggested as a novel area for probiotic use of PAB. rhamnosus LC-705. This group is associated with disease (see below) and is therefore not appropriate. PAB have been observed to survive gastrointestinal transit in relatively high levels. These observations suggest that certain combinations of PAB and other probiotics may be synergistic. but their b-galactosidase activity decreased during prolonged exposure to bile. exhibited the highest adhesion both in vitro and in vivo.[31 – 33] Although potential probiotics often originate from the intestine. while others may be antagonistic.[32] Mice fed P. Good correlation between in vitro adhesion and adhesion in mice for six strains of PAB has been observed.[34. Survival of gastrointestinal transit is an important selection criterion for probiotics. which simulates passage through the stomach. some strains of classical PAB have been observed to adhere well to keratin. allowing more lactose to enter the cell. indicating a competition for binding sites. Some reports describe the detection of relatively high levels of mainly cutaneous PAB.[30] while others report low or nondetectable levels of PAB. In addition. shermanii JS adhered to Caco-2 enterocyte-like tissue culture cells at levels similar to L.[41] Some PAB have been assessed for their ability to adhere to intestinal mucosal models.to threefold through prior adhesion of other probiotics. this adhesion could be increased two. the classical PAB have a number of properties that make them good probiotic candidates.[35] Despite their resistance to bile. However. indicating that PAB present in a food matrix may exhibit better survival. a probiotic strain known to adhere well to the intestinal mucusoa and intestinal mucosal models.[37] How this preadaptation can be maintained in a product to facilitate better survival of gastric transit remains to be determined. Exposure to bile also stimulated the production of b-galactosidase.[36] The ability of PAB to survive low pH can also be significantly improved by a short exposure to a nonlethal pH (e. acidipropionici CRL1198.[41] Interestingly. further increasing the activity.

[45] They observed that PAB and several intestinal bacteria. PAB appear to fulfill the major selection criteria for probiotics and may therefore have a potential to be used as such. L. A. Bougle and coworkers[32] observed that feeding 5 Â 1010 CFU of P. At that time.[48] Although bifidobacteria are considered to be the main beneficial members of the intestinal microflora.Hoang-Dung TRAN and friends Thus.4-naphthoquinone (MW 217 Da). such as Bacteroides. B. freudenreichii DPC 3801 in milk. However. This may be due to the production of lactic acid from hexoses that cannot be utilized by PAB. a mere increase in their numbers does not necessarily contribute to a beneficial health effect. shermanii has been observed. C. such as a reduction in the level of coliforms. Also. 15 strains of PAB were observed to inhibit the growth of some bifidobacteria. . and it is therefore unlikely that this increase is due to the previously consumed PAB. freudenreichii ssp. penta-. released growth-stimulating factors for bifidobacteria in the growth medium.[50] This could indicate that dairy products that contain growth-promoting peptides for PAB will retain their functionality and may be able to stimulate PAB growth in the intestine. which are produced by cell wall – associated proteinases of L. However. other mechanisms may also be involved. Inc. helveticus strains were found to be growth stimulating. helveticus DPC 4571 stimulated the growth of P. fulfilling probiotic selection criteria does not guarantee probiotic efficacy. The stimulatory activity was purified and identified as 2-amino-3-carboxy-1. an increase in the level of fecal bifidobacteria in children upon consumption of P. This will have to be assessed in vivo in the target host. peptides from casein produced by other L. Changes in Intestinal Microflora Composition Feeding of PAB to mice has been observed to cause changes in the composition of the fecal microflora. Stimulation of Propionic Acid Bacteria Growth by Lactobacilli Selected lactobacilli have been observed to stimulate the growth of propionic acid bacteria. It is not certain if ´ these bifidogenic properties of selected PAB strains are expressed in vivo. The produced lactic acid will subsequently serve as a carbon and energy source for the PAB. However.[47] Other workers have observed that PAB stimulation of the growth of bifidobacteria appears to be a relatively common phenomenon. Warminska-Radyko and coworkers[35] observed that all 27 PAB tested stimulated the growth of at least some bifidobacteria. although this would need to be assessed in vivo. no PAB were detected in the feces. The specific health benefits of increased fecal bifidobacteria levels will need to be determined. and in particular propionic acid.[51] Sidorchuk and Copyright © 2004 by Marcel Dekker. and Enterococcus. All Rights Reserved. helveticus DPC 4571. and hexa-peptides from casein. Stimulation of Bifidobacterium Growth Organic acids. However. This appeared to be due to the production of tetra-. However. Enterobacter. freudenreichii SI26 and SI 41 daily increased the fecal Bifidobacterium one week after stopping the consumption of PAB. casein-derived peptides are apparently able to survive digestion since selected peptides retain their ability to lower blood pressure upon consumption.[46] The ability of PAB to stimulate the growth of bifidobacteria was first observed by Kaneko and co-workers.[49] The health implications of this are uncertain. have been observed to stimulate the growth of bifidobacteria.[45] Propionate is therefore also often added to media for the selective enumeration of bifidobacteria.

though it has been hypothesized to relate to the absorption of cholesterol to the bacteria whereby the cholesterol would be excreted from the body without being reabsorbed.Hoang-Dung TRAN and friends Bondarenko[48] observed an increase in fecal bifidobacteria and lactobacilli and a decrease in enterobacteria and staphylococci in children with microflora abnormalities. Bacterial enzymes involved in the production of these harmful substances are azoreductase. However. D. Growing cells Copyright © 2004 by Marcel Dekker. P. E. carcinogens. in combination with L. shermanii JS and L. with conflicting results. Acetate. and b-glycosidase.[53] Although the observations that PAB can reduce fecal enzyme activity and remove aflatoxins are promising. Cholesterol-Lowering Effects P. Whether this will also contribute to a protection against pathogens remains to be determined. may in that way contribute to lowering of serum cholesterol. In a screening it was observed that three out of the six tested PAB were able to form CLA.[55 – 57] CLA consists of a mixture of isomers of octadecadienoic acids with conjugated double bond of which the cis-9. It therefore remains to be determined whether PAB will perform better. The ability of certain probiotics to reduce the level of these enzymes in feces is considered to be a desirable trait. Inc. F. All Rights Reserved. it remains to be determined whether this will actually lead to a reduced risk for cancer in humans.[51] The activity of other fecal enzymes was not changed upon feeding either of the five tested PAB. shermanii JS. acidipropionici has been shown to reduce serum cholesterol levels in mice. acidipropionici CRL1198 reduced the fecal b-glucuronidase in mice. which could be counteracted by the presence of propionate.[58] CLA is mainly produced by growing cells. and tumor promoters from dietary and endogenously produced precursors. dairy products and beef are the main source. It was observed that P. PAB have been observed to be able to form CLA from LA.trans-11 configuration is considered to have highest biological activity. b-glucuronidase. which is produced by bacterial fermentation. rhamnosus LC-705 was found to cause a reduction in the level of fecal azoreductase in elderly subjects.[56] Because CLA is formed in the rumen by microbes such as Butyrovibrio fibrisolvens through conversion of linoleic acid (LA).[54] PAB. freudenreichii ssp. In addition to absorption or metabolism of cholesterol.[52] In addition to altering the fecal enzyme activity. Production of Conjugated Linoleic Acid and Other Biologically Active Components Conjugated linoleic acid (CLA) has attracted extensive interest because of its potential beneficial physiological effects and anticarcinogenic and antiatherosclerotic properties. freudenreichii ssp. nitroreductase. while none of the tested lactobacilli or lactococci were able to do so. Fecal Enzyme Activity The intestinal microflora is able to generate mutagens. The reduction of serum cholesterol levels has been assessed for many probiotics. nitrate reductase. has been observed to cause an increase in serum cholesterol. but nongrowing cells are also able to convert LA to CLA. which are main propionate producers.[40] The mechanism by which this occurred is not known. Consumption of a combination of P. rhamnosus LC-705 has also been observed to be able to reduce the fecal level of the carcinogen aflatoxin B1 in volunteers. . the production of propionic acid may contribute to the lowering of serum cholesterol.

Inc.[63] However. coli O157:H7 in ruminants. depending on the microbiota present. PAB can accumulate varying amounts of trehalose. Trehalose is a low-energy sugar because it is only partially digested in the human gastrointestinal tract and is poorly metabolized by many microbes.[61] Although green vegetables are the main dietary source of folic acid. Tsuchida and coworkers[66] observed that injection of P. although the former will produce a larger fraction of the biologically important cis-9.Hoang-Dung TRAN and friends may accumulate 80 – 87% CLA of the initial LA present in the culture.[64] Because PAB are normal members of the rumen microflora. Mn. Pulverer and coworkers[67] showed that injection of P. Because some dairy starters produce folic acid. Another potential nutritional contribution of PAB is their mineral content. All Rights Reserved. Some dairy products are richer in CLA then others. it has to be determined if such CLA-enriched products indeed have additional health benefit over the traditional products. coli O157:H7. and Co. avidum into mice reduced the number of liver tumors induced Copyright © 2004 by Marcel Dekker. Indeed. It is not likely that PAB will produce appreciable amounts of CLA in situ in the human intestine due to low local levels of LA. since the biomass of PAB that is consumed is in general low. milk does contain reasonable amounts of folic acid (50 – 100 mg/L). In the formation of propionic acid. Immunomodulation Cutaneous PAB have been observed to modulate the immune response upon injection of whole cells or cell fragments in animals. . the precursor dimethylbenzimidazole can be added to the fermentation mixture.[65] it could be hypothesized that trehalose-excreting PAB would have a similar effect. coli. certain fermented dairy products like Brie and Camembert cheeses may contain 10 times higher levels of folate than milk. In response to external stresses. It is. It has been suggested that the isomerization is a detoxification reaction for LA.[60] To further increase the yield of the vitamin. This leads to a displacement of the latter by the commensal E.[61] Trehalose has been observed to be utilized by commensal Escherichia coli but not by enterohemorrhagic E. PAB use biotin as a cofactor in their propionic acid synthesis. however. In addition. the feeding trehalose has been observed to reduce the carrier level of E.[56] Inclusion of PAB may enrich these products with CLA. it remains to be determined whether this represents a significant source of these minerals. It can therefore be used as a dietetic sugar. and biotin. PAB have been found to contain relatively high amounts of Mg. although their inclusion should not affect the taste of the product negatively. acnes into mice activated macrophages and significantly reduced systemic metastasis formation after tumor transplantation. folic acid. not known how much their biotin content contributes to the dietary intake of this vitamin from foods containing PAB. Nor is it certain whether the biological production of CLA will be able to compete with the chemical production. trans-11 isomer.[58. G. This may provide an additional control method for this pathogen. Similarly. Feasible applications may be in the field of CLAenriched fermented dairy products. PAB use enzymes that contain several specific cofactors such as vitamin B12. PAB have therefore long been known to be efficient producers of these vitamins and are used to commercially produce vitamin B12.[62] It can be expected that PAB-containing cheeses are good sources of folate.59] The practical implications of this remain to be determined. The production of vitamin B12 is a two-stage process involving anaerobic growth followed by aerobic incubation in the latter phase of the production.

not the common method of application for probiotics and may provide a safety concern for applications in human medicine (see below). Oral administration of P. freudenreichiii ssp. granulosum KP-45 in patients with colorectal carcinoma resulted in a reduced number of postoperative wound infections and significantly increased the survival of patients treated for stage I and stage II colorectal cancer. All Rights Reserved.[72] In humans. Oral administration of P.Hoang-Dung TRAN and friends by RAW 117-H110 lymphosarcoma. The general contribution of PAB to protein digestion therefore remains to be determined. immune modulation after oral administration of PAB has been observed. injection of selected PAB appears to result in a potentially beneficial modulation of the immune response in animals and in humans. Treatment of the mice with P.[69] Injection of P.[73] The main immunomodulatory activity has been obtained through injection of cutaneous PAB. granulosum or its cell walls into mice prior to infection with herpes simplex. The recurrence rate was significantly less in the treatment group as compared to the control group after 76 months. shermanii JS to mice stimulated the proliferative activity of B and T lymphocytes. it is not certain whether proteolytic activity in the colon is a desirable trait. Interestingly.[30] However. Protease Activity Dairy propionibacteria are known to possess membrane-bound proteases that are able to hydrolyze casein. the enzymes are released when the cells undergo autolysis during the ripening of the cheese. Although the proteolytic activity was in general low.[75] In particular. It is therefore important that research on the immune-modulating effects of PAB be more focused on oral administration and the use of classical PAB instead of cutaneous PAB. Extracellular proteases were found to be responsible for the activity. acnes and Brucella vaccine has been shown to improve the response to the vaccine.[68] Simultaneous administration of P. avidum KP-40 was found to counteract the immune depression caused by intensive sport activities and normalized lymphocyte counts and activities. however. not all investigations have reported high levels of PAB in human feces. P. similar results have also been obtained in human studies.[71] It can be hypothesized that oral administration of PAB may induce apoptosis in colorectal carcinoma cells via the production of short-chain fatty acids. acidipropionici CRL 1198 was observed to increase the phagocytic activity of macrophages[40] in mice. Proteolysis will yield free amino acids. vaccinia. There is some evidence that oral administration of PAB may provide an immunomodulatory response. Inc. . both through activation of macrophages and through T-cell –mediated cytolytic activity. the cutaneous PAB have been observed to be able to produce inflammatory substances such as tryptamine and Copyright © 2004 by Marcel Dekker. or mouse hepatitis virus led to a significant reduction in mortality among the treated mice. This will provide safer products for assessment in humans. This is.[6] PAB have been suggested to be the predominant proteolytic bacteria found in human feces. In addition. the authors concluded that PAB may make a significant contribution to proteolytic activity in the colon due to the high levels of PAB observed.[74] Proline iminopeptidases have also been isolated from PAB. Preoperative injection with P. H. granulosum on the same day as the virus infection did not always result in a protective response. which can be decarboxylated to biogenic amines or give other substances that are potentially harmful to the host when absorbed from the colonic contents.[70] Thus. in particular acetate and propionate. as stated above.

[79. Although dairy PAB are consumed in relatively large amounts in cheese (108 CFU/g) without any known ill effects. SAFETY No data are currently available concerning the possible acute or chronic toxicity of PAB. strains belonging to this group do not appear to be particularly suited as probiotics. as mentioned above. . safety assessments should be considered even for strains from this group. it would be advised that toxicity studies be performed prior to widespread use of PAB as probiotics. All Rights Reserved. nose. giving rise to a large number of both long-chain and short-chain free fatty acids. acnes may cause opportunistic infections in patients after surgery and implantation of foreign bodies.[81] This could relate to the strong nonspecific immune modulation exerted by many PAB. VII.80] Treatment involves intravenous antibiotic therapy and removal of the infected tissue/foreign body. Although mainly cutaneous PAB have been shown to modulate the immune response. unclear whether the organism is directly involved in the etiology of gallstone formation. It is. In addition to having an unpleasant smell. the use of PAB as probiotics would involve the consumption of larger numbers. Inc.[77] P. Nor did it exhibit any cytotoxic effects on tissue culture cells. Nevertheless. shermanii T-73 was not observed to cause any ill effects on the general state of health of mice. P. Because of the potential link between cutaneous PAB and infections. immune deficiency. Dairy strains have so far not been found to be associated with disease[79] and appear to be better candidates for probiotic use.[78] Cutaneous PAB and in particular P. or trauma. VI. however. or rabbits upon intraperitoneal administration of 109 – 1010 CFU.[79] Cutaneous PAB have also been tentatively linked to rheumatoid arthritis. Other predisposing factors include diabetes. although it probably dues not initiate it. P. In particular.Hoang-Dung TRAN and friends histamine. they also function as important signaling substances for mosquitos[84] and may thus indirectly contribute to the development of disease spread through mosquitos. acnes is also responsible for the breakdown of skin triglycerides. CONCLUSIONS The above overview suggests that selected PAB strains have a good potential for use as probiotics. freudenreichii ssp. guinea pigs. P. acnes may be involved in development of acne vulgaris. acnes has also been found to be associated with gallstones. cutaneous PAB have been linked to different diseases.[76] The health consequences of in vivo proteolytic activity of PAB need therefore to be carefully examined. and throat infections have been indicated to be the main predisposing factors. malignancy. A succes rate of 80% has been observed. where ear.[83] Infections in which PAB have been reported to be involved in the etiology have without exception been caused by cutaneous PAB. some dairy PAB Copyright © 2004 by Marcel Dekker. PAB have also been connected to bacteremia in association with endodontic therapy[82] and meningitis in children. Unlike dairy PAB. Members of this group are known to carry virulence factors such as hyaluronidase activity.[48] Nevertheless. members of the group of dairy PAB may offer a range of organisms that could be beneficial to the health of the consumer and can be considered to be safe due to their long history of safe use in fermented dairy products. even though it seems unlikely that any serious risk exists. However.

J. Appl.. 14.U. Stepaniak.. A. 44. ¨ ¨ ¨ ¨ Freudenreich.A.H. The classical propionibacteria: their past. Microbiol.M. Nes. Glatz. REFERENCES 1. 1906..D. Centralbl. S. Bernard. Detection of the bacteriocin propionicin PLG-1 with polyvalent anti-PLG-1 antiserum..D. J. J.W.D. Faye. 529. C. J. Jack. Glatz.. 2235– 2239. Anaerobe 2002. P. L. A number of important issues remain to be resolved. Appl. nov. present. Ray. 2. Glatz.-T.. 79. US patent 4. Casey. T. Antimicrobial and autolytic systems of dairy propionibacteria. Interaction between propionibacteria and starter/non-starter lactic acid bacteria in Swiss-type cheeses.R. even by oral administration. 58.. Prog.. H. Tagg. Hyg. Lait 1999. 6. 710 – 716. and future as industrial organisms. 1996.A. D. II. 44.L. Leversee. 59 – 68. A.. M. Dairy Sci. Odegard. Thierry. 41–47.. 8. 1 – 15.. B.F.. 15. F. Microbiol.. O.. Shuttleworth. Maubois.. 22. but much work remains to be done. 7. Sci. 6. Uber die im Emmentalerkase stattfindende propionsausegarung. Microbiol. 2000. Appl. Norton. . C.H. Fed-batch fermentation with and without on-line extraction for propionic and acetic acid production by Propionibacterium acidipropionici. containing PAB. because PAB produce a rather strong flavor. Abt. K.. Die hauptlinien des naturlichen bakteriensystems.. Centralbl. Nahrung 2000. M. Biotechnol. ¨ Jensen. 9. Lait 2002. Rev.Hoang-Dung TRAN and friends have been shown to have immunomodulatory activity. Fornili. M. 17.N. I.. Hyg. 5....C. L.. thesis. Ghoul. The production of volatile fatty acids and carbondioxide by propionic acid bacteria with special reference to their action in cheese. Physiological peculiarities of propionibacteria—present facts and prospective applications. Isolation and characterization of proline iminopeptidase from Propionibacterium freudenreichii ATCC 9614. L. Partial purification of the bacteriocin produced by Propionibacterium jensenii B1264. Ozadali. 1999. Sherman. Brede. 303– 309. Mays. Ph. R. O. Prince. Lait 2002. 4. 10. 83. 90. 16.A. E. B. L. S.. Jensen.-B. T. other than cheese. Glatz.E. 1994. 82. Thomas. 305– 346..A. 82..A. J. 779– 787. I.794. Environ. Microbiol. Pitt. J. I.. 2001. 2001. T.M. 1909. ETH: Zurich. II Abt. Ratnam. Production of cheese flavour compounds derived from amino acid catabolism by Propionibacterium freudenreichii. ¨ Miescher. 102 – 106. Munro. Bachmann. T. Thus. Maillard. ASM News 1992. McCaskill. derived from granulomatous bovine lesions.. Langsrud. ¨ Frohlich-Wyder.080. Vorobjeva. ¨ ˚ Holo. 17. J. Bacteriocins of gram-positive bacteria. 67.F. N. Bakteriol. 197– 201. 1923. 8. R.. it will be a challenge to produce functional foods. 1988. 11. J. 171– 200. Bacteriocins of propionic acid bacteria. D. Inc. 125 – 136. M. Microbial co-culture production of propionic acid. Propionibacterium australiense sp. Barefoot. A. Nilsen. Bakteriol. Finally.. 12. Exopolysaccharide production by Propionibacterium acidipropionici on milk microfiltrate. B. 82. Copyright © 2004 by Marcel Dekker. Shaw. there is considerable potential for the probiotic use of PAB. 277 – 301.-P. 3. This is encouraging because the use of cutaneous PAB is less desirable as they have been associated with disease and elaborate safety investigations will be needed to guarantee their safety and include them in novel functional foods.. Engasser. H.H. Gorret. To what extent are PAB members of the normal intestinal microbiota and what is their ecological function? The proposed health effects discussed above need to be verified in well-conducted clinical studies.G. P.. Brendehaug. 17 – 32.B. J. 13. B. All Rights Reserved. J. 59. Forbes-Faulkner. Bodine. Lait 2002.

Nader de Macias. 75. XLI. 28. 82. Pattison. 1992. ´ Perez-Chaia. Soc. 40.Hoang-Dung TRAN and friends 18. Exp. W. Preserving Foods using Metabolites of Propionibacteria other than Propionic Acid.. Ayres... Production of bacteriocin jenseniin G by Propionibacterium is pH sensitive. G. 33. 1975. L. 435 – 445. ´ Bougle.. 32. Moore. Lait 2000. ¨ ¨ Ouwehand. Purification of propionin. Lait 2000. Macfarlane. 30. and use of Said Strain and Preparations for the Controlling of Yeast and Moulds. Food Prot. Barefoot. Composition for Extending the Shelf Life of Processed Meats. shermanii. S. Effect of propionibacteria supplementation on fecal bifidobacteria and segmental colonic transit time in healthy human subjects. Oliver. U. Meijeritiet Aikak 1983. L. Matrozza.. P. Sandine. Ecol. Gonzalez. Soc. Die Propionibakterien der Molkereiindustrie als Darmkanalmikroben. Laniewska-Moroz. 1647– 1656. 215 – 220.. Wolynec. Proc. 41.F.. Gastroenterol. Warminska-Radyko. patent 5. J. Cummings. Health Dis.. In vitro adhesion of propionic acid bacteria to human intestinal mucus. C. U. A. 1999. S.847.A. G. 27. Appl. 63. 80. 1– 95.378. ´ ´ Zarate. Effect of selected natural antimicrobials on baker’s yeast activity.E. G. Ramanathan. Jenseniin... S.. 80. Cutting. A heat stable bacteriocin produced by Propionibacterium jensenii P126. Microbiol. patent 6. Scand. Composition and Method for Treating Bacterial Infection. Viability and b-galactosidase activity of dairy propionibacteria subjected to digestion by artificial gastric and intestinal fluids. All Rights Reserved.. U. L.H. Sandine. Arhan. 34. Lett.981. J. S. 193– 207. I. patent 5. Ekinci. 12. Maubois J-L. W. McFarland. 20... Normal flora: diversity and functions. Inc. 1992.. S. 132. Effect of bile on the b-galactosidase activity of dairy propionibacteria.. Roland. M. T. Oliver. Forsch... A. Read. G. Microbiol. Allison. N. 1993. G. J.. J. 2001. S. F. C. an antiviral agent from propionbacteria. S. T. Lait 2002. Babuchowski.E. On the survival of a Propionibacterium freudenreichii .. 37. A.F. Microbiol.culture during in vitro gastric digestion. J. Cult. A. D. 271 – 273. Lactobacillus casei ssp. 33. Grinstead. Med. 211– 215. Tomes. G. Environ.. Appl. 28. Antiviral principles of propionibacteriaisolation and activity of propionins B and C. U. A. 129... K. S. Holdeman.W. 38. S.S. Lebensm. 23. Barefoot. 1974. Oliver. Mantere-Alhonen.P. 34.E. A.V. G. Weber.V. 176 – 180. Gonzalez.219. 123. S. Proc. Cutting. 127 – 129... von Holly. Possibilities for stimulation of Bifidobacterium growth by propionibacteria. 1986. Salminen. 113 – 121. Suomalainen. Bacterial Treatment to Preserve Silage.C. ¨ ¨ ¨ Mayra-Makinen. S. 73– 77. P. Z. Jan. Dairy Prod... 35. 36.603. Appl. D.E. 19. Human fecal flora: the normal flora of 20 JapaneseHawaiians. Lett. G. 4 – 13. Protein degradation by human intestinal bacteria.J. Characterization of propionibacterial growth metabolites inhibitory for gram negative bacteria. 25. 1966. 123 – 130. 29. Microbiol.T. Bacterial Preparations Comprising Said Strain. Lait 2002. Remmert. Mantere-Alhonen. 27.Y.C. Ramanathan. J... Barefoot. 961 –979. Tolkko...S.718. Rouault. 158. 19 – 23. A. Lebeurrier. Perez Chaia. 24.. G.S.S. Gen. ´ ´ ´ Zarate. patent 5. 31. 1993.. Biol. Perez Chaia. . Copyright © 2004 by Marcel Dekker. J. F.H.096. Appl. W.F. Unters.705. 29. 58. W. U. Suomalainen. Microb.W. Propionibacteria in the gut: effect on some metabolic activities of the host. Biol. On the nonexistence of propionic acid in various kinds of breads. N. 1999. 2001.. 82.H. 26. Oeser. Exp. 2000.. H. N. 1995. 1968.A. Luck. rhamnosus. patent 4.. Sabel. E. M. Med.458. Boudreaux. Ratnam. 40. 267 – 276. W.S. 21. 325 – 336. Al-Zoreky.221. 1991. Meijeritiet Aikak 1982. A. Lait 1995. Acid stress susceptibility and acid adaptation of Propionibacterium freudenreichii subsp. T. Microbiol.-L. Ayres. 2000. D. 22.. 39. 1214– 1221. J. G. 144–148..

T. 16. Suomalainen. A.. Y. T. 63. Conjugated linoleic acid: a review. 91 – 101. Kaneko. 367 – 382. H. Nakamura. Hyg. V. R. Bondarenko. Gonzalez. Mantere-Alhonen.S. Selection of a biologically active mutant of Propionibacterium shermanii and the possibility of its use in complex therapy of enteral dysbiosis. Belury. Lett. S.C. Bjorck. Witthoft. H. Taketomo. Rombouts. 681 – 687.. 59.. H. Food Prot.C. Salminen. MacDonald. Am. Nutraceutical production by propionibacteria. Microbiol. Sato. 65. Mykkanen. Kaneko.M. A. 159–162. 327–332. G. Adhesion of propionibacteria to intestinal epithelial tissue in vitro and in vivo. T. Vuorinen.. Oliver.I. J. 85. 82.. Annu. 1998. C. S. Spadafora. Santos. 52. Hata. 2002. 1999. Wigertz. Dietary conjugated linoleic acid in health: physiological effects and mechanisms of action.B. J. 44.. P. M.. Kelly. Eshuis. 80.G. 2002. 40. ´ ´ Zarate. 2000. Isolation and structural identification of bifidogenic growth stimulator produced by Propionibacterium freudenreichii. J. Growth stimulator for bifidobacteria produced by Propionibacterium freudenreichii and several intestinal bacteria. Am. 95– 102. Hugenholtz. 1996.Hoang-Dung TRAN and friends 42. L. Morita de Ambrosini.M. Metab. Nutr. 50. A. 45.... K. A. 19. J. 55.. V. 103– 112. 175– 185. ´ ¨ ¨ Forssen. 48.. Rev. Fonden.. 82.I...M... Suomalainen. Appl. H. Ann. 2000. On trace elements in Propionibacterium freudenreichii mass. M. Batsman.. Nakajima. M. Condon. 28. J.. ´ ´ Perez Chaia. S. O’Callaghan.M. Wolever. Nutr. Lehto. 82. P. 1959– 1964. Clin.. Inc. Interaction between colonic acetate and propionate in humans. J.. 54.. Ahokas. Characterisation of the stimulants produced by Lactobacillus helveticus in milk for Propionibacterium freudenreichii. Coll.. All Rights Reserved. A. 53 – 59.. S. G. 49. T. J.. lactobacilli and total anaerobes from faecal samples. Microbiol. Meth. faecal azoreductase activity and faecal mucins. J.. T. 1991. J.. Med. Hunik. Lait 2002. Yamamoto. Ability of a mixture of Lactobacillus and Propionibacterium to influence the faecal aflatoxin content in healthy Egyptian volunteers: a pilot study. Clin. H. Perez Chaia.M. Biosc. E.. Meijeritiet. 77. Altern. 6. Appl. 41– 45. 505 – 531... The effect of probiotics on constipation. 46. 22.. 62. Lait 2002. T. Lagstrom. 53.M.. 1997. Meguro. M. S.. J. Lait 2002. Laakso.. 60.M. ¨ ¨¨ El-Nezami. ¨ Ouwehand. 1982.N.. Suomalainen.. Sidorchuk. H. N. Iwata. Salminen. R. Am. S.. Y. 767–771. K. Hunik... 46.. I. S.. A. Y. Probiotics for the skin: a new area of potential application?. A placebocontrolled study of the effect of sour milk on blood pressure in hypertensive subjects. F. Hartemink. H. ˚ Ouwehand. Yoshiyama. T. Nutr. 56... Microflora. Lait 1999. Mori. P. Dairy Sci. Copyright © 2004 by Marcel Dekker. Conjugated linoleic acid and disease prevention: a review of current knowledge. J. Production of conjugated linoleic acid by Propionibacterium freudenreichii ssp. Jagerstad. Nutr. one Lactococcus and one Propionibacterium to cultured human intestinal Caco-2 cell line. Ohni. H. Microflora. 69– 80. T. Immunol. 36. Folates and dairy products: a critical update. S. J. shermanii.A. ¨ Rainio. E.. ´ ¨ Jiang. Sato. Mori... Nutr. Improved Process for the Production of Vitamin B12. 331 – 338. A. Kankaanpaa. 2001. Takano. Dairy Sci. J. 64. Microbiol. Piveteau. J. M.I. Vahvaselka. 1994. Smid. T. 53. 13– 17. 111S– 118S. Lyons. Biosci. 1984. 51. Cogan. Meguro. 19. Aikak. Kamiyama. Zarate G. G. J. . 36. Production of conjugated linoleic acid by dairy starter cultures. patent WO 00/37669. Coll. M. 2000. Salminen. 19. 1997. S. 2003. Comparison of media for the detection of bifidobacteria.. The probiotic properties of propionibacteria. H. Y. 181 – 192. 47.S. 43.H. Nutr. 534 – 539. Epidemiol. 79. S. K. 2002. Salminen. 58.. B.. 57. Am. Rev. 393– 404. 61. Adhesion of two Lactobacillus strains. J.. Microbiol. 100S– 110S..

Krause.. I. Meniningitis and shunt infection caused by anaerobic bacteria in children.. M.. Morohashi. 13. Ko.L. Immunopathol.J. I. 80. A 1980. Pichlmaier. J. D. Ko. 23.Hoang-Dung TRAN and friends 64. 819– 822. 491 – 494... Health Dis.. L. L. M. 1996. Haouzi.. 11– 13. Tungga. 117.P. H. Rinta-Koski. X.. Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria.. S. Infect. G. 77. Kirjavainen. 2000. Dis. C. A.. ¨ Isenberg. 1987. Osborne. C.. Milchwissenschaft 2001. Immunomodulation with killed Propionibacterium acnes in guinea pigs simulaneously vaccinated with Brucella abortus strain 19.. G. Bakteriol.. Electron. Neurol. 281.. H.Q. 1995. Greenam. 1986. V. Mantere-Alhonen. Merilahti-Palo. Frazier. L. Proteinase activity of dairy propionibacteria.. Zentralbl.. 799– 802.. de Vaux.. 205– 212. Gastroenterol. R. ´ Jan. Zentralbl. Pulverer. 1991.S. 74. Clin. All Rights Reserved. J. Y.T. 67.. Helander. Copyright © 2004 by Marcel Dekker. Belzacq. M. 1999. 9. O. 26. Severe infections caused by Propionibacterium acnes: an underestimated pathogen in late postoperative infections.. 234– 237. T. Pediatr. J. J. T. 248. Grossberg. E. S. M. 1994. Wright PFA. Brook.. I. Lehtonen. 68. Highfield. A.. I. Salminen. 477– 482. 65. R. 69. 83. G. 13. P. Rowland. Xiao. J. Mitra. D. Microbiol.-S. Schultz. H. Stewart. D. Haynes. Microbiol... 2001. Zbinden. Protection and therapy of mice with acute and chronic experimental virus infections with Propionibacterium granulosum KP-45. H. Diag. Toyoda. C. 519 – 524. Immunol. R. D.H. Ahokas.. Manner. R. 77 – 80... 79.V.. 1399– 1404. Med. Detection of bacterial DNA from cholesterol gallstones by nested primers polymerase chain reaction. 179– 185.... 1302– 1304. S. A. Bacteraemia caused by periodontal probing. 68. T. Microsc. 71. 4. Bakteriol. Lab.D. Tsuchida. 1998.. Appl. E. E.. J... J.... J.. Vet. Montonen.. 1994. 71 – 84.T. Li.. C. Dupuis. Brenner. I.S.H. Influence of propionibacterium avidum KP-40 on the cellular immune system after intensive sport activity. Yale. 42..J. Pathogenesis of acne. Dent... von Graevenitz. 99– 105. R. Effects of orally administered viable Lactobacillus rhamnosus GG and Propionibacterium freudenreichii subsp. Hamaoka. Propionibacteria isolated from rumen used as possible probiotics together with bifidobacteria. Brook. J.. Allaker. Rouault. Pottkamper. Br. Randerath. Morrison. J.. Anticancer. Ecol. Metabolic activities of the gut microflora in relation to cancer. 34. 367– 371. Daly. T. P. Death Diff. Appl. Res. J. Pulverer. 286 – 295..P.. 73. The production of inflammatory compounds by Propionibacterium acnes and other skin organisms. Boyaval. 11. Immunol. C.. Biol. X.. A. R. Inc. Li. P..W. J. Rev. Metivier. Beuth. Biotechnol. Wu. Szmigielski. 7. 1998. 14. Microb.. Pulverer. Combined immunomodulation (Propionibacterium avidum KP-40) and lectin blocking (D-galactose) prevents liver tumor colonization in BALB/c-mice. Jakab.. Mitchell..R. El-Nezami. M. Corr. shermanii JS on mouse lymphocyte proliferation.. Tsuji. A. Aust. 66. 78. 76. Propionibacterium acnes isolated from sternal osteitis in a patient with SAPHO syndrome. Gann 1981. H. 2002.B. G. O. Displacement of Escherichia coli O157:H7 from rumen medium containing prebiotic sugars. G. 6. Rintala.. R. Cell. Kobus. S. Jeljaszewicz. Med. Dermatol. Panangala.. M. Infections caused by Proionibacterium species. 179 – 188. J. Inhibitory effect of Propionibacterium acnes-activated macrophages on the tumor metastasis enhanced by tumor-specific immunosuppression. Zentralbl. 750– 755. 75. Rheumatol.. Fujiwara. 2002. Mottonen. World J. 29 – 40. Gubler.. 175 – 183. Beasley.. 1996. R. Hughes. 72. H. Grundmann. 72. M. Kroemer.S. Jeljaszewicz. 1996. Preoperative immunostimulation by Propionibacterium granulosum KP-45 in colorectal cancer. Pulverer. 69. J. Kotilainen. 284. Beuth. . 2002. Environ. Ruef. 56. 82. 81. Bakteriol.. Stutzer.. D. J. Gil. G. 70. Hutkins.

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and use of starter cultures. Thus. genetics. and bacteriophage resistance. Fermented milk can be used in more than 1000 products for and affects flavor. and industrial starter production are discussed. France I. All this is reflected in enormous demands for starter cultures. its effect on the final product. stable quality. texture. production. physiology. The art of making cultured food products by using the former day’s whey or fermented product for today’s process has been changed to a science with exact knowledge of the factors influencing the specific starter species and strains. Montpellier. their activity. INTRODUCTION Since the early 1900s there has been a marked worldwide increase in the industrial production of cheeses and fermented milks. shortening processing times. Many outstanding reviews have been published about the metabolism. in this chapter the general view of the most important factors concerning starter activity. Finland MARC BIGRET BioSav LTD. Helsinki. Inc. 175 Copyright © 2004 by Marcel Dekker. All Rights Reserved.Hoang-Dung TRAN and friends 5 Industrial Use and Production of Lactic Acid Bacteria ¨ ¨ ¨ ANNIKA MAYRA-MAKINEN Valio LTD. and processing larger quantities of milk. and final product quality. Process technology has progressed toward increasing mechanization. The discussion of industrial production of starters is mostly based on the author’s experience. increasing factory sizes. .

L. Inc. Raw mixed cultures are widely used in traditional cheese making in Switzerland.[1] Thermophilic starters are used for yogurt and for cheese var` ieties with high cooking temperatures (Emmental. while P cultures propagated in nonaseptic dairy conditions attained the optimum balance between phage-sensitive and -insensitive strains. in pairs. The raw mixed cultures can be natural whey cultures or produced by macerating dried calf vells in the previous day’s cheese whey.[11] About 85% of all cheddar cheese was produced with these starters in 1985. All the categories are known as cheese starters. In spite of the use of these unknown mixed cultures. helveticus.[13] Thermophilic cheese cultures can also be divided into two categories: raw mixed starters and defined strain starters with multiple or single strains. During the 1970s defects in secondary fermentation occurred in Emmental cheese. Hoang-Dung TRAN and friends MESOPHILIC AND THERMOPHILIC STARTERS USED BY THE DAIRY INDUSTRY The starters used in dairy products can be divided into mesophilic and thermophilic starters according to optimum growth temperature. Under aseptic conditions the L cultures became dominated by phage-sensitive strains. and they are distributed weekly to cheese factories by the Research Institute. L. Differently composed starters can be categorized as follows: Single-strain starter: one strain of a certain species Multiple-strain starter (defined strain starter): different known strains of one species Multiple-mixed-strain starter: different defined strains of different species Raw mixed-strain starter: species and strains partly or all unknown The traditional raw mixed-strain starters are widely used. fermentum. are used in the production of many cheese varieties. or in multiples. Comte. The mixed cultures contain Streptococcus thermophilus and different species of Lactobacillus: L. France.[3] During the 1970s the big cheese plants started to use defined phage-insensitive strain systems with good success. and Italy.[12] In the Netherlands a totally different starter system is applied based on the difference in phage sensitivity between the starters propagated in the laboratory (“L cultures”) and in practice (“P cultures”). lactis. and the trend is toward multiple-strain starters. the multiple-strain starters have become popular in Australia. . Grana). which stimulated propionic acid fermentation and production of CO2. The multiple strains are used singly. L. bulgaris.[8 – 10] and Ireland. with the optimum around 308C. The starter cultures are usually composed of different species or of several strains of a single species. and their use was pioneered in New Zealand by Whitehead[2] to avoid open-texture defects in cheddar cheese caused by flavor-producing organisms in mixed cultures. the need to know the exact properties of starter strains is well recognized in Switzerland. Mesophilic cultures are either raw mixed-strain or multiple-strain cultures in Europe and North America. acidophilus. Mesophilic cultures grow in temperatures of 10 –408C. especially in fermented milk products and ripened cream butter. caused by starters that were too proteolytic.II.[7] the United States.[14] After research on this defect the variety of starter cultures decreased. Thermophilic starter cultures have their optimum growth temperature between 408C and 508C. and L. and ripened cream butter.[4 – 6] Since this development. All Rights Reserved. Gruyere. composed of acid-forming lactococci and often of flavor producers. The defined strain cultures have led to the understanding of strain interaction with phages. The cultures used are P cultures kept in The Netherlands Dairy Research Institute and distributed to the cheese factories for large-scale production. Mesophilic starter cultures.[15] Copyright © 2004 by Marcel Dekker. fermented milk products.

Dutch: Mixed cultures. are propagated. cremoris are acid-producing microorganisms. 1. is the only aroma producer. Single-strain starters have been used in Finland for Emmental cheese. Lc. lactis ssp. All Rights Reserved. in order to keep a composition as close as possible to the original culture. . Lactobacillus is a large genus with over 50 homo. lactis and Copyright © 2004 by Marcel Dekker. Because of the decrease of natural flora in raw milk during the 1970s and 1980s. cremoris. lactis and Lc. diacetylactis. Mesophilic Species and Types of Starters The species composition of most mesophilic starters include Lactococcus lactis ssp. helveticus and propionic acid bacteria were also used. and Leuconostoc cremoris. 2. without isolation. lactis var. Lactococcus lactis ssp. thermophilus and L. a secondary resistant strain is derived to replace the original strain. lactis ssp. Australian: This system consists of a single strain. the culture is of D type.Hoang-Dung TRAN and friends Single-strain starters containing Str.and heterofermentative species. replaced as soon as possible in case of a bacteriophage attack. Either the same culture containing two to six strains is used alone for a long time. by the 1950s single strains of L. Leuconostoc lactis. Lb. thermophilus strains. A combination of these two last systems has been successfully used in the United States and Ireland by selecting secondary resistant strains and including them afterwards in multiple-strain cultures. In the latter case. the culture is of B (or L) type. lactis var. it is called BD (or LD) type. When both aroma-forming species are in the culture. When the only citrate-fermenting species present is Lc. Inc. the cultures have to have different bacteriophage sensitivity profiles. Thermophilic Species and Types of Starters Thermophilic organisms belong to two genera: Lactobacillus and Streptococcus. B. lactis var. or several cultures are used in rotation in order to prevent bacteriophage attacks. diacetylactis. When starter cultures contain only these species. The various starter systems used in the dairy industry are either mixed cultures in which the composition of the mixture is not defined or cultures containing well-defined single or multiple strains. A. coming from dairies or butter plants. Several combinations of single or multiple strains of lactic acid bacteria are currently used in cheese making. lactis ssp. they are characterized as O type. more species of lactobacilli and propionic acid bacteria were needed for Emmental cheese to accelerate the ripening time and to improve the taste and eye appeal. very few bacteriophage attacks are unnoticed. New Zealand: This system is used in many Anglo-Saxon countries and utilizes multiple-strain cultures composed of a small number of defined strains. lactis ssp. From the sensitive strain screened in the factory. Lc. Lactococcus lactis ssp. helveticus have been ` developed in France[16] and used for Gruyere and Emmental cheese as frozen concentrated cultures for bulk starter or direct inoculation of cheese milk. delbrueckii ssp. 3. Only a few of these are involved in milk fermentations. When those cultures are propagated under aseptic conditions. starting in the 1930s with Str. When the Leuconostoc sp. diacetylactis and Leuconostoc sp. are citric acid – fermenting bacteria. lactis.

Lactobacilli are used in combination with Str. Lb. partially. yakult. thermophilus.[17] A symbiotic relationship exists between S. Lb. C. Starter Function Among the physiological functions of lactococci are several of great importance in cheese manufacturing and maturation. salivarius ssp. thermophilus and Lb. For this reason only galactose-fermenting lactobacilli should be used as starters together with Str.[18 – 21] Some data have been published about the symbiotic relationship existing between Str. while the latter produces formic acid –like compounds. acidophilus is of intestinal origin and is widely used in different kinds of milk products because of its believed beneficial effects on human and animal health. casei and Lb. glucose. At the end of the growth phase. which promote the growth of Lb. The bacterial transport of lactose.5% of the lactose is used by lactococci. 1984). Many outstanding reviews have been written in this area describing the symbiosis in detail. thermophilus by releasing amino acids. is not further treated here. Two different mechanisms have been found: the permease and the phosphoenol Copyright © 2004 by Marcel Dekker. less than 0. Sugar Transport Across the Cell Membrane. consequently. casei produce diacetyl from citrate. and Lb. and galactose across the cytoplasmic membrane has been well characterized. Lb. thermophilus and Lb. Some strains of Lb. thermophilus do not metabolize galactose. it has no major influence on the technology and. bulgaricus (formerly known as Lb. bulgaricus. helveticus. bulgaricus) is a component in yogurt together with Str.[22] The lactobacilli with lower optimum growth temperatures. 1. but the actual compounds involved are not known yet. and thus lactose metabolism by Str. helveticus are the starter lactobacilli for cheeses with high cooking temperatures. . thermophilus results in galactose accumulation in the medium (Thomas and Crow. but this species is used as a starter only by the Japanese in making fermented milk. Inc. influencing the final organoleptic qualities of the cheese: Fermentation of sugars. Lb. Lb. All Rights Reserved. bulgaricus. plantarum. grow in cheese as natural contaminants. Acid Production Lactose is the major fermentable sugar found in milk at levels of 40– 50 g/L. bulgaricus stimulates S. lactis. This combination is naturally selected because of the high temperatures used in the fermentation of certain cheeses and yogurt. and Str. taste of cheese Synthesis of flavor compounds Synthesis of texturing agents. The glucose moiety of lactose is used faster than the galactose moiety by lactococci. thermophilus. delbrueckii ssp. which may influence the consistency of the product Production of inhibitory components Since the lipolytic activity of lactococci is very low.Hoang-Dung TRAN and friends Lb. leading to a pH decrease important in the clotting phenomenon and reduction or prevention of the growth of adventitious micro-flora Protein hydrolysis which causes the texture and. bulgaricus. delbrueckii ssp.[23] The fermentation product of lactococci is L (þ)-lactic acid. thermophilus.

Carbohydrate metabolism is controlled by both repression and retroinhibition. Both permease and PEP/PTS systems [24] and only permease [25] have been suggested. is PEP-dependent. Lb. Repression is a mechanism that controls enzyme synthesis.. Contradictory results have been published about the transport of lactose in Str. b-casein being most susceptible. helveticus has been shown to attack as-casein and partly b-casein. There are relatively few studies on the transport of lactose by thermophilic cultures. After transportation. In the PEP/PTS system lactose is transported into the cell via a complex system by which lactose is phosphorylated to lactose phosphate and thus transported across the cell wall. This system.[31] Several studies describe three cell wall – associated proteinases. The permease system is found in thermophilic species and the leuconostocs. or corresponding free sugars.[26] implying that permease is the most important transport system.29] The proteolytic activity of Str. Inc.Hoang-Dung TRAN and friends pyruvate-phosphotransferase (PEP/PTS) systems. Inside the cell. and the PEP/PTS system in the lactococci. Lactobacilli contain more b-gal than p-b-gal. while galactose-6-phosphate is metabolized along the D -tagatose-6-phosphate pathway. Lactic acid bacteria are only mildly proteolytic compared to.[30] The proteinases of lactococci involved in the first step of casein breakdown are high molecular weight proteins located primarily in the cell wall. the sugars can be either lactose phosphate.g. Lactose can also be transported by a permease system. The internalized peptides are hydrolyzed by cytoplasmic peptidases. Lactose is transported via PTS. Bacillus and Pseudomonas. e. Proteolytic Activity All starter culture species are nutritionally fastidious.5 –6. the glucose moiety is catabolized through the glycolysis pathway (Embden-Meyerhof-Parnas pathway). This system. uses the ATP of the cell..[28. their isoelectric point is between 4.55.5. and they are either activated or stabilized by Ca2þ ions. All milk proteins including whey proteins are available for hydrolysis in starter strains.4 and 4. One of these is thought to be responsible for the bitterness of cheese. All Rights Reserved. Their optimum pH is around 5. Galactose is used in the Leloir pathway. lactose is hydrolyzed to glucose and galactose by b-galactosidase (b-gal). Then. Copyright © 2004 by Marcel Dekker. Carbohydrates Catabolism. It could be expected that more accessible proteins in the casein micelle. Lactic acid bacteria utilize the polypeptides generated by milclotting enzymes and by bacterial cell wall proteinases and therefore are responsible for the casein degradation. Then peptides and amino acids are transported across the membrane via specific transport systems. Proteinases. . thermophilus. glucose phosphate. requiring many amino acids and growth factors for adequate growth. are hydrolyzed before as-casein. k. composed of two enzymes and a soluble factor together with a thermoresistant protein. 2. e. which requires energy. The combined action of proteinases and peptidases provides the cells with peptides and free amino acids. and Lb.g. The lactose phosphate is hydrolyzed by a b-gal to give glucose and galactose-6-phosphate. bulgaricus degrades all the major caseins.and b-casein. thermophilus is lower than that of lactococci and does not affect casein hydrolysis in cheese. galactose phosphate. and retroinhibition controls enzyme activity. These molecules can be metabolized by three different pathways.[27] This has been shown in lactococci. The lactose phosphate is hydrolyzed to glucose and galactose-6-phosphate by phospho-b-galactosidase (p-b-gal).

cremoris. diacetylactis and Leconostoc ssp. The lactococcal citrate metabolism pathway consists of enzymes the genetic determinant are in plasmids.[33] This is supported by the fact that in cheese where BD cultures are used. Casein degradation initiated by a milk-clotting enzyme and proteinases. the studies carried out on this topic have shown that citrate-negative variants of a citrate have always lost a 5. is not yet understood. Aminopeptidase activity is especially high in Lb. and Lb. A number of peptidases has been described. lactis. lactis ssp. lactis and Lc. Lb. these species could have a considerable effect on proteolysis. plantarum. All Rights Reserved. prolinase and prolidase. proline-iminopeptidase. The aroma compounds in fermented milk are mainly organic acids. It is likely that the known number of different proteinases may change due to improved knowledge of their specificity and their genetic back-ground. bulgaricus strains. endopeptidases. Pyruvate is degraded in the cell in the presence of Mg2þ. Unfortunately. The removal of this anchor results in the release of the proteinase. cremoris. Lactobacilli exhibit a wide range of peptidase activity.[32] The identification and characterization of cell wall – associated proteinases are under investigation. 3-butylene-glycol from citrate. the amino acid nitrogen level is higher than in the control cheese prepared only with Lc.Hoang-Dung TRAN and friends These observations suggest an active role of the lactococcal proteinases in producing bitter peptides of medium size (tri. The role of this system. Second. and the compounds present mostly in matured cheeses.[35] Several peptidases with broad specificities have been isolated from Lb. Inc. 3. the involvement of plasmid DNA has been studied. Because spontaneous irreversibly proteinase-negative variants appear with high frequency. X-prolyl-dipeptidyl aminopeptidases. lactis var. . under the control of a gene called prt M. helveticus. It has been suggested that citrate and other carboxylic acids affect peptidase activity. produced by Lc. lactis ssp. Aroma Formation The flavor compounds produced by lactococci can be divided into two categories: the compounds in products of fermented milk.[36] Being natural contaminants of cheese. Peptidases. acetoin. lactis ssp.[34] but dipeptidase and caseinolytic activities do not vary much between Lb. and carboxypeptidases have been found in various lactococci.[23] Copyright © 2004 by Marcel Dekker. and 2. and taste. aminopeptidase P. Na2þ. Indeed. Plasmids ranging in size from 13.to hexapeptides) during cheese maturation. an arylpeptidyl-amidase. The pyruvate metabolized from citrate by citrate lyase (or citritase) is toxic to the cell when its intracellular concentration is too high.5 to 100 kilobases (kb) are involved in proteinase production. di. diacetyl. lactis ssp. It has been suggested that these arome compounds prevent pyruvate accumulation in the cell. continues with peptidases. Aminopeptidase.[31] In addition.[37]. which produce smaller peptides and amino acids.and tripeptidase. and thiamine pyrophosphate.5 megadalton plasmid. lactis and Lc. lactis ssp. A majority of peptidases are metal enzymes. produce acetaldehyde. casei NCDO 15 and 2 strains of and Lb. lactic and acetic acid. it seems that a membrane-located lipoprotein is involved in the activation of the proteinase. Lc. The location of peptidases in the cell has not been determined. the various studies cannot be compared because the methods used differed. which produce large peptides. Studies on proteinase localization have shown that they are attached to the cell wall by an “anchor” present at the C-terminal end of the protein. texture. helveticus.

bulgaricus. Exopolysaccharide Formation (Ropiness) Many strains of lactic acid bacteria produce exopolysaccharides (EPS).[50] Toba et al. cremoris. Inc. casei. At the end of the 1980s the production of thermophilic viscous yogurt starter cultures became more common. [51] found that glucose.[39] Many varieties of cheese contain these species. This property has been utilized in Finland since the nineteenth century. dihydroxyacetone. These starters are used. analysis of enzymatic systems of lactococci. and Propionibacterium shermanii. glucose. Production of EPS in mesophilic lactococci has been shown to be plasmid encoded.43] The role of EPS in this phenomenon has yet to be elucidated.Hoang-Dung TRAN and friends The impact of lactococci on the production of flavor compounds in ripened cheese is much more difficult to determine. thermophilus. to replace stabilizers in yogurt.[33] One of the reasons for this is that the lactococci play an indirect role in cheese flavor production. and galactose but not glycerol. and evaluation of different strains used in cheese production might allow us to establish a better correlation between lactococcal activity in cheese and flavor development. 4. S. especially in higher temperatures. The form of EPS can be as a capsule closely attached to the bacterial cell or as loosely attached or excreted slime. The starters of this product contain mesophilic. All of them have been shown to contain galactose and glucose and sometimes hexose-like components and rhamnose (reviewed by Cerning. although they do not contain plasmids. There have also been some attempts to investigate the antitumor activity of slimeforming lactic acid bacteria. galactose. The aroma compounds in Swiss cheese have been reported to be produced by reactions between dicarbonyls and amino acids. Nevertheless. [52] isolated a phosphoruscontaining polysaccharide that contained rhamnose. but the amino acid composition of this protein is similar to that of the whey.[49] The chemical composition of EPS of mesophilic and thermophilic lactic acid bacteria varies from strain to strain. mozzarella. However viscosity is unstable in thermophilic starter strains. rhamnose. even though it is known that the former influences the latter.[41. which are further metabolized to volatile compounds.[42.47] EPS-forming bacteria are often considered to be more resistant to bacterio-phages than nonencapsulated ones. and analytical methods are inadequate. Nakajima et al.or tripeptides and free amino acids. Copyright © 2004 by Marcel Dekker. Lb. glyoxal. better knowledge of proteolysis and peptidolysis in cheese. In some isolated capsular materials protein has been found. and phosphorus form a capsular polysaccharide of Lactococcus lactis ssp. as these viscous strains are hosts for many phages [48] and a certain phage can also dissolve the capsular material of even nonhost strains. and cheddar cheeses and in cultures of Lb. and diacetyl have been found in Swiss. All Rights Reserved.[40] A comprehensive review has been published by Cerning. The peptidases generate di. . methylglyoxal. slime-forming lactococcal strains together with aroma-producing lactococci and leuconostocs. It was thought to be a deacylated lipoteichoic acid.[41]).[38] The dicarbonyls. Some key flavor compounds are present at nanogram concentrations. villi.[41] Slime-forming lactic acid bacteria has been increasingly used in the dairy industry. This is not the case among mesophilic lactococci. glycerol. in some cases. especially in the production of a thick viscous fermented milk product. They are widely used to increase the rheological quality of yogurt and to inhibit syneresis of the coagulum. No direct relationship has been established between the amino acid nitrogen and cheese flavor.[44 – 46] This may explain the instability of slime production.

[64] The two bacteriocins produced by lactococci. Nisin is effective against gram-positive species and also against Clostridium botulinum spores. but it is also well recognized for its anti-microbial action. even at low temperatures. Bacteriocins produced by lactobacilli have been characterized from Lb.[57] To make this system efficient.[62] Lb. certain components have to be present in milk. Production of Inhibitory Components The observation that lactic acid bacteria have some preserving effect dates back to the turn of the nineteenth century.[66] Copyright © 2004 by Marcel Dekker. attributed to lactic acid bacteria and linked to hydrogen peroxide production. diacetyl.Hoang-Dung TRAN and friends 5.[56] The second system of inhibition. Recent investigations have also shown nisin to be inhibitory toward Listeria mono-cytogenes. although in much smaller amounts. is detected in milk at a concentration varying from 1 to 10 ppm. They are thermostable. only general remarks are presented here. reacts with two substrates: thiocyanate and hydrogen peroxide. helveticus.[53] Bacteriocins of lactic acid bacteria have been the subject of wide research. which inhibits microorganisms. reduction of pH and production of organic acids (lactate. Hydrogen peroxide can react with thiocyanate in a reaction catalyzed by lactoperoxidase to form an oxidation product.[65] and it was found to be effective in prevention of blowing (butyric acid fermentation) caused by clostridia. Hydrogen peroxide (H2O2) is produced in milk by lactic acid bacteria. fermentum. All Rights Reserved. lactoperoxidase. Bacteriocins are a heterogeneous group of antimicrobial substances in respect to bacteria production bacteria. . nisin and diplococcin. Inc. gram-positive bacteria.[53] and accumulation of hydrogen peroxide in growth media can occur because lactobacilli do not possess catalase. Thus.[63] and Lb. Since many reviews have been written on this topic and there is a chapter in this book about the subject. Lactic acid bacteria produce other inhibitory substances. and thus the use of it in foods may be problematic. Few other bacteria are able to grow at pH values achieved by the action of lactic acid bacteria. acetate) are the primary inhibitory actions of these bacteria. Thiocyanate (SCN2).. plantarum. and chemical properties. are well characterized. hypothiocyanate. antibacterial spectrum. Nisin was found in 1928 by Roger and Whittier.[61] Lb. mode of action.[58] Diacetyl imparts butter aroma. which has made it useful in thermally processed foods. widely distributed in animal secretions. The inhibitory level by Jay [59] is 200 mg/mL for yeasts and gram-negative bacteria and 300 mg/mL for nonlactic. The first application in Swiss cheese was done 1951 by Hirsch et al. acidophilus. bacteriocins. A relatively large amount is needed for inhibitory action. and secondary reaction products such as hypothiocyanate generated by the action of lactoperoxidase on hydrogen peroxide and thiocyanate. According to the early research the organic acids from sugar fermentation were responsible for the good keeping quality of fermented foods. These include hydrogen peroxide. Hydrogen peroxide is generated by different mechanisms by certain lactobacilli.[54] Antagonistic effect has been demonstrated against Staphylococcus aureus [55] and Pseudomonas spp. The bacteriocins are defined as follows:[60] They generally have a narrow range of action. An enzyme. is the lactoperoxidase system (LPS). Part of the molecule is a peptide and therefore they are sensitive to proteases. a pathogen of great concern. The concentration of lactoperoxidase in milk is 10 –30 mg/mL.

9 g/L Other (vitamins. Lb. and Lb. Nonprotein nitrogen represents 5– 70% of the total nitrogen in milk.[68] B.[69] These antimicrobial substances were later referred to as red protein. Endogenous or exogenous factors can affect the starter activity in the starter tank or during the dairy process. pantothenic acid (B5). . on the one hand. it is suggested that its targets include both RNA and DNA. lactis ssp. lactis ssp. The vitamin requirements vary from species to species. inhibitory bacteria. milk is not an optimal growth medium. In addition. citrate. some derived from cow’s blood. 35 g/L Protein. pyridoxine (B6). Lactic acid bacteria have a very limited effect on milk fats. niacin (PP) and riboflavin (B2).71 – 74] Copyright © 2004 by Marcel Dekker. For instance. lactoferrin. the geographic location. enzymes. lactis and L. Lactococci require niacin (PP). the lactoperoxidase system. Inc.. but not until 1927 were the agents identified by Jones and Little as lactenins.[6. addition of yeast extract can stimulate the production of lactic acid. The concentration of these components (containing fewer than eight amino acids) is usually too low to provide the required nutrients. 49 g/L Lipids. The average composition of cow’s milk is: Water. Thermophilic streptococci require pantothenic acid (B5) riboflavin (B2). etc. 905 g/L Lactose.e. lactis. bulgaricus. The constitutive molecules of this fraction have an important role in the nutrition of lactic acid bacteria because of their direct uptake by the cell.) traces The impact of lactic acid bacteria on the main components of milk involves uptake of fermentable sugars. FACTORS INFLUENCING STARTER ACTIVITY Milk as a Growth Medium Even though lactic acid bacteria are able to grow in milk. Inhibitory Compounds in Milk The antimicrobial effects of milk have long been known. cremoris. lactis ssp. free amino acids (including free methionine. nonprotein nitrogen (NPN). 34 g/L Salts.[70] Many antimicrobial factors have been identified since then. It is well known that the origin of the cows. Consequently. lactoferrin. A. and pyridoxine. biotin. on the other hand. As diplococcin is more active against cells in the exponential growth phase than in the stationary phase. vitamins and. and the stage of location all cause variation in milk components. is active only against L. and their corresponding enzymatic systems. cremoris. produced by L. All Rights Reserved. acidophilus require cobalamin (B12).Hoang-Dung TRAN and friends Diplococcin.[68] Variations in milk lead to modification of the physiological reactions of the microorganisms. dissolved oxygen. niacin (PP). and biotin (H). thiamine (B1).[67] III. lysozyme. and bacteriocins. free fatty acids. Lactobacilli require calcium pantothenate (B5).7. proteins and peptides. These include variations in milk composition caused by mastitis or seasonal changes. residual sanitizers. agglutinins. The remaining components of great importance in the nutrition of lactic acid bacteria are. and afterwards divided into lactenin 1 (L1) and lactenin 2 (L2) by Auclair and Hirsch. Lb. i. an essential amino acid) are not present in milk at sufficient levels to allow satisfactory growth of the cells.

[77] The most widely used group is b-lactam antibiotics and their derivatives. thermophilus strains tested. individual differences between cows are known to exist.3– 0.80] This can be seen in Table 1. In an experiment to test the actual effect of low antibiotic levels on cheese produced by mesophilic (Edam cheese) and by thermophilic (Emmental cheese) starters. and sulfa drugs.3– 0. However. mg/mL Streptomycin.3 2. lactis/cremoris/diacetilactis and three mixed cultures (DL) tested. they are not reviewed here in detail. The results are presented in Table 2.2– 0. allergic reactions. and thus general withdrawal times are not always valid. Strict penalty rules and improved testing systems have reduced the residue levels significantly. and development of resistant bacteria.5 Mesophilicb 0.0 0.[78. ¨ ¨ ¨ Source: A. tetracyclines.05– 0. and it causes huge economic losses to dairies.0 Penicillin. thermophilus starter strains have been found. Antibiotic Residues Antibiotics are the most important group of exogenous inhibitory factors because of their common use in the treatment of mastitis in dairy cows.0 0. IU/mL Tetracycline. The most important sources of problems in the dairy plant are antibiotic residues and bacteriophages. These are discussed further below. All Rights Reserved. 1.g.01 0.5– 5.76] To avoid residues in milk. Single strains of Lactococcus lactis ssp. Inc. Antibiotics were introduced in the 1940s for mastitis therapy.5 0. but streptomycin-sensitive S.004– 0. Table 1 Sensitivity of Thermophilic and Mesophilic Starters to Different Antibiotics Starter cultures Antibiotic Thermophilica 0. Copyright © 2004 by Marcel Dekker. Today mastitis is still the most serious problem affecting cows. different antibiotics were added to the cheese vat.0– 4. Mayra-Makinen. varying from country to country. macrolides.2 0. intestinal disorders.. either alone or in combination. There are also several health aspects associated with the problem.01 0. IU/mL a b Thirty-two S. unpublished data. mg/mL Cloramphenicol. the manufacturers of veterinary products are generally compelled to specify a withdrawal period for any product.[12.Hoang-Dung TRAN and friends Heat processing of milk and good manufacturing practices combined with intensive quality-control procedures have minimized the effect of most of the factors mentioned above.[81] Little information is available on the levels of antibiotics required to inhibit leuconostocs or propionic acid bacteria used in hard cheeses. . e.0 0.5– 1.[75.79] The levels of antibiotics required to inhibit different starter strains are very strain dependent. mg/mL Spiramycin. The number of antibiotics used is huge and still increasing.5– 1. Mesophilic cultures are less sensitive to penicillin and spiramycin and more susceptible to streptomycin and cloramphenicol than thermophilic cultures. Other common antibiotic groups are amino-glycosides. Therefore.005 – 0.

butyric acid fermentation Not tested Smell of butyric acid. and structural protein profiles. strong off-flavor Off-flavor Not tested Tasteless Tasteless. serotyping. In order to differentiate the phages. unpublished data.01 IU/mL Spiramycin 1. Bacteriophages Bacteriophages are viruses that specifically infect bacteria.Hoang-Dung TRAN and friends Table 2 Effect of Different Antibiotics at Low Concentrations on Edam and Emmental Cheese Quality Cheese quality Antibiotic Edam No defects Tasteless Bitter. slimy surface Abnormal cheese. wet surface Strong off-flavor. Host Range: Strain rotation is more easily handled when the host range of a phage is known.003 IU/mL 0. All Rights Reserved. uneven body. they use the cells’ enzymes to grow. morphology. DNA/DNA hybridization. 2.008 IU/mL 0. .7 mg/mL a b Brown spots in the body.005 IU/mL 0.3 mg/mL 0. various taxonomic criteria have been proposed. uneven texture. Taxonomy. phages of lactic acid bacteria are of considerable economic importance because they represent one of the main causes of fermentation failure. are host range. The most important of them. It can be concluded that low levels of antibiotics cause different kinds of defects in cheese: off-flavors. But as this criterion cannot be correlated with others. severe offflavor. uneven body. uneven body Strong off-flavor. In the dairy industry. proposed for a taxonomic classification. In fermented milk products the effect of antibiotics is seen in slow or inhibited acid formation and in a decrease in aroma formation. ¨ ¨ ¨ Source: A. After infecting bacterial cells. abnormal eye formation Strong off-flavor. Copyright © 2004 by Marcel Dekker. Inc. Effects on propionic acid bacteria could be seen in eye formation of Emmental cheese and in browns-pot defect caused by streptomycin. 1. uneven eye formation. it is a practical parameter rather than a real taxonomic characteristic. strong off-flavor a Emmentalb No defects Off-flavor. Evaluation at 3 months.01 IU/mL 5.0 IU/mL Streptomycin 1. uneven eye distribution Not tested Penicillin 0. strong smell on surface Evaluation at 14 weeks. Mayra-Makinen.0 mg/mL Tetracycline 0. and butyric acid fermentation. much work has been done to improve our knowledge of phage infection and bacterial phage mechanisms. Due to their economic importance. uneven body Tasteless. After some time cells are lysed and bacterial growth is stopped.

based on structural protein profiles. Second. the phage injects its chromosome inside the bacteria. . First. In phages of lactic acid bacteria. In the next step. All Rights Reserved.[84] Phage Development and Bacterial Resistance. various morphologies are observed. 3. The head. therefore. These groups have been compared with host range classes.2. or large isometric shape. Group II contains both virulent and temperate changes. bacterial DNA is hydrolyzed. DNA/DNA Hybridization and Structural Protein Profiles: It has been possible to determine five groups of homology for a large number of phages of Lc. Serotyping: Antibodies have been prepared against pure phages in order to classify them in various groups. indicating that the serological criteria are not sufficient to classify the majority of the phages. and the bacterial metabolism is used by the phage genome to develop new phage particles. As a rule. may be transferred from one cell to another. The DNA penetration is Ca2þ-dependent and energy-requiring. 4.. small isometric. Copyright © 2004 by Marcel Dekker. Several. The phage fails to adsorpt on cell surface and therefore does not infect the strain. 1990). a lytic enzyme or lysin is synthesized by the phage to make the cells burst. allows the grouping of phages corresponding to the five DNA homology groups.[85] Most of the known mechanisms are coded by plasmids and.[83]. This receptor allows the phage to adsorb on the cell surface. Phage resistance mechanisms developed by lactic acid bacteria are correlated to the various steps of phage infection.[82] The results of these studies have been variable. Groups I and III contain almost 80% of phages and include only virulent phages. with either prolate. lactic acid bacteria have developed mechanisms to resist phage attack. Hoang-Dung TRAN and friends Morphology: Phages are submicroscopic particles consisting of a head containing the DNA and a tail. Inc. has a size of 40– 70 nm. an average of 100 phages per bacteria are released into the milk. Adsorption: Phage-resistant strains have mutated cell-wall structures. recognized as receptors by phages. the phage recognizes a molecular structure on the cell wall. The details of these five groups are given in Table 3. The classification. Morphological classification was proposed on the basis of electron microscopic observation. Table 3 Classification of Lactococcal Bacteriophages by DNA/DNA Hybridization Percent of phages of the group 29 21 48 1 1 Head morphology Prolate Small isometric Small isometric Large isometric Large isometric Genome size (kb) 19 – 22 30 – 40 30 – 35 53 134 DNA homology group I II III IV V Source: Ref. Four mechanisms are well described: 1. The tail measures between 100 and 500 nm. lactis by DNA/DNA hybridization (Prevots et al. In the last step.

is degraded. laborious vulnerable. Inc. Therefore. They can be stored for several months at 2258C. Lysogenic Immunity: This is observed when the bacterial chromosome harbors the DNA of a lysogenic phage. besides the increasing use of concentrated starters. . But. In 1970 the freeze-drying technique was developed to make use and transportation still easier. each attacked cell releases very few phages in the medium. USE OF STARTERS IN THE DAIRY PROCESSES There have been considerable changes in the cultivation of starters in dairy processing. and the desired volume is reached by successive subcultures. Abortive Infection: In the case of abortive infection. with a modification enzyme.2. There are several advantages to the use of concentrated and concentrated freezedried starters[86. So. Hoang-Dung TRAN and friends Restriction-Modifications: The restriction-modification system combines restriction enzymes. The production technology of concentrated frozen starters was developed during the 1960s. General practical steps in the preparation of starters are discussed here. The unmodified phage chromosome is hydrolyzed by the restriction enzymes as soon as it enters the cytoplasm and. All Rights Reserved. 3. Freeze-dried cultures can also be used for producing a mother culture or bulk starter in cases where a small amount of starter is needed. The prophage probably codes for molecules. IV. the burst size is very low. especially where local special products are made or where the transportation of starters from a central laboratory is easy and regular. activity is good and can be Copyright © 2004 by Marcel Dekker. The starters are easily contaminated during the numerous inoculations or infected by bacteriophages. Still. since they are still in practice. the quality is even. These starters require low temperatures during shipment and storage. Starters are the most important factors determining the final quality and properties of the product. capable of specific endonucleolytic activity. and thus the plant can use the same production lot for months. and needs skilled personnel to manage it. In the case of mixed-strain cultures the strains should be freeze-dried separately in order to avoid changes in balance. The starters available are sold in different forms by several starter producers. 1. generally exhibiting specific DNA methylation activity. due to an unknown phenomenon. The procedure is thus expensive. The four alternative commercially available forms of starter cultures are shown in Fig. all the phases of the infection occur. The modern systems of concentrated frozen and concentrated freeze-dried cultures have made it possible to directly inoculate the bulk starter or the production process itself. the selection of starter type and form is very important for the individual dairy plant. This causes significant savings in labor and material costs in the dairy. In the traditional liquid starter system the starter is first cultivated as a liquid stock culture. liquid starters are widely used. 4. This methylation protects the DNA from the corresponding restriction enzyme. including the traditional systems (liquid starters). consequently. which inhibit the development of other related phages and render the strain resistant. The stock culture comes weekly from a central laboratory or from the plant’s own culture.87] they are easy to use. Little or no disturbance is observed in the growth rate and acidification during cheese making.

directly-to-vat starters. Inc. among other bacteria. the storage temperature is critical. they require less labor and are easily adapted to a five-day production week. Not all good traditional milk starters are suitable for production as concentrated freeze-dried starters. The faster development of lactic acid bacteria and the pH decrease due to acid production lead to microbiologically stable fermented products. fermentable sugars. and thus the activity has to be controlled in the dairy plant. The first incubation period (time and temperature adapted to the Copyright © 2004 by Marcel Dekker. water activity. Despite these disadvantages. The selection of starters with respect to quality of the final product is not made by the dairies but by the starter producers. On the other hand. and Staphylococcus carnosus are generally inoculated. there are also disadvantages. and pH) allow growth. and bacteriophage control is easier to manage. beside the meat slurry. there is a competition between various species. All Rights Reserved. These basic phenomena of bacterial ecology have been used to produce meat products such as sausages and fermented vegetables. When the physical conditions (such as temperature.Hoang-Dung TRAN and friends Figure 1 Alternative starter types used in dairy product manufacture and cultivation steps in the dairy process. For sausage preparation. pediococci such as P. Lb.[87] The shipment of frozen starters is precarious since temperature changes affect the starter activity. salt. tested prior to use. V. . starter research is strongly oriented to production techniques and strain selection in order to produce active. in several niches. plantarum. STARTER CULTURES FOR FERMENTED MEAT AND VEGETABLE PRODUCTS The growth of lactic acid bacteria in milk to produce fermented dairy products is based on a few simple principles: lactic acid bacteria are present. acidilactici. and spices.

Fermentation of Starter Cultures To produce active and storage-stable concentrated cultures. and effect on the harvesting methods. or Lb. P. Industrial-scale production of cultures involves batch fermentations. The combination of NaCl and lactic acid makes the fermented vegetables stable for a long period of time at room temperature. complex equipment is associated with difficulties in production schedules. plantarum. Only about 25 – 50% of traditionally used strains are suitable for the production of concentrated. pickled cucumbers.[91] Starter concentrate production can be divided into the following general steps. Kosikowski[89] and Bergere and Hermier[90] were the first to use neutralization to increase the bacteria count in fermentations about 100-fold in concentrated cultures. There are several problems with using continuous fermentations: undesirable contamination is possible. pentosaceus. Thereafter the sausages are cooled and aged to obtain the final product. PRODUCTION OF STARTERS ON AN INDUSTRIAL SCALE The interest in producing concentrated frozen starters began during 1960s with research on mesophilic single strains and especially cheddar cheese starters.Hoang-Dung TRAN and friends technology) allows the bacteria to grow.[86] It is generally Copyright © 2004 by Marcel Dekker. Widely available fermented vegetables include sauerkraut. thereby influencing the quality of the end product. cerevisiae.[87] The process is very stressful for the starter strains and new selection criteria must be used. the following aspects must be considered: cost. .[92] Gilliland. brevis can also be used. NaCl brine is added. Methods to produce concentrated cultures differ in several ways from traditional ones. Growth Medium To choose the growth medium. All Rights Reserved. The salinity also controls the growth of lactic acid bacteria. Leuconostoc mesenteroides. ability to produce a high number of cells (about 10 –15 times higher cell densities that in liquid milk culture) with high activity. which are simpler and more convenient than continuous fermentation processes.[87] and Tamine and Robinson:[80] Preparation of the inoculum Preparation of the media Fermentation at constant pH Harvesting the culture Adding the cryoprotectant Freezing Freeze-drying Packing and storing A. Usually. and olives are fermented with Lb. Inc. freeze-dried cultures. VI. cabbage. which have been described in detail by Porubcan and Sellars. several strain-dependent factors have to be checked concerning the growth medium and growth conditions. Starter strains are grown under strictly controlled conditions in a medium from which the cells are easily harvested into a smaller volume.[93] 1. P. and green olives. and bacteriophage problems have also been reported.[88] During the preparation of these products. and the pH decrease is measured. cucumbers.

Inc.[97] Most fatty acids of bacterial cells are located in the cell membrane. and it can be assumed that the membrane composition is very important for the cells to survive freezing. survival at 2178C has improved. Manganese and magnesium did not have this effect. pH.[97] The addition of 100 mM—1 mM calcium has been shown to influence the freezing resistance of lactobacilli and change the cell morphology from long chains to short individual cells. Bauman and Reinbold[103] reported better freezing stability at 2208C when a temperature of 328C had been used during fermentation. Copyright © 2004 by Marcel Dekker. . Porubcan and Sellars. compared to the optimum growth temperature. With certain nutrients or additives the resistance of cultures against subsequent concentration/freezing/drying processes can be improved. unpublished data). This could be because lactococci naturally contain higher levels of C19 cyclcopropane fatty acid. In addition.[99.[87] It has also been noted that lactococci survive freezing at 21968C better than lactobacilli. The methods are strain-dependent. When growing mixed cultures. bulgaricus after freezing at 21968C increases considerably if the cells have been grown in an appropriate medium supplemented with Tween 80. All Rights Reserved.[98] The ratio of unsaturated to saturated fatty acids in cell membranes also seems in some cases to be related to the ability of lactococci and lactobacilli to survive freezing.[94] A variety of different nutrients are needed for starter strains in the culture medium.102] but few research results have been published on the effect of temperature on process stability. Growth Conditions The conditions during the fermentation affecting the growth and activity of cultures are temperature. Oleic acid in Tween 80 was identified as the effective component to increase process stability by raising the levels of a C19 cyclopropane fatty acid in the lipid fraction of cells. yeast extract. the optimum cooling/harvesting time relative to the growth curve has to be considered. a decrease or increase in the growth temperature strain dependence affects the dechaining of cultures and thus also process stability in freezing and freeze¨ ¨ ¨ drying (A.95] Skim milk has been the most common medium for lactococci. whey. Mayra-Makinen. and peptones—are used to satisfy the complex demands of starters if there is no way to determine the exact growth factors during the fermentation process. and clarification might be necessary to avoid this material in concentrated starter culture. and the first group of complex nutrients—skim milk.Hoang-Dung TRAN and friends accepted that some milk solids should be included in the growth medium to ensure the synthesis of necessary enzymes for starters to perform well in milk and to maintain a balance between strains in multiples-strain starters when fermented as a mixed culture. With thermophilic cultures it has been noticed that. mixing (oxygen content).[94] The use of cheese whey and whey permeate as growth media has been investigated primarily because they are inexpensive and contain nutrients that are used by starter strains for growth. By increasing the ratio. Also.[101. perhaps by increasing membrane flexibility.[96] The same results have been reported for cultures frozen at 2178C. The activity and the bacterial count of Lb.[93. Usually the optimum growth temperature of the species is used in fermentation. regardless of the growth medium. and much of the research has involved supplementing whey with extra nutrients. and the difficulties in harvesting have been solved by adding sodium citrate to solubilize milk proteins. and type of neutralizer used. improper heating causes precipitation in the medium.[92] divided them into four groups.100] 2. Whey alone is not rich enough for maximum growth.

[101. on the accumulation of H2O2 can be prevented.106] It can be concluded that the basis for active. Lb. process-stable culture is built during the fermentation by modifying the growth media and conditions so that they are straindependent. Porubcan and Sellars[92] also reported industrial-scale ultrafiltration Copyright © 2004 by Marcel Dekker. thermophilus cultures lose activity fast if cooling and harvesting is delayed to the stationary phase.[93. In order to maintain constant pH continuous agitation is needed. lactis. Str. which can be autoinhibitory. On the other hand.105] Both the optimum pH of the culture and type of neutralizer used are of importance. For lactococci. in addition to the type and amount of nutrients adequate for optimal growth. depending on the strain.[108] The optimum pH of thermophilic lactobacilli. helveticus. bulgaricus. Concentration of Fermented Cultures Centrifugal separation or membrane processes can be used for harvesting cells from the medium. bulgaricus.Hoang-Dung TRAN and friends Maintaining the pH of the growth medium at the optimum level increases the number of cells. lactis. Centrifugation is mostly used on an industrial scale because the low viscosity of the medium. oxygen toxicity has been observed in culturing some lactococci.[109] Oxygen can also cause the production of hydrogen peroxide by some starter strains. By adding catalase or other reducing agents. Especially important factors to be checked for industrial production are: Dechaining effect of certain components in medium Temperature optimum to produce process-resistant strains (not always the growth optimum) pH optimum for growth and further processing Harvesting time at certain point of growth curve Process resistance as selection criterion for starter strains B. unpublished data).107] Ammonium hydroxide seems in general to be the best neutralizer in order to achieve higher cell yields in mixed cultures[93] and more freezing-resistant cultures at 2308C. and Lb. According to Porubcan and Sellars. . Most research has been done with lactococci.8.[92] To reach the maximum bacterial level in the fermentation. acidophilus from milk-based cultures by centrifugation. many lactobacilli can be harvested irrespective of ¨ ¨ ¨ the growth phase without losing activity (A. even with the addition of citrate. the factors limiting growth have to be considered. and higher temperatures favor this technique. unpublished data). Inc.[110] Other toxic metabolites can be formed in the growth medium. Higher yields of ¨ these species are obtained with ammonium hydroxide as the neutralizer (A.Mayra-Makinen. As a result. and Lb.[111] Sparging carbon dioxide can be an effective way to avoid the toxicity of oxygen and is actually needed for optimal growth of some starters. the large cell size.106.Mayra¨ Makinen. Lb. Cooling and harvesting at certain stages of the growth curve are critical for some cultures. D -leucine formation has been reported in lactococcal cultures by Gilliland and Speck. the properties of the cells.[104. is 5. of which the formation of lactate salts is considered to be the most important. harvesting is recommended at the end of the logarithmic growth phase.5.4 – 5. depending on the strain. Increasing the growth seems to be a combination of several factors. whose optimum pH is in the range of 6 – 6. All Rights Reserved.[92] it is difficult to concentrate Lb. The temperature is usually kept between 5 and 158C.[92] Very little has been published about concentration of thermophilic lactobacilli. Lb.

Frozen concentrates are stored at 2408C. Freeze-dried products are stored at 220 to 2408. An approximate 12-fold concentration can be reached by ultrafiltration without any cell damage caused by heat developing during the process. but there seems to be a variation among cultures. thermophilus cultures. frozen storage. but for some cultures again no effect has been seen. in many cases 2408C freezing and storing temperature is also used. The most efficient and widely used method is fast-freezing in liquid nitrogen in the form of pellets.5%) has been used with good results. All Rights Reserved. The studies carried out in this area have been done mostly with pure strains. FUTURE TRENDS As explained in this chapter. and activity remains good for at least 6 months. on the other hand. Studies must focus on the relationship between the constitutive strains of Copyright © 2004 by Marcel Dekker. The activity of a mesophilic mixed-culture concentrate with a pH of 5.6 after frozen storage. The shelf life depends on the form of starter culture (frozen or freeze-dried) and the storage temperature: the lower the temperature. unpublished data). Glycerol is widely used in frozen cultures.18C/s). the longer the shelf life.[113] further research is needed to improve the “freeze-drying resistance” of cultures.Hoang-Dung TRAN and friends of lactococci.2 was lower than that of a concentrate with a pH of 6. VII. Therefore. as far as technology is concerned. especially in concentrating processsensitive cultures.2 –6. the important characteristics of the bacterial strains are being increasingly well understood and measured.. Inc. Knowledge needs to be improved in the understanding of the global behavior of a culture composed of several strains. lose activity easily if the pH of the concentrate is ¨ ¨ ¨ below the optimum 6. The extensive report of Morichi[112] concerning the mechanisms and cryoprotectants involved in freeze-drying gives a view of this complex area. The resistance of cultures against the deleterious effects of freeze-drying can be improved by cryoprotectants. The activity of a culture can be maintained if storage is carried out under recommended conditions. A lower pH does not seem to have any effect on the activity after freeze-drying. which are easy to use as a frozen concentrate or to freeze-dry.Mayra-Makinen. Str. there are great differences in storage stability between cultures.[117] The optimum concentrate pH for lactobacilli is 5. Although possible mechanisms of cryoprotection have been proposed by Fennema et al.[112] A variety of different cryoprotectants are used[113] but the most common ones in industrial production are lactose or sucrose (7%).[105. The pH of the concentrate affects the activity during storage. monosodium glutamate (5%).[107] According to several reports it seems that cryoprotectants are not needed if the concentrate is active. C. Again. and freeze-drying. . and short-term refrigeration does not usually decrease activity.8. Handling of the Concentrate Cryoprotectants have long been used to improve the ability of culture concentrates to survive freezing.4 – 5. freezing is fast (. Most of the research on freeze-dried cultures has been done with lactococci. and ascorbate in a milk or water base. Microfiltration processes with ceramic filters are becoming an important method.6 (A. and storage is at 21968C. and for some cultures glycerol is not effective.116] The use of liquid nitrogen is expensive.115] Freezing can affect the activity of cultures strain-dependently.[22.[114] Lactose (7.

Heap. L.. 1980. Aust. NZ J. 227. 5.. Mesophilic starters. Ernstrom. Sandine. 1977. 7. Dairy Sci.K. 13. 241.E. Valles. Neth. A. R.D. Whitehead. 1976. S. Bull. Steffen. G. 70. Spontaneously developed mixed-strain cheese starters. Inc. Fox. Dairy Sci.R. 157. 48. 1953. 17. C.R. Vassal. silage.C.A. 12. 1980a.. 15.. H.. G. Le Lait 1968.C.. P. R.L. C. For example. P. 179 pp. R. 61. 8. Cheese starters under control. Dairy Sci. Lawrence.. 1978.M. 109. 3. Limsowtin..F. Their behaviour towards phages and their use in the Dutch cheese industry.K. 1979.A. G. L. 14. Heap. 38.. such as sausage. Dairy Ind.C. Stadhouders. Physics and Microbiology.Y. 6. Selection and characterization of phage-insensitive lactic streptococci. 63. 11. 101. C.J. and where the contaminant level could be reduced by the action of modified lactic acid bacteria. Vol. 1. Sandine. 9. W.. Lawrence. Das neue Kulturenkonzept fur die schweizerischen Kasereien. Heap. Dairy Technol.Y. C. C. H. H. Cheddar cheese starters: current knowledge and practices of phage characteristics and strain selection. Auclair. Understanding the symbiotic and inhibitory phenomena between strains is of prime importance for the control of the cultures used in fermented products. one must achieve the following: Identify the corresponding genetic determinant of certain physiological characteristics Use methods that allow a stable transfer of genes Dispose of vectors donor and recipient strains of the same species in order to obtain a GRAS microorganism Solve regulatory problems and constraints related to genetically modified microorganisms REFERENCES 1. The use of mesophilic cultures in the dairy industry. Limsowtin. Pearce. it would be useful to have strains producing bacteriocins in order to manufacture fermented products in which raw materials should not be heat treated. 1977.. Defined single strains of lactic steptococci in bulk culture for cheddar and Monterey cheese manufacture. Hong. All Rights Reserved. 1987. H. Rousseaux. H. 10. The use of concentrated frozen suspensions of thermophilic lactic acid bacteria in making Gruyere cheese. 11. Cheesemaking and cheese research in Switzerland. 4. NZ J.. Leenders. 62. Deutsche MolkereiZeitung 1980b. 297. A multiple starter concept for cheesemaking. 126. . Danielle.R.E. Jarvis. R. 16.A. Daly. These programs aim at implementing new characteristics in technologically interesting strains. Milk Dairy J. Hull. To make these techniques feasible. J. R. G. 1979. G. 37.Hoang-Dung TRAN and friends a mixture. Mocquota. Int. J.. Technol.W. 1988. 16.. J. 19.H.C. 2. T.. J. 49.E.. Int. besides acidification. W.. A. 101. Richardson. 65. Rev. IDF Doc. 32. Dairy Fed.. or vegetable products. Antonie van Leewenhoek 1983. Copyright © 2004 by Marcel Dekker.E. 1181. Cogan. 70. J.. Lawrence. The selection of starter strains for cheesemaking. J. 16. 12. Technol. Cheese Starter Culture Cheese: Chemistry. 73.M.A. Huggins. Dairy Sci. J. Daly. Ed. Lawrence. E. Dairy Sci. The second main direction followed by researchers is in genetics. 1972. ¨ ¨ Steffen. 1984. 1981. Bacteriol. Bacteriophage in cheese manufacture. 62. Commercial use of a multiple strain starter of known composition in cheddar cheese manufacture. Dairy Sci. G. Control of bacteriophages in cheese factories. Petersson. 1186.

R. FEMS Microbiol. Effet des acides amines et peptides sur la croissance de Streptococcus thermophilus. Mills.. 595. 55. J. Portalier.G.. Atlan..J. N.W. 1982. 1990. 1989.. 15.. 1989. 40. 604. Technol.G. J. Laloi.. His et Met. Milchwissenschaft. Le Lait 1991.. Argyle.F.. 1717. Le Lait 1971. C. M. Inc. 37. Premi. 131. mainly in cheese manufacture. 1971. Veaux. 24.. Bednarski. 36. El-Doda. F. P. 20.. Dairy Sci. 34. T. Dairy Sci. J. Galactose fermentation and classification of thermophilic lactobacilli. en relation avec la fabrication des fromages a pate cuite. F. Yogurt I. Copyright © 2004 by Marcel Dekker. Etude des interactions entre diverses bacteries lactiques ´ ` thermophiles et mesophiles. 65. 71. plantarum.. Appl. I. Utilization of lactose. Nitrogen sources for growth of lactic streptococci in milk. 1982. E. Dairy Sci.. 140.. 1408. N. Proteolytic cuzymes of dairy starter cultures. T. III. H. ´ Accolas.. R. L’etat des connaissances en matiere de nutrition des bacterins lactiques. Lolkema. Peptides comportant Glu. P. P. Pette. M. 39.. Peptidases of Lactobacillus casei and L. 37. El-Doda. Aust. J. Botazzi. 60. E. Simard. Lactose-hydrolyzing enzymes of Lactobacillus species. Zevaco. FEMS Microboil. 36. 1950. G..E. 38. 27. Blanchard.G. Olson. Lemieux. Evolution of free amino-acids during the ripening of cheddar cheese containing added lactobacilli strains. Basic Microbiol. D. Cell wall associated proteinases in Lactobacillus helveticus. Isolation and characterization of aminopeptidase-deficient Lactobacillus bulgaricus mutants. R. 245.. Milchwissenschaft 1985. Dairy Sci. Peptide hydrolases from thermobacterium group of lactobacilli. 4. E.. 35. 1987. 54. Pritchard.Hoang-Dung TRAN and friends 17. 1989. Action of Lactobacillus bulgaricus proteinase preparations on milk proteins. Appl. Desmazeaud. Ezzat. 87. M.J. 26. The impact of lactic acid bacteria on cheese flavor. A. 267– 316. Driessen. bulgaricus and Str. Thomas. 29. 24. Ezzat. 33. 249. Elliker. 8. Le Lait 1980. 28. Environ. J. D. 1981. glucose and galactose. 1987. Hillier.. 23. 87. Nikolov.. Kingma.. Continuous production of yogurt cultures and stimulation of Lactobacillus bulgaricus for formic acid. A method for determination of a-dicarbonyl compounds. Vescovo. Rev.G. 27.D... 34. Ismail..-P. 32.. Simard.. 26. J.. Food Sci. 1972. J. 18. L. thermophilus. 51. M. III. Appl. All Rights Reserved.. Battistotti. P. M.R. Jette. A review of the factors likely to influence its development. Milk Dairy J. L. Milk Dairy J. Turner. Beta-galactosidases et phospho-beta-galactosidases de Strepococcus thermophilus. Desmazeaud. Neth. 1982. Tinson. F. A.. K. L. 43. 1983. W. M. D. Lactobacillus bulgaricus and Lactobacillus lactis. 1989. N. J. Hammond. Microbiol. B. NZ J. Nardi. 31. Microbiol. Genetics of the proteolytic system of lactic-acid bacteria. . Jago.W. Characterization of the intracellular exopeptidases.R. Griffith. Le Lait 1986. Auclair. 25. Microbiol.M. Z. Abo-Elnaga. Kok. Mathison. Hammond. 19. 21. Boullane. I.C. R. V. Milchwissenschaft. 676. Hemme. 1990.. 63. FEMS Microbiol Rev. R. G. W.. D. D. G.E.D. W. 46.E. Chandan. Sandine. Metabolism of Streptococcus thermophilus. 214.L.. Rev. J. 45.E. Technol.G. J. R. Plapp. 1979. Stadhouders. Lorient. P. R. M. 30. 2474. 22. 135.. Martley. 123. 885. 66.J. 51. J. Generation of Swiss cheese flavor components by reactions of amino acids with carbonyl compounds.. Puchades. Dairy Sci. Gedrychowski. 72. Evidence that Lactobacillus bulgaricus in yogurt is stimulated by carbon dioxide produced by Streptococcus thermophilus. Symbiosis and antibiosis in mixed cultures of Lb.. Bracquart. Le Lait 1983. Biter flavour in dairy products. L. Thomas. Environ.E. 445. Neth. 16. Lemieux. 197. 599. 1932.. 72.

J. Toba. 1991. M. Daeschel.. J. 45. application of human lymphocyte culture techniques. Chem..C. Kandler. 27 pp. V. Vol 2.Y. Appl.. L. H.L. 43... Kambe.. 51. Cerning. Microbiol. Nurmiaho. 149. All Rights Reserved.. T. 58.W. Plasmid-encoded functions of ropy lactic acid streptococcal strains from Scandinavian fermented milk. Immunobiological effects of Streptococcus cremoris from cultured milk “villi”.. Le Lait 1987.. ´ Forsen. 309. I.. Environ. Rev. D. Microbiol. Boquien. F. 59. 1982. Hydrogen peroxide formation by lactobacilli and its effect on Staphylococcus aureus. Food Technol. Production and mode of action of Lactocin 27. J.L... 67.D. Inhibition of Pseudomonas species by hydrogen peroxide producing lactobacilli. Komatsu. A. E. 1986. 1568. J. R. 42. H. Int. Price. Phenomenes de co-operation et ´ d’inhibition entre les bacteries lactiques utilises en industrie laitiere.. 167– 172.. 61. Desmazeaud. J. Biol. Plasmid-encoded ropiness production in Lactobacillus casei ssp. FEMS Microbiol. 1983. Agric. Bottazzi. T. Copyright © 2004 by Marcel Dekker. Dairy Sci. 44. 1984. 771. Sutherland. In Surface Carbohydrates of the Procaryotic Cell. Tynkkynen. Itoh. 18– 30. C. 709 – 712.W. R. M. 25.R. V. 50. 11. J. Forsen. Hasegava.S. Nakajima. Vescova.A. Neve. 67 (2).. 46. Dairy Sci. M. H.. their nature and production. S. Toba. Appl.S. 1989. 525. Lee. 54.J. 1472. ´ ¨ Saxelin.. H. Oda. 52. M. Properties of a Lactobacillus fermenti bacteriocin.. Antitumor polysaccharide from Lactobacillus sp. 1990.. Microbiol. 1975. I. G. 7. Desmazeaud. 52. Gen. T. V.J. 49. S.. 1182. Inc.. J. Ed. 70. A novel phosphopolysaccharide from slime-forming Lactococcus lactis subspecies cremoris SBT 0495. 43. Streptococcus lactis subsp..H.C.. N. 525. Merilainen.. Toyoda. Hinsdill. cremoris LAPT 3001 isolated from Swedish fermented milk “langfil”. Milk Food Tech. Environ.M.. Le Lait 1991. 1967. 41.... A. Appl.L. Macura. Kitazawa. 1968.A. 164. Geis. Ultrastructure and host specificity of bacteriophages of Streptococcus cremoris. J..P. H.M. Adachi. Genus Lactobacillus.. M. Sundman. 73. ¨ Bjorck. Tsuchiya. Jay. R. H. 1623. Scandinavian ropy milk-identification and characterization of endogenous ropy lactic streptococci and their extracellular excretion. Vedamuthu. Microbiol. Weiss. S. 139. Biochemie. 57. R. J. Herva.M.. M. Von Wright. J. M.C. 1988. Juillard. M. Kotani. 87. 51. Williams and Wilkins: Baltimore. 44. Teuber. J.. H.A. 1989. IDF: Brussels. 53. Dahiya.. . 13.. Antimicrobial properties of diacetyl. 1990. 60. 1985. 41. Arvilommi. T. and Leuconostoc cremoris from Finnish fermented milk “viili”. Nurmiaho. Piard. Heiska. Antimicrobiol. 55. Int. Korhola. In Bergey’s Manual of Systematic Bacteriology. M.. MD. Speck. I. Partial characterization of a new C3type capsule-dissolving phage of Streptococcus cremoris. Mukai. L. 1385. Saxelin..Hoang-Dung TRAN and friends 40. J. 1970. V.J. 62. In Natural Antimicrobial Systems. T. The lactoperoxidase system.L. Exocelular polysaccharides produced by lactic acid bacteria. G. De Klerk. Dairy Sci. 71. Scolari.L. Can.. Adachi. 735. 48. E. 33. Lett. E. Neville. Inhibiting factors produced by lactic acid bacteria. 437. Biotechnol. 56. casei. Construction of Streptococcus lactis ssp lactis strains with a single plasmid associated with mucoid phenotype. Appl. Bacteriocin from a homofermentative Lactobacillus. Involvement of a plasmid in production of ropiness (mucoidness) in milk cultures by Streptococcus cremoris MS. 1987. 47. O. Microbiol. J. 113– 130. Environ. J. Microbiol. Capsular polysaccharide of a slime-forming Lactococcus lac˚ tis ssp. Ed.. P. 1977. 12. 1987.L. Sutherland. S. E. Smit. S. Agents Chemo-ther. Environ. Bacterial exopolysaccharides. R. 1986. 1979. Food Microbiol. 677. 5. Oxygen metabolites and catabolism end products. 1986.. Spinner. E. 51. Townsley..E. M. Upreti. Food Microbiol. 53. 47. diacetylactis.. P. Antimicrobial substances from lactic acid bacteria for use as food preservatives. 48.

329. M. M... 64.. 67. Academic Press: New York. 1968. 1985. Am. 70. 1984. Aikak. A. Med. Le lait: milieu de culture. A note on the inhibition of an anaerobic sporeformer in Swiss-type cheese by a nisinproducing streptococcus. 86th Ann. Yamada. Assoc. M. 2nd Symp. FL. Auclair. M. CRC Press: Boca Raton. Dairy Technol. 1953. Scand.J. 1122.G. .. P. Koritz. 29 (6).. 20. Anti-microbial activity of lactic acid bacteria against Listeria monocytogenes. 1974.. In Proc.E. 176. Persistence in milk of active antimicrobial intramammary substances.. 1987. Daeschel. 199. Dairy Inf. 83. E.-L. 1988. Veterinary Pharmacology and Toxicology. 411. Harris. 1975. Microbiol.H. 1990. M. 65. 56 (7). McKenney. Swedish University of Agricultural Sciences. 1983.E. M. Microbiol. 1988. 80. Gilliland. Sanders. 77. Method of estimation. Hydrogen peroxide excretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxide. Environ. Bacteriocins of lactic acid bacteria. Rosen. J. 1977. Molecular characterization and the comparison of 38 virulent and temperate bacteriophages of Streptococcus lactis. Meeting. 77. Copyright © 2004 by Marcel Dekker. Aliment Nutr. Med.F.E. P. Immunology of the antibiotics. Milchwissenschaft. Mata. Ruckebusch.C. S. V. F. ¨ ¨ ¨ Mayra-Makinen. In The Antigens IV. ˚ Carlsson. 51.. Biochemie. R.. 86. Microbiol. Eds. Relano. Environ. Mata.. Yoghurt Science and Technology. 337.A. Stiles. 79. Mattick.. J. Toutain. 36.. 313 – 325. Inc. 3053. Dairy starter cultures.C. Sela.R. 75. M. 74. G. Soc. European Assoc. 277 pp.K. All Rights Reserved.. 4 (2). P. Pergamon Press: Oxford.. Daeschel. 1977. T.. M. In Bacterial Starter Cultures for Foods. J. 866. Klaenhammer.E. 1986. Mata. 68. H. J.. The occurrence of inhibitory and stimulatory phenomena. Ed. 18. T. Dairy Res. L. Ashton. Muriana. Present state of lactic acid bacteria phage taxonomy. P. Korhonen. V. Reiter. Iwami. J.. Microbiol. Microbiol. Detection of Inhibitory Substances in Milk. P. Bonneau..Y. Penicillin hypersensitivity—is milk a significant hazard? A review. 87. Thesis.R. 1973. 70. D.. 32 pp.. 8. J. Dewdney. H. J. University of Helsinki. J. P. Tamine. S. 71. 78. 66. The manufacture of starters by batch fermentation and centrifugation to produce concentrates.Hoang-Dung TRAN and friends 63. Availability and usage of new antibacterial drugs in Europe. Phage resistance in lactic acid bacteria. Dairy Res. R. Hirsch. 85.E. Desmazeaud. Dewdney. 133. MTP Press: Lancaster. 81.R. Taxonomic differentiation of 101 lactococcal bacteriophages and characterization of bacteriophages with unusually large genomes. A. 145 pp. R. M. J. L. 842. Antibacterial activity of the lactoperoxidase system in milk against Pseudomonas and other gram-negative bacteria. Food Protect. ¨ Bjorck. Appl. Conjugal transfer of plasmid-encoded determinants for bacteriocin production and immunity in Lactobacillus acidophilus 88. Chapman. Klaenhammer.. Robinson.D..R.. G.A. 70.. Stanley. 1980. Dairy Sci. Milk components inhibitory to Bacillus stearothermophilus. 1985. 1991. Marshall. Soc. 205. Y. M. L.R. 553. Concentrated starter cultures. Infect Immun. 2180. A. A. 384. 70. T. Gen. J. 1989. Am. Characterization of a bacteriocin from Lactobacillus plantarum. Ziv. Biochemie. Ritzenthaler.M.. P. 52. Untersuchungen zur Bakterizidie der Milch und Immunisierung der boonier Milch driise. 30. 1951. 40. Biochemie. Abstracts. Klaenhammer. Soc.. Microbiol. 84. A. 76. Vet. 395. 45. 82. Grinsted. J. 1987. 69. 73. B.T. Hirsch. 1990. M. Busta. G. J. Ritzentmaler. 1990...M.-G.M. Appl. Edwards. 70. Appl. Archimbaut. Prevots. 1988. 72. Ritzenthaler. 30.. Thesis Report 6. Ed. F.. The inhibition of micro-organisms by raw milk. G. 1983. McDonald. J. Stadhouders. T. T101-test for antibiotic residues in milk. A. 53.. Gilliland. Meijeritiet. 38. Carlsson..

. H. 100. 56th Ann. S. 104. IFST Annual Symp. 57. J... Jansen. Bergere. 92.. Gilliland. 135. 13. Arbetsutskottet for ˚ livsmedels.. Holloway. Jakubowska. Gilliland. Session of IDF. 1980.W. 40. Dairy Sci. 1971. M. 1966.P. R. Environ. Speck. Microbiol. Speck. 90. Microbiol. 111... 22. J. Microbiol. J.E. Appl. .-E. 97. 1972. An experimental continuous culture unit for the production of frozen concentrated cheese starters. 91. 149. E. 106. Mechanism and presentation of cellular injury in lactic acid bacteria A subjected to freezing and drying.E. D. Sellars. 98. 23. Hermier. 165. 107. II. 48. 1973. Pol. Speck. Speck. R. 149.T. Piatriewicz. 17. T.. Morris. 108. P. Neth. J. Gilliland. Sverige. I. Environ. Inc. R. 1983a. M. 112. 151. La production massive de cellules de Streptocoques lactiques. Wright. 39. 33. A new approach to the production of cheese starter. 471– 472... Microbiol. 93. A.R. All Rights Reserved. 1981.G. Porubcan. A. G. F. 1982. Selection of bacterial strains for the food industry and their applications in various food products. H. S. Kemicentrum.D. Observation on continuous growth behavior in reconstituted skim-milk.T. J. S. Appl. 89. T. Growth studies on the lactic streptococci.. Stability of lactic acid bacteria to freezing as related to their fatty acid composition. Eschar. Growth and preservation parameters for preparation of a mixed species culture concentrate for cheese manufacture. Int. 1573. 1972.L. Lactic starter culture concentrates. J. Preservation of starters and mass production of starter bacteria. 403. Wright. Some preliminary investigations.E. Bacteriol. 4.E. Influence of calcium and manganese on dechaining of Lactobacillus bulgaricus.. Cogan. 1972. Zottola.. T. Copyright © 2004 by Marcel Dekker. Halvarskrift no 2. Stadhouders. Food Sci.. 1972. Mitchell. S. Aust. Gilliland. Appl. Milk Food Technol. 17. Pont. Peebles. S. 1983b. Van Nostrand Reinhold: Princeton. Morichi.T. Dairy Res. III.E. Biological response of lactic streptococci and lactobacilli to catalase. 99. 785.. Hup. Freezing of lactic cultures.A. 38. Elliker. Dairy Res.E.Hoang-Dung TRAN and friends 88. J. Speckman.A. D -Leucine as an auto-inhibitor of lactic streptococci.-L..... E..L.S. 551. MI. Appl. W.L. 109. Priidak. Relationship of cellular components to the stability of concentrated lactic Streptococcus cultures at – 178C.A. 96... Le Lait 1968. Milk Dairy J. Dairy Res. J.D. Acta Microbiol. J.A. 1969.M. 1983.L. 27. S. 95. H. 805. 24.. J. 357. D. M. 259.J. Dairy Technol. 444...G. Sandine. Death of Lactobacillus bulgaricus resulting from liquid nitrogen freezing. Dairy Congr. Gilliland. Condon. Gilliland. Goldberg. Lyophilized lactic acid starter culture concentrates preparation and use in inoculation of vat milk for cheddar and cottage cheese.L. Peppler. Dairy Res. Reinbold. L.. 51.. Lloyd. Preparation of concentrated lactic streptococcus starters. 1968. Keen. H. C. McKay. J. Speck. J.. Cheese and Fermented Foods. Appl. 29. Smittle.L. G. 489. Boisen. J. 20 pp. L. 101. 182. J. 94. Evaluation of lactic acid streptococci for the preparation of frozen concentrated starter cultures. 110.A.L. 23. ¨ ¨ Petterson. 1974.R. Survival of Lactobacillus bulgaricus during freezing and freeze-drying after growth in the presence of calcium. J. S. G. Microbiol. Efstathiou. 34.B. 1975. Optimum growth parameters of lactic streptococci used for the production of concentrated cheese starter culture.. Behaving av bakteriemassor for meijeri-industriellt bruk. Croissance de “Streptococcus lactis” dans un milieu A pH constant. Lund. 793. C.L. Dairy Sci. Bannikova. 1964. 103.. Pepsinized sweat whey medium for growing Lactobacillus acidophilus for frozen concentrated starters. Bauman. Klaenhammer.M. L.E. 773. Libudzisz. G. L. In Microbiology.R. NJ. Buckley. Jorskning. Kosikowski. Selection of lactic acid bacteria for production of cultured concentrate. 1974. 797. 1969. Appl. M. E. 1969. Appl. T. 1977. Japan. Pont. 102. C.R. Ed. Microbiol. Klaenhammer.A. 40. J. M. S. 59 pp. Z. 49. 1968. 105.. M. Edwards Brothers: Ann Arbor. 1979.T.

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P.84. HOLZAPFEL Federal Research Center for Nutrition and Food. enterococci have emerged as important nosocomial pathogens. on the other hand. They are ubiquitous microorganisms. 199 Copyright © 2004 by Marcel Dekker. Inc. they give rise to concern because of the potential transfer of antibiotic resistance. either where they are purposefully added to the product as starter cultures[165] or where their presence results from environmental contamination. more importantly.Hoang-Dung TRAN and friends 6 The Genus Enterococcus: Biotechnological and Safety Issues CHARLES M. FRANZ and WILHELM H. the fact that certain Enterococcus strains can cause human disease. As a result of their natural association with the gastrointestinal tract as well as functional and technologically desirable properties. and clinical microbiology. INTRODUCTION The enterococci are lactic acid bacteria (LAB) that are important in environmental. Over the last two decades. Karlsruhe. Enterococci are typical opportunistic pathogens that may cause infections.117] The detrimental activities of enterococci are related to spoilage of foods. A. Enterococci are also of technological importance in the production of various European fermented foods such as sausages and cheeses. while. desirable for use as starter cultures in food production or as probiotics. the possible presence of virulence factors.[6.[6. . but have a predominant habitat in the gastrointestinal tracts of humans and animals. Germany I. Institute of Hygiene and Toxicology.[84] They occur as natural contaminants on meats as a result of contamination from the gastrointestinal tract at the time of slaughter. some strains are also used successfully as probiotics. especially in the nosocomial setting in patients who have underlying disease. All Rights Reserved. and their role in human disease. food. and this rise in their association with human disease can be explained in part by their increasing resistance to antibiotics as well as their promiscuity regarding transfer of genetic material. especially meats and. on the one hand.302] This ambiguous nature of enterococci makes them.

thermophilus and. E. the species epithet faecalis was suggested.Hoang-Dung TRAN and friends II. chemotaxonomic and phylogenetic studies have resulted in the assignment of more than 20 species to the genus Enterococcus (for reviews. This explains why the history of enterococci cannot be separated from that of the genus Streptococcus. but the actual number fluctuates from time to time as individual species are reclassified or new taxa are discovered.[5.14] Phylogenetically. and Descheemaeker et al. azikeevi. the so-called clostridial subdivision of the gram-positive bacteria. gilvus.16]). and serological studies. A 16S rRNA-based Copyright © 2004 by Marcel Dekker. Streptococcus.[21] could not distinguish between the two using either protein analysis or PCR-based typing. and Weissella. Lactococcus.[14] Since 1984.13. Aerococcus.[5. porcinus is a junior synonym of E. and lactobacilli are more distantly related. see Refs. Pediococcus. the 16S rDNA sequences of a possible new species. casseliflavus. canis. This correlated with the grouping of Sherman. and enterococcus. together with the other genera of LAB: Aerococcus.[7 – 9] and the separation was confirmed on the basis of 16S rRNA sequence analysis. lactococci.24] De Graef et al.[5] The fecal streptococci that were associated with the gastrointestinal tract of humans and animals with some fermented foods and with a range of other habitats constitute the new genus Enterococcus. modern classification techniques. The enterococcus group included Streptococcus faecalis. Streptococcus faecium. AJ309563). Streptococcus bovis. lactic. Lactobacillus. Carnobacterium. and Abiotrophia. Lancefield[3] developed a serological typing system for streptococci in which those of fecal origin possessed the group D antigen. Oenococcus.14. Melisococcus. and Streptococcus equinus as the enterococcal or group D strains. Globicatella. more recently. Vagococcus.[14] Enterococcus solitarius [22] was shown to be more closely related to the genus Tetragenococcus. Tetragenococcus. Inc. and E.[23. Moreover. Leuconostoc. E. Because of their close resemblance to strains isolated from the human intestine. For example. and Enterococcus.[4] who proposed a new classification scheme for the genus Streptococcus that separated it into four divisions designated pyogenic. This was first based on DNA : DNA and DNA : rRNA hybridization studies. which has nomenclatural priority. Enterococci belong to the Firmicutes with low mol% G þ C content in the DNA. . Lactococcus. Tetragenococcus.[11. E. with the exception of S. All Rights Reserved. viridans.[17 – 19] The species Enterococcus flavescens [20] appears to be identical to E.12] were separated from the nonpathogenic and technically important species of the new genus Lactococcus. TAXONOMY The genus Enterococcus was proposed by Thiercelin and Jouhaud[1] for gram-positive diplococci of intestinal origin.[18] showed that E.[10] The typical pathogenic species remained in the genus Streptococcus and. Andrewes and Horder[2] classified potentially pathogenic bacteria from a patient with endocarditis as Streptococcus faecalis. Globicatella. The phylogenetic relationship of the different species within the genus Enterococcus has been determined by comparative sequence analysis of their 16S rRNA genes. pallens.6] Members of the genus Streptococcus formerly grouped as faecal streptococci or Lancefield’s group D streptococci were subdivided into three separate genera: Streptococcus. Streptococus macedonicus. but this species has not been further described. phoeniculicola were only described recently.13. the closest relatives of the enterococci are the genus Vagococcus followed by Carnobacterium. Dolosigranulum. villorum. has been submitted to GenBank (GenBank accession no.[15] while the streptococci.[5. Alloiococcus. Facklamia. E.

E. dispar group: E. E. 1. E. avium. raffinosus. pallens E. E. E. E. hirae. avium group: E. the following species groups can be distinguished: 1. villorum. sulfureus Figure 1 Distance matrix tree showing the phylogenetic relationships of Enterococcus species based on 16S rRNA sequence comparisons. dispar. E. canis. solitarius was used as the outgroup. asini. gallinarum. . durans. Based on these data. E. E. 4. E. gallinarum group: E. E. E. faecium. azikeevi E. faecium group: E. mundtii. 5. 2. Copyright © 2004 by Marcel Dekker. casseliflavus. saccharolyticus group: E. and the bootstrap probability values (%) are indicated at the branch points (200 tree replications). E. gilvus E. flavescens E. Inc. malodoratus. E. E. E. 3.Hoang-Dung TRAN and friends phylogenetic tree of Enterococcus species is depicted in Fig. E. saccharolyticus. pseudoavium. All Rights Reserved.

[30] were able to accurately identify Enterococcus species by PCR amplification and sequencing of a conserved internal fragment of the D-alanine : D-alanine ligase genes (ddl). E.[18. pseudoavium. Tetragenococcus. gallinarum.[27] A variety of genotypic methods have been used successfully to identify enterococci to genus or species level. De Graef et al.[18] showed that these tests are not universally applicable. all others are nonmotile. gram-positive cocci which are arranged in pairs or short chains. with the increasing number of newly described species. E. avium and E. and E. in the presence of 40% bile.26] The Voges-Proskauer (VP) reaction and acid production from ribose have been suggested to have high differential value. E. and at pH 9. Williams et al.[28] For differentiation between enterococci and lactococci on the genus level. although with some species.. The traditional or “old” Enterococcus species (E. while sequencing of the 16S rRNA gene yields accurate species identification and can aid in the description of new Enterococcus species (see above).[34] Gelsomino et al.[35] and Vancanneyt et al.[29] described a rapid PCR-based method based on amplification of a region of the 16S rDNA gene.14] However. durans. cecorum.. E.6 (Table 1). e. pediococci.. .[36] showed that Enterococcus species can be differentiated quite well by RAPD-PCR. E. the traditional phenotypic identification using genus-specific characteristics has become exceedingly difficult.. E. and Vagococcus within the family Enterococcaceae. pallens. E. especially because in their study on E. E. many of the more recently described or “new” Enterococcus species vary in their physiological properties from those of the typical enterococci (Table 1). mundtii) can be easily distinguished from other gram-positive. in 6. and lactobacilli.[21] Quednau et al. Endospores are absent.[33] Andrighetto et al. IDENTIFICATION The genus Enterococcus is found together with the genera Melissococcus.. All enterococci are facultatively aerobic chemo-organotrophs with a fermentative metabolism. E. canis the strains from this species were mostly VP-negative. especially in discriminating enterococci from streptococci. homofermentative cocci such as streptococci and lactococci by their ability to grow both at 108C and 458C. Ozawa et al. E. like those of the genera Streptococcus and Lactococcus. Reliable identification of the genus Enterococcus and its species thus ultimately relies on the use of a combination of phenotypic. 7.5% NaCl. faecalis. Hoang-Dung TRAN and friends E. with L (þ)-lactic acid as the predominant end product of glucose fermentation. cecorum group: E.6. gallinarum and E. However. the cells are frequently arranged in pairs and are elongated in the direction of the chain. Inc. faecium. ratti III. catalase-negative.[24] Descheemaeker et al.[25] Members of the genus Enterococcus. hirae. such a differentiation was not possible. haemoperoxidus. Tyrrell et al. E. and these are reviewed by Domig et al. and phylogenetic information in a polyphasic taxonomy approach as described by Vandamme et al. mundtii. All Rights Reserved. faecalis group: E. if not impossible. Copyright © 2004 by Marcel Dekker.[32] used tRNA intergenic spacer PCR for the identification of enterococci species. are catalase-negative. columbae E.[5. gilvus are yellow-pigmented. faecalis. lactococci. casseliflavus. They showed that using this method they could accurately separate enterococci from streptococci. sulfureus. Deasy et al.. Baele et al. Thus. E. E. genotypic.g. moraviensis.[31] used restriction fragment length polymorphism of the 16S/23S intergenic spacer region to distinguish the Enterococcus species. They have a homofermentative lactic acid fermentation. E. casseliflavus are motile. E. Within the chains.

casseliflavus E.d. differing reports in literature. modern taxonomic studies based on molecular biological techniques for classifi¨ cation and species identification by Ott et al. n.d. variable. n.. n.d. avium E.. haemoperoxidus E. columbae E.d. n. villorum 108C (þ) V þ 2 2 þ þ þ þ V/2 þ þ þ þ þ þ þ þ n. n. i. n.5% NaCl 2 V V/þ 2 2 þ/2 þ þ þ þ þ þ þ þ þ þ þ þ 2 þ þ/2 þ þ (þ) þ þ þ 40% Bile þ V/þ þ (þ) (þ) þ þ þ þ þ þ þ þ þ þ þ þ þ 2 n. in human foods.d.d.d. n. not determined.d þ þ (þ) þ þ þ þ n. A. þ þ þ þ 0. enterococci occur in a truly epiphytic relationship. pallens E. Inc. All Rights Reserved.d. solitarius E. on plants. n.d. phoeniculicola E.[38] The early studies on the characteristics of the epiphytic life of enterococci (fecal streptococci) on plants by Mundt et al. þ Esculin Group D hydrolysis antigen þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ n. asini E. V/þ V/þ n.d. surface waters.Hoang-Dung TRAN and friends Table 1 Characteristic Physiological Properties of Validly Described Enterococcus Species Growth at Growth in the presence of 6.d. þ 2 2 2 þ þ þ þ þ n. n. ratti E. sulfureus E. pseudoavium E.d.d. (þ). moraviensis E. saccharolyticus E. pH 9.d. þ/2.d.d.e.[39] and Muller et al. n.d þ þ þ þ þ þ 2 n. IV. dispar E. Species E. n. HABITAT Environment Enterococci occur in a wide variety of environmental niches.d.d. faecium E.d. mundtii and E.d. raffinosus E.d.04% Sodium azide n. including soil.d.[6] Certain Enterococcus species are known to be typically associated with plants.[38] were ¨ performed before the genus Enterococcus was redefined by Schleifer and Kilpper-Balz. þ þ n. n.d. þ þ n.d. þ þ (þ) n.[7] however. cecorum E.d. porcinus E. þ þ þ n. casseliflavus.d. as a result of association with plants and animals.d. 458C (þ) þ þ þ n.d. the yellow-pigmented E. hirae E. durans E. gallinarum E.[37] On plants.d. þ þ n. 2 þ þ þ V/þ þ þ 2 þ 2 2 þ þ n. n.d.d. þ þ þ þ þ þ þ þ þ þ þ 2 2 2 (þ) þ V þ þ þ þ V þ þ þ þ n. þ þ n. in the gastrointestinal tract of warm-blooded animals (including humans).d. and municipal water treatment plants. malodoratus E. faecalis E.d. and. n. gilvus E.d.[40] validated this epiphytic Copyright © 2004 by Marcel Dekker. . n.d. weak positive.d. mundtii E. n. n. waste waters. þ 2 n. þ n. (þ) 2 þ 2 n. V.d. flavescens E. þ n.6 n.d. n.

but can be associated with processed meats. bovis was the predominant group D organism isolated from feces of dairy cows.[50] These pig carcasses from three different slaughter plants contained mean counts of 104 –108 enterococci per 100 cm2 of carcass surface throughout processing. faecium. Enterococci were also consistently isolated from beef.45] C. and E.46] The intestinal microflora of young poultry contained principally E. but E. E. faecium. durans has been isolated from humans. in the human bowel. faecalis predominated among the gram-positive cocci isolated from chicken samples collected at abattoirs. faecalis. In a study on poultry. while E. hirae and E. faecalis.[49] while in another both E.43] Although E. In raw meat products. chickens.72 CFU/g of chicken carcasses from five retail outlets in Spain.[51] showed that E. faecalis in human faeces range from 105 to 107/g compared with 104 to 105/g for E. they are less prevalent in livestock such as pigs. 1. isolated from pig carcasses. faecalis. avium species group (E. faecium. S. casseliflavus. cats. E. E. and calves. E. and E. faecalis was shown to be the predominant isolate from beef and pork cuts in one study. faecium predominated in fecal samples. while E. possessed a 16SrDNA genotype uncommon to Enterococcus species described at the time of the study. and to a lesser extent E. sulfureus. hirae. poultry. faecalis. and E. columbae is an important member of the gut flora of pigeons. Gastrointestinal Tract Enterococci are well known to occur as part of the natural microflora of the intestinal tract of warm-blooded animals and humans and constitute a large proportion of the autochthonous bacteria associated with this ecosystem. and E.[54 – 58] Enterococci not only may contaminate raw meats. E. cecorum were the enterococci most frequently isolated from pig intestines. E. E. faecium. or pig carcasses or fresh meat cuts in studies of antibiotic resistance of enterococci.[14. and E. All Rights Reserved. faecalis. E. and sheep.[5.[52] Capita et al.41] Numbers of E.[44] In a study by Devriese et al. and enterococci occurring on plants were identified as E. faecium outnumbers E. cattle. faecium. Inc. The habitat of the members of the E. malodoratus. hirae frequently occurs in the intestine of pigs but may also occur in the gut of poultry.[45] E. and dogs. and E. mundtii. faecalis is often the predominating Enterococcus sp. faecium from pre ruminant calves.[50] Devriese et al.[40] however.[44.[42. Cooking of processed meats may confer a selective advantage on enterococci as these bacteria are known to be among the most thermotolerant of the Copyright © 2004 by Marcel Dekker. Foods Meats The presence of enterococci in the gastrointestinal tract of animals clearly leads to a high potential for contamination of meats at the time of slaughter. faecalis was isolated from feces of pre ruminant calves and ruminating young cattle and dairy cows. durans occurred in meat and prepared meat products. durans are frequently isolated from human feces. faecium and E. faecium. cattle. B. E. avium. E. E.[47] E. raffinosus.Hoang-Dung TRAN and friends relationship. E. pseudoavium) otherwise is largely unknown.[53] found enterococci to occur at a mean count of log 2. E. faecalis and E. although in some individuals and in some countries. malodoratus is often found in the tonsils of cats. The majority of the isolates in the study of ¨ Muller et al. faecalis were the most predominant Enterococcus spp. cecorum predominated in the intestine of chickens over 12 weeks old. . E. but not from ruminating young cattle or dairy cows.[48] E.

Inc.75. Salami and Landjager were shown in one study[70] to contain enterococci at numbers ranging from 102 to 105 CFU/g. faecalis is a frequent contaminant.[61 – 64] Enterococci are also isolated from certain types of fermented sausages.[59. have also been associated with African fermented sorghum Copyright © 2004 by Marcel Dekker.[97] It has also been suggested that enterococci can use the antimicrobial compound oleuropein in olive grapes as a growth substrate. contaminated water.90 – 95] 3.89] and contribute to the ripening and aroma development of these products due to their proteolytic and esterolytic activities. not all of the lactic acid bacteria are suited to grow at the relative high pH conditions resulting from alkaline treatment of the olive grapes to hydrolyze the bitter glucoside oleuropein.81. thereby preventing the outgrowth of Listeria monocytogenes and slime-producing lactic acid bacteria. De Castro et al. thus promoting the thermotolerant bacteria present. faecalis. Enterococci can grow in the restrictive environment of high salt content and low pH of the cheese[72. durans are most often isolated from such cheeses (Table 2).86 – 88] Numbers of enterococci in cheese curds range from 104 to 106 CFU/g.87. particularly the dry fermented sausages known as chorizo[65 – 67] and espetec[68] produced in Spain. natural milk starters are made by pasteurising milk at 42– 448C for 12 –15 hours. Cheese Enterococci occur in many traditional European cheeses manufactured in mostly Mediterranean countries from raw or pasteurized milk.[96 – 101] in which E. Because of their tolerance to the high pH values and salt concentration used in the olive brine. and these may contribute to ripening and product flavor. the enterococci may survive and contribute to the fermentation of meat products.[72 – 83] The source of enterococci in milk and in such cheeses is thought to be the feces of dairy cows.[69] or in sausages such as salami ¨ ¨ and Landjager produced in many European countries. some enterococcal strains have the ability to produce enterocins harboring antimicrobial activity against pathogens and spoilage microorganisms of meat concern. thermophilus strains and Enterococcus spp. Thanks to their competitive advantage over other microbiota in meat fermentations. .[34. Such enterocinproducing enterococci or their purified metabolites may be applied as extra hurdles for preservation in sausage fermentation and in sliced-vacuum packed cooked meat products.60] After surviving the heat-processing step.[72. the enterococci.[85] Strains belonging to the species E. faecalis strains. the enterococci appear to be well suited for growth at these conditions. faecium. In addition. All Rights Reserved. or milking equipment and bulk storage tanks[84] as well as natural milk starters.[97] suggested that lactic acid bacteria growing at the beginning stages of the olive fermentation are important for improving the hygiene of the product.77.76.[102] thus lowering the toxicity of the fermentation medium for growth of other LAB. as well as the production of diacetyl. However. in Italian sausages.[71] 2.[85] The isolation of enterococci from natural milk starters can be explained by their heat resistance. and they frequently occur in retail fermented olives. faecalis and E. In addition. faecium have been implicated in spoilage of cured meat products such as canned hams and chub-packed luncheon meats. and in the fully ripened cheeses from 105 to 107 CFU/g (Table 2). Fermented Vegetables Enterococci occur in a variety of fermented vegetables.Hoang-Dung TRAN and friends non-sporulating bacteria. especially E. which include S. E. but it is often not clear whether they originate from the plant material itself or as environmental contaminants. Enterococci have been isolated also from Spanish-style green olive fermentations. both E. and E.80 – 82.

2%) Lactobacilli Leuconostocs Enterococci Lactococci.r.7 Cheese White-brined cheese Country of origin Greece Milk source Raw goat’s milk or mixed goat’s and ewe’s milk ewe’s milk. n.r. 86 Orinotyri cheese La Serena ewe’s milk cheese Manchego cheese Greece Spain Raw ewe’s milk Raw ewe’s milk n. in Cheeses from Mediterranean Countries Enterococci in curd (log CFU/g) 4.8 75 Teleme cheese Greece Pasteurized ewe’s milk n.2 6. plantarum (47%)b E. paracasei (10%) E.4%) L. Inc.2%) Pediococci (9. leuconostocs Lactobacilli Leuconostocs Enterococci Enterococci Ref.a n. faecium (12%) L.0 Enterococci at end of ripening (CFU/g) 6.9 5.r.2 83 74 Spain Raw ewe’s milk n.r. Table 2 Numbers and Predominance of Enterococcus spp. faecalis (9%) E. 72 .8%) E.8 7. enterococci. paracasei subsp. cow’s milk or mixed ewe’s and goat’s milk Predominant bacteria in end product (% of isolates) L.6%) L. 86 Kefalotyri cheese Greece 4.r.Hoang-Dung TRAN and friends Copyright © 2004 by Marcel Dekker. casei (15. faecium (35. casei subsp. 6. durans (9. plantarum (18. All Rights Reserved.

E. L. E. mesenteroides subsp.3 Caprino d’ Aspromonte Serra cheese Italy Portugal Raw or heated goat’s milk Raw ewe’s milk 4 –6 n.Hoang-Dung TRAN and friends Copyright © 2004 by Marcel Dekker. casei subsp. ¼ Lactococcus. E. Lact.r. spp. Lact. faecium. Cebreiro cheese Spain Raw cow’s milk n. Leuc. E. n. 5– 7 n. E. mesenteroides Enterococci. E. faecalis. durans. L. Staph. faecalis (var. L. 6. lactis. faecalis. mesophilic and thermophilic cocci Leuc. lactis (19. faecalis (30.9%) Leuc. E. faecium E. ¼ Enterococcus.r.8%) E. Micrococcus spp. lactis. liquifaciens) (11. paracasei 81 301 82 80 76 87 n.1%) E. casei. E.r. mesenteroides subsp. faecium. . All Rights Reserved. ¼ Staphylococcus. Leuc. mesenteroides (6.r.r. faecium (4. L.9%) Lact. durans. plantarum. Picante da Beira Baixa cheese a b Portugal Mixture of raw goat’s and ewe’s milk n. lactobacilli.r.r. 6– 7 7.5 ´ San Sımon cheese Tetilla cheese Spain Spain Raw cow’s milk Raw cow’s milk 5 –6 n. ¼ Leuconostc. Leuconostoc mesenteroides subsp. ¼ Lactobacillus.0%) W. faecalis.3%) E. ¼ Weissella.) paramesenteroides (7. mesenteroides/ dextranicum. Inc. Staph. W. ¼ not reported. (Leuc.

BIOTECHNOLOGICAL IMPORTANCE OF ENTEROCOCCI Bacteriocin Production Many enterococci isolated from the environment or from foods were shown to be bacteriocinogenic.[122] The two structural subunits are encoded by two open reading frames (ORFs) (cylLL and cylLS). faecium strains. and secretion mechanisms have been reviewed previously. enterolysin A. Enterocin Classification and Characteristics Bacteriocins are microbially produced. A.68. It consists of two peptides. Q. mode of action. L50. faecalis. and generally these exhibit activity towards listeriae. heat-stable bacteriocins which were divided into three subgroups by Nes et al. monocytogenes has prompted a great amount of research for using bacteriocin-producing enterococci as “protective cultures” or their purified bacteriocins in the biopreservation of foods (see below). class IIb includes the two component bacteriocins. AS-48.[111]: class IIa comprises the pediocin-like bacteriocins that contain a conserved YGNGVXC amino acid motif at the N-terminus and which generally have a strong anti-Listeria effect.[107 – 112] The currently most widely used classification system of bacteriocins produced by LAB is that of Nes et al. and E. All Rights Reserved. and most isolates consist of E. Inc. immunity. heat-labile proteins. The bacteriocins from enterococci are usually referred to as enterocins.[121.[121] Cytolysin has bacteriocin as well as haemolytic activity and thus is active against both eukaryotic cells (erythrocytes) and gram-positive bacteria. 1.114] Class III bacteriocins are typically large (.[105] V. the enterococci contribute only a minor part of the microbial population associated with the fermentation (approximately 10% of the isolates from Hussuwa).[111] but some have unusual structural or genetic characteristics that do not allow grouping into this classification scheme (Table 3).122] The genetic locus for cytolysin production is located on the 58 kb pheromone-responsive plasmid (see virulence factors below) pAD1.104] In our own studies on traditional fermented African foods.111] Class II bacteriocins are small (4 –6 kDa).[111] who grouped these bacteriocins into three classes. mundtii have been described and include the well-characterized enterocins A. B. Cytolysin is the only known enterocin that can be classified as a class I bacteriocin. usually against closely related strains[107] (see also Chapter XX). E.[113.[103. enterococci were also associated with the fermentation of products such as Hussuwa made from sorghum in the Sudan and Okpehe made from locust beans in Nigeria.[66. membrane-active peptides with antimicrobial activity. Lantibiotics contain the unusual amino acids lanthionine and b-methyllanthionine.Hoang-Dung TRAN and friends foods.10 kDa). P. The preprotein encoded by these Copyright © 2004 by Marcel Dekker. . EJ97. Class I bacteriocins are ribosomally synthesized lantibiotics that undergo extensive posttranslational modification to produce an active peptide. Their genetics. and class IIc consists of bacteriocins that are secreted via the sec-pathway or preprotein translocase. production.[106] The food origin of many enterocin producers and the activity towards the important foodborne pathogen L.115 – 120] Some of these enterocins can be readily grouped into one of the bacteriocin classes as defined by Nes et al. Class I Enterocins.[107. Enterocins produced by Enterococcus faecium. 1071. both of which contain lanthionine residues. bacteriocin 31. In these foods. and mundticin KS. mundticin.


All Rights Reserved.[125] Cintas et al. . and Micrococcus spp.[118] Mundticin KS is identical to the previously described mundticin produced by E. and it is active against strains of Enterococcus.98 and 2031.125] Enterocin L50 consists of two structural subunits. innocua. including Clostridium tyrobutyricum. L. (Table 3) and is produced as a prepeptide bearing an 18-amino-acid leader peptide of the double-glycine type. The genetic locus for enterocin L50 was cloned and sequenced and contained the EntL50A and B structural genes encoding the 44-amino-acid (EntL50A) and 43-amino-acid (EntL50B) peptides.[118] Thus. durans. respectively. Enterococcus spp. peptides L50A and L50B. monocytogenes. mundtii ATO6[124] (see below). Enterocin L50 exhibits a wide antimicrobial spectrum. The two peptides have homology only to the a and b peptides of the two-component bacteriocin lactococcin G.Hoang-Dung TRAN and friends ORFs are posttranslationally modified to yield the active subunits CylLL00 and CylLS00 with a molecular mass of 3437. Inc.[99.122] (Table 3)..and 34-amino-acid mature enterocin 1071A and 1071B peptides.[125] suggested that although enterocin L50 may be considered a class IIb bacteriocin according to the classification systems of Nes et al. It is produced by E. respectively (Table 3).[125] The A and B peptides have 31-amino-acid residues in common.[65. Enterocin 1071 (Ent 1071) is produced by E. E.[117] The molecular mass of the purified bacteriocins is 4285 and 3899 Da for enterocins 1071A and 1071B. Pediococcus pentosaceus. monocytogenes and B. Mundticin KS produced by Enterococcus mundtii NFRI 7393 also belongs to the class IIa bacteriocins and exhibits activity against Lactobacillus spp. Lactobacillus. as well as the foodborne pathogens L. as well as L. respectively. faecalis strains BFE 1071[116] and FAIR-E 309[117] as a two-peptide bacteriocin from the two structural subunits enterocin 1071A and B. and they are 72% identical. faecium strains L50 and 6T1a.[117] Both bacteriocin subunits are encoded as prepeptides. CLASS IIB ENTEROCINS .117] and DNA sequencing revealed two ORFs encoding the 39..81 Da. however.[99. as well as strain BFE 900 isolated from black olives. as the structural genes do not encode prepeptides but encode the mature bacteriocin without an N-terminal extension.123] EntA consists of 47 amino acids with a theoretical molecular weight of 4829 Da. This enterocin has antimicrobial activity against a broad range of gram-positive bacteria.68. both of which have antimicrobial activity and which exhibit synergism when combined. monocytogenes.125] EntL50A and B are different from other class II bacteriocins.[118] The mature bacteriocin contains 43 amino acids (Table 3) and is produced as a preprotein bearing a 15-amino-acid leader peptide of the double-glycine type. except that the last two C-terminal amino acids are inversed in position when comparing the amino acid sequences of mundticin and mundticin KS. while mundticin contains Ser42 Lys43. Enterocin A (EntA) is a class IIa bacteriocin that contains a N-terminal YGNGVXC motif and is active against L. faecalis. cereus.[116] The genetic determinants for this two-peptide bacteriocin are located on a plasmid DNA. E.[117] Enterocin L50 (Ent L50) is produced by E. mundticin KS contains Lys42 Ser43. which have theoretical molecular weights of 5190 and 5178 Da (Table 3). Class II Enterocins CLASS IIA ENTEROCINS .[116] and Franz et al. L. Lactococcus lactis.[111] it has Copyright © 2004 by Marcel Dekker.[116. salivarius.[116] Chemical and genetical characteristics of enterocin 1071 were studied by Balla et al. respectively[121. faecium strains CTC492 and T136 (both isolated from Spanish fermented sausages). characteristics that allow grouping of this bacteriocin as a class IIa bacteriocin. each bearing a double-glycine-type leader peptide of either 18 amino acids (Ent1071A) or 24 amino acids (Ent 1071B).

and sakacins A and P. and it is thus not known whether this bacteriocin is secreted by dedicated transport proteins or by the bacterial pre protein translocase[111]. Enterolysin A is inhibitory towards Enterococcus.[126] Alignment of the bacteriocin 31 and enterocin P signal peptide amino acid sequences showed that they share sequence identity of 12 amino acids. Enterocin CRL35 produced by E.[127] has strong sequence similarity at the N-terminus with mundticin (Table 3). mundtii strain isolated from processed vegetables[124] consists of 43 amino acids and contains a YGNGVXCconsensus motif at the N-terminus of the bacteriocin molecule. both isolated from Spanish fermented sausages. botulinum. monocytogenes.501 Da). . it is not clear whether mundticin belongs to the class IIa or class IIc bacteriocins as defined by Nes et al. botulinum. this bacteriocin has a YGNGVXL motif at the N-terminus.[111]. cereus. For this reason. Lactobacillus. and L.[66. haemolyticus. and sakacin P. and the genetic determinants involved in production for this bacteriocin are located on a pheromone-responsive plasmid.. the characteristic cysteine that is located two amino acid residues from the valine is not present. It is a large (calculated molecular weight of 34. and it is active against Enterococcus spp. therefore. Class III Enterocins. Inc.[127] The full amino acid ´ sequence of this bacteriocin has not been determined. and Pediococcus spp. Enterolysin A is a bacteriocin produced by E. C. The genetics of mundticin production have not been studied.[66] Bacteriocin 31 is produced by E. leucocin A.[126] Bacteriocin 31 is also secreted by the bacterial preprotein translocase and contains a 24-amino-acid signal peptide. however.[66] The mature enterocin P bacteriocin consists of 44 amino acids and has a theoretical molecular weight of 4493 Da (Table 3). OTHER CLASS II ENTEROCINS . faecium CRL35 shows strong sequence homology to other class II bacteriocins. lugdunensis. faecalis LMG 2333.[66] which is a determinative characteristic for grouping this bacteriocin into class IIc bacteriocins according to the classification scheme of Nes et al. curvacin A. occurs by the bacterial preprotein translocase. carnobacteriocins BM1 and B2. monocytogenes and C. heat-labile bacteriocin[128] and therefore fits the general characteristics of class III bacteriocins. Mundticin has a molecular weight of 4287 Da as determined by mass spectrometry and has activity against Enterococcus.[128] The bacteriocin is encoded as a prepeptide consisting of a 316-amino-acid protein bearing a 27-amino-acid signal Copyright © 2004 by Marcel Dekker. and S. Clostridium perfringens. and Pediococcus spp.Hoang-Dung TRAN and friends more in common with a group of staphylococcal peptide toxins. monocytogenes. leucocin A. aureus. The gene encoding the prepeptide of enterocin CRL35 has not been cloned and sequenced. Instead. Lactobacillus. S. SLUSH A – C and AGS1 –3 produced by Staphylococcus aureus. S.[111] Enterocin P has sequence similarity with the other class II bacteriocins. and B. produced by an E. All Rights Reserved. CLASS IIC ENTEROCINS . Secretion of enterocin P.115] Its antimicrobial spectrum includes activity against Enterococcus. Leuconostoc. and Lactobacillus spp. It also contains a YGNGVXC motif near the N-terminus (Table 3). Enterocin P is produced as a prepeptide of 71 amino acids with a 27-amino-acid signal peptide (Table 3). faecalis YI717. The genetic determinant for enterolysin A was cloned and sequenced. which include d-lysin.[66] The mature bacteriocin 31 consists of 43 amino acids (Table 3).. Lactococcus. therefore it is not yet clear whether this bacteriocin belongs to the class IIa or class IIc bacteriocins. as well as L. faecium strains P13 and L50. The sequence reported by Farıas et al. Enterocin P (EntP) is produced by E. mesentericin Y105. Pediococcus. Mundticin. and L. such as sakacin A.

[12] Enterocin Q (EntQ) is produced by E. and Staphylococcus.[134] showed that the as-48 gene encoded a 105-amino-acid prepeptide consisting of a 35-amino-acid signal peptide and a 70-amino-acid mature peptide (Table 3). S. Enterocin Q also lacks the YGNGVXC consensus sequence at the N-terminus.[120] Enterocin AS-48 is produced by a clinical isolate of E. like Enterocin L50. bacteriocin 21 and enterocin 4 was subsequently reported for other strains of E.[120] A search of the protein databanks did not reveal homology to any of the bacteriocins described to date.[12] It is possible that EntB is secreted by the dedicated transport proteins of EntA or some other transport system available in the cell. monocytogenes. T136. Cintas et al.65] This indicated that EntB was secreted by a dedicated-type secretion mechanism. Inc. which makes it different from the class IIa and class IIb bacteriocins.129] which also does not contain the YGNGVXC-consensus motif.[115] suggested that enterocin Q is transported from the cell by a presently undetected ABC transporter. Genes encoding putative transport proteins such as an ABC transporter and a putative accessory protein were found in close proximity of the Ent EJ97 structural genes and may be involved in transport and possibly immunity.[12. Enterocin EJ97 (Ent EJ97) is a bacteriocin produced by E. Enterococcus. Amino acid sequence comparison indicated homology to cell wall –degrading proteins such as lysostaphin produced by Staphylococcus simulans biovar.[114] Enterocin Q is a unique bacteriocin. Listeria.133] The structural genes for enterocin AS-48 ´ are located on a pheromone-responsive plasmid. is problematic. as has been demonstrated for other bacterial proteins that do not contain an N-terminal leader or signal peptide. and Martınez-Bueno et al. which are all endopeptidases belonging to the M37 protease family.[12. faecalis.[130] The genes for enterocin EJ97 production were detected on a 60 kb conjugative. staphylolyticus and to ALE-1.65] The bacteriocin consists of 53 amino acids with a molecular weight of 5463 Da (Table 3).[131] Production of identical bacteriocins. Enterocin B shares sequence similarity only with carnobacteriocin A. faecalis strain S-48. and the structural gene encodes a 44-amino-acid bacteriocin (Table 3) that. which is inhibitory to species of Bacillus.[128] Atypical Enterocins. faecium L50 in addition to the bacteriocins enterocin P and enterocin L50. AS-48 forms a 70-aminoacid cyclic molecule that results from a head-tail linkage of the N-terminal Copyright © 2004 by Marcel Dekker. an assignment of this enterocin into one of the class II subclasses. and BFE 900. This feature (absence of a N-terminal extension involved in bacteriocin transport) actually makes both the enterocin L50 and enterocin Q atypical enterocins. involving the ABC transporter and accessory proteins. and C. similar to EntL50 and EntQ.[132. perfringens. and the foodborne pathogens L. Enterocin B (Ent B) is produced in addition to EntA by E. Genetic analysis showed that the prepeptide contained an 18-aminoacid leader peptide of the double-glycine type. faecium BFE 900 that contained the structural gene for EntB did not contain ABC transporter or accessory protein genes.65. pheromoneresponse plasmid pEJ97.[12. Enterocin B is active against Enterococcus and Lactobacillus spp. is also not produced as a preprotein. LytM. due to the lack of a leader or signal peptide.Hoang-Dung TRAN and friends peptide. aureus.[12. and zoocin A. does not contain a N-terminal extension. All Rights Reserved. However. faecalis EJ97. faecium strains CTC492. . which cannot be grouped into any of the class II bacteriocin subgroups.[120] Again in this case.68] Enterocin B does not contain the YGNGVXC-consensus motif at the N-terminus of the mature peptide (Table 2). a 12. as it has a relatively small size of 34 amino acids (Table 3) with a theoretical molecular mass of 3952 Da[115] and.0 kb DNA fragment cloned from the chromosome of E.

faecalis. strains producing the enterocins A. which are otherwise generally produced by LAB. in which they are found together with a complex associative microflora.115.135] have been found in raw milk and dairy products. Inc. in part. also explain the success of these bacteria to successfully compete in such various niches such as the gastrointestinal tract and various foods. lactis and Lactobacillus paracasei added to milk prior to Camembert cheese making or sprayed onto the surface of the cheese. the decrease in L. but to which no bacteriocin was added. especially L. innocua. also isolated from cheese. As shown above. However.[140.5 days after brining.134.[150] Laukova and Czikkova[146] added the semi-purified bacteriocin enterocin CCM 4231 to bryndza cheese that was experimentally contaminated with L.[145] Sarantinopoulous et al.Hoang-Dung TRAN and friends methionine (Mþ1) to the C-terminal tryptophan (Wþ70). The effect of this bacteriocin appeared to be mostly bacteriostatic during the first 24 hours of cheese making. faecium EFM01. innocua growth. which may. 2. faecium. the cyclic nature of AS-48 makes this bacteriocin molecule quite unique when compared to the linear bacteriocins. Thus.[136 – 139] E. and non-Enterococcus starter cultures may influence bacteriocin production and Copyright © 2004 by Marcel Dekker.[134] Thus. Production of antilisterial bacteriocins by various enterococci from Spanish-style dry fermented sausages were proposed to make these suitable for addition to meat as co-cultures to improve food safety.[145] used an E.[146] Garcia et al.145 – 150] A bacteriocinogenic E. E.141] Another strain. monocytogenes. enterocins produce an impressive array of antimicrobial substances.[142] Similarly.[65. innocua counts during the ripening of the cheese appeared to be mostly due to the low cheese pH values.143] and espetec[68] produced in Spain.[93] used a bacteriocin-producing E. monocytogenes. faecium WHE 81 isolated from cheese produces enterocins A and B that also inhibit L. when surfaces were contaminated with ´ ´ Listeria not later than 1. no enterocin activity could be detected during cheese ripening. E. and activity could be detected in the cheese until the end of the ripening period. totally inhibited Listeria spp. nisin-producing L. and Q were isolated from certain types of fermented sausages. Only a slight reduction (1 log unit) was noted when comparing this cheese to a control inoculated with L. when pH was not low enough to inhibit L. particularly the dry fermented sausages known as chorizo[65 – 68. faecalis strain INIA4 which produces AS-48 to study inhibition of a mixture of L. L50. Use of Enterocins or Enterocin-Producing Enterococci in Biopreservation Bacteriocin production by enterococci isolated from dairy products has been investigated. has stimulated interest in using these bacteria or their purified bacteriocins for use in food production as biopreservatives.[93.[106] The bacteriocin was produced during drainage of the whey. P. and food-spoilage bacteria such as Clostridium tyrobutyricum. B. faecium strain was tested on a laboratory scale for use in combination with a commercial starter culture for Taleggio cheese making. faecium FAIR-E 198 strain as adjunct starter culture in feta cheese-making. which is involved in the “blowing” defect of cheeses.144] Enterocins or starter cultures containing bacteriocin-producing enterococci have been used in model studies to improve safety of the cheeses. All Rights Reserved. . varying levels of success were achieved and it was suggested that rennet.[106] An inhibitory starter culture consisting of bacteriocinogenic E. innocua at a similar level. innocua strains during manchego cheese making. while growth and acidifying activity of the thermophilic commercial starter culture was not inhibited. Bacteriocin activity of enterococci against foodborne pathogens. Strains producing enterocin AS-48[131. CaCl2. was shown to produce enterocin A.

[88] showed that some enterococci isolated from cheese produced acid at sufficient levels for production of artisanal cheeses. with E. as the complex food constituents may interfere with bacteriocin production levels.[34.158] confirmed the poor acidifying activity of enterococci in cheese production as only a small percentage of strains could decrease the pH below 5.151.[93] Enterocin-producing strains or purified enterocins have also been used for biopreservation of meats in model studies.Hoang-Dung TRAN and friends hence the successful inhibition of target bacteria.94] are traits that enable enterococci to occur in and grow in cheeses and meats. faecium.156] Morea et al.160. Inc.161] Although there are exceptions. Acidifying Activity As members of the lactic acid bacteria.[144.[144] showed that two bacteriocin-producing strains of E.145. durans isolates in particular were able to reduce the pH of skim milk below pH 4. faecalis strains possessing higher activity than other Enterococcus species.[157] showed that the pH of milk 24 hours after inoculation with enterococci strains isolated from mozzarella cheese did not decrease below pH 5.156. enterococci with desirable technological and metabolic traits have been proposed as part of defined starter cultures or as adjunct starter cultures for different European cheeses. faecalis strains isolated from Italian cheeses.[156.5 after 24-hour fermentation was observed for E. and as such these may already be considered as technological traits.[93. which is important for any food fermentation.0 – 5. Other investigations[34. faecalis generally appears to be a stronger acidifier than E.152] B.[151] In contrast. 1. Other Technological Traits of Enterococci for Use as Cheese Starter Cultures Because of their role in ripening and flavor development in cheeses. Conflicting reports on proteolytic activity of enterococci suggest a marked strain-to-strain variation of this phenotypic trait.[156] Delgado et al.4 after 24 hours at 228C. deboned chicken breasts. The bacteriocin-producing strain itself was not used as a starter culture. Lipolytic. because bacteriocin production and growth of the strain were inhibited by low temperatures and the salt and pepper ingredients used in the sausage recipe. minced pork meat.[88. Callewaert et al. faecium effectively inhibited a strain of L. and espetec. innocua in model Spanish-style dry fermented sausage. All Rights Reserved. can be considered as an additional technological trait.[88] E. ˆ ´ pate. Two E. Therefore.[90.2 after 16– 24 hours of incubation at 378C. enterococci produce lactic acid as end product of metabolism. generally the proteolytic activity of enterococci appears to be low.159] 2. showed a marked antilisterial activity in model meat and meat products such as cooked ham. Proteolytic.153 – 155] The ability of enterococci to grow at low pH and high salt concentration and their relative heat resistance[85. and this acidifying activity. .[151.149] Thus in vitro production of bacteriocins is no guarantee for in situ inhibitory efficiency.163] Copyright © 2004 by Marcel Dekker. Enterocins A and B from E.90. and lowering of skim milk pH to about pH 4. faecium CTC492. However. it was suggested that particular enterocins could be considered as additional biopreservative hurdles for successful prevention of listerial growth in fermented sausages. and Esterase Activity Proteolytic activity of enterococci for breakdown of milk casein is important for cheese ripening. when added as semi-pure preparations. in milk and in meats the enterococci generally exhibit only low acidifying ability.

When present as a sole carbon source in MRS broth without glucose. faecium. as this compound occurs in many natural food substrates such as milk. and fruits. . All Rights Reserved. which have importance as cheese flavor compounds.[87] indicated that citrate in milk was metabolized by E. Citrate Metabolism Citrate metabolism plays an important role in many food fermentations involving lactic acid bacteria. ethanol. vegetables.Hoang-Dung TRAN and friends Esterases are arbitrarily defined as enzymes that hydrolyze substrates in solution. Since citrate is a highly oxidized substrate.156. For this reason it would probably be better to use enterococci in food fermentations as adjunct starter cultures in combination with established starter strains. color. Sarantinopoulous et al. faecalis strains appearing to be most active. enhanced the water-soluble nitrogen fractions. faecalis. As shown above. have distinct aroma properties and can significantly influence the quality of fermented foods. Copyright © 2004 by Marcel Dekker.163] 3. faecium as adjunct starter cultures.[158] showed that strains of E. Sarantinopoulous et al. Inc. Sarantinopoulos et al. citrate was not catabolized but stimulated the growth of the E. thus having an effect on cheese texture.[156] Hydrolysis of triglycerides by enterococci has been reported. faecalis and to a lesser extend by E. It was shown that the presence of the enterococcal starter strains positively affected the growth of nonstarter LAB. the results of these study supported previous suggestions that enterococci indeed positively influence cheese fermentations. either single or combined. faecium strains appear to be more esterolytic than other enterococci species.[93] Clearly. faecalis strain. Lipolysis. Nevertheless. on the other hand. faecium. faecalis FAIR-E 229.[162] Some of these end products. such as NADH.[156] E. and positively affected taste.[162] showed that in skim milk citrate and lactose were co-metabolized by E. while minor amounts of lactate. E. rather than using these bacteria as defined starter cultures by themselves.[156] Esterases have been linked to the flavor development and cheese texture by lipolysis of milk fat and subsequent conversion of the free fatty acids produced to methylketones and thioesters. on the microbiological. aroma.162. no reducing equivalents. acetaldehyde. which can contribute to the texture of some fermented foods. and their proteolytic and esterolytic properties may not be high. and sensory characteristics of feta cheese in a well-defined study.[162] Freitas et al.[161 – 164] while the esterolytic system of enterococci appears to be complex and more efficient than their lipolytic system. physicochemical. increased the proteolytic index and free amino group concentration. and acetoin were also detected.[93] studied the technological properties of two strains of E. enterococci are not good acidifiers of milk and meats. while lipases hydrolyze substrates in emulsion. For example.[162] indicated that Enterococcus strains have the metabolic potential to metabolise citrate and thus actively contribute to flavor development of fermented dairy products. the breakdown of citrate also results in the production of CO2. durans varied in their ability to utilize citrate or pyruvate as the sole carbon sources. are produced during its degradation. The work of Sarantinopoulous et al. and E. the effect of the technological properties of the enterococci is not negligible and should not be underestimated. with E. and acetoin.[95. while in MRS broth containing citrate and lactose or glucose as the sole carbohydrate. In addition. such as diacetyl. faecalis FAIR-E 229 with the main end products being acetate and formate. structure. is not directly involved in cheese rheology but partial glycerides are tensio-active and influence molecular organisation. and the overall sensory profile of the cheese. which results in the formation of metabolic end products other than lactic acid. citrate was catabolized by E.

.[168] The wide distribution and association of enterococci with traditional fermented foods may to some extent explain the elevated levels of tyramine that may be found in these products. strengthening of the gut mucosal barrier. Administering the probiotic occurred at the time at which a vaccine for canine distemper virus was given. As a result of high metabolic activities. This probiotic has been claimed to be hypocholesterolaemic in the short term. thermophilus and one of E. Within the LAB.[181] but long-term reduction Copyright © 2004 by Marcel Dekker. Hoang-Dung TRAN and friends Production of Biogenic Amines During Fermentation Numerous bacteria. Enterococcus spp. D. and especially tyramine. Enterococci as Probiotics Functional effects claimed for probiotics include inhibition of pathogenic microorganisms. faecium. Inc. All Rights Reserved. It could be shown that fecal IgA and canine distemper vaccine – specific circulating IgG and IgA were higher in the treated puppy group when compared to the control group Furthermore.[175. it has been shown that levels of 1 –2% NaNO2 also result in increased production of tyramine in a model system with a strain of E. faecalis. mature B cells (CD21þ/MHC class IIþ) were greater in the group fed the probiotic. are also occasionally used as probiotics. including strains of LAB. and lowering of blood cholesterol levels.[175 – 178] The use of E.[179] Benyacoub et al.[169 – 174] Most probiotic cultures are of intestinal origin and belong to the genera Bifidobacterium and Lactobacillus. has been reported particularly for cheese and fermented sausages. concomitantly with an increased production rate.176] Several placebo-controlled. especially in protein-rich substrates such as meat and milk.[179] showed that turkey poults fed the probiotic showed a continuous increase in lactate concentrations in the small intestine. the tyrosine decarboxylase activity of enterococci was reported to increase with increasing NaCl concentrations of up to 10% (and with elevated production levels even at 20% NaCl).[180] studied the effect of the probiotic E.[180] Another probiotic for human use that contains enterococci is the Causidow culture that consists of two strains of S. vomiting. stimulation of the immune system. faecium SF68 was successful for both adults and children. This stimulatory effect of lactobacilli was suggested to benefit the animal as it could repress potentially harmful bacteria during the early stages of life. associated with food fermentation are able to form biogenic amines (BA). It decreased the duration of diarrheal symptoms and the time for normalization of patient’s stools. amino acid (and particularly tyrosine) decarboxylase activity appears to be quite common among be the enterococci. the production conditions for BA-positive enterococci appear to be optimal. and increased blood pressure. which in turn stimulated the growth of other LAB. especially lactobacilli.C. antimutagenic and anticarcinogenic activities.[85. Ingestion of high levels of biogenic amines such as histamine and tyramine may result in allergic reactions or cause intoxication symptoms such as headache. Vahjen et al.165 – 167] Within 25– 378C and pH 5 –7. faecium SF68 as an animal probiotic was also investigated. faecium SF68 has been used to treat diarrhea and is considered as an alternative to antibiotic treatment. double-blind clinical studies have shown that treatment of enteritis with E. Contrary to the general expectation for BA-positive LAB. faecium SF68 on young dogs and found that the probiotic enhanced specific immune functions when compared to an untreated control group. however. amino acid decarboxylase positive strains may produce relatively high amounts of BA. Their ability to produce BA. In addition. E.

. probably because of the emergence of vancomycin-resistant strains.192] They rank among the most prevalent organisms encountered in nosocomial infections.80%).[193] E. faecium (50%) and E.[185. All Rights Reserved.24) deconjugates bile salts by liberating the glycine and/or taurine moiety from the side chain of the steroid core. 48 E. gallinarum. this could be interpreted as a beneficial technological trait.[189] For a probiotic strain. The highest incidence of BSH active strains was observed for E. 3.188] In a study on enterococci isolated from foods. ENTEROCOCCI IN HUMAN DISEASE Infections Caused by Enterococci Enterococci are typical opportunistic pathogens and usually cause infections in patients who have severe underlying disease.Hoang-Dung TRAN and friends of low-density lipoprotein (LDL) cholesterol levels was not demonstrated. durans (44%). and Lactobacillus.[194] A shift towards E. Bifidobacterium. bile salt hydrolase activity appears to be a fairly common trait among enterococci. It was hypothesized that deconjugation of bile salts may contribute to lower cholesterol levels because free bile acids may more likely be excreted from the gastrointestinal tract than conjugated bile salts. casseliflavus. or who are immunocompromised. VI. generated by the enzymatic conversion of primary bile salts by other intestinal bacteria.1.C. and urinary tract and other infections. Peptostreptococcus. Thus. are cytotoxic and co-carcinogenic. Bacteroides. In contrast to the desirable cholesterol-lowering effect. the clinical relevance of this effect is uncertain.[191] They are usually associated with hospital-acquired infections and cause bacteremia. faecium is associated with the majority of the remaining infections. faecalis predominates among enterococci isolated from human infections (. 2 E.[182.[187. and 1 E. BSH activity was also suggested to allow bacteria to survive conditions and grow in the gastrointestinal tract. who have received surgery. A. including members of the genera Enterococcus. faecalis (81%) followed by E.186] The conjugated BSH enzyme (E.5.[195] Copyright © 2004 by Marcel Dekker. Inc. 16 E. which in turn may lower cholesterol levels. we screened 117 Enterococcus strains isolated from food (47 E. malodoratus) for BSH activity. endocarditis. it should be mentioned that BSH activity may also have adverse effects in that extensive deconjugation of bile salts in the human small bowel can lead to steatorrhea and that secondary (dehydroxylated) bile salts. faecalis. BSH activity is mediated by various gram-positive intestinal bacteria. the emergence of antibiotic-resistant strains of enterococci and the increased association of enterococci with human disease (see below) have raised concern regarding their use as probiotics. While the probiotic benefits of some strains are well established. it may increase the demand for cholesterol as a precursor for de novo synthesis of bile salts. while E.[190] The use of enterococci as probiotics remains a controversial issue. faecium. durans.183] hence.[184] Cholesterol-lowering effects of probiotics have often been linked to bacterial bile salt hydrolase (BSH) activity. Clostridium. The fear that antimicrobial resistance genes or genes encoding virulence factors can be transferred to probiotic strains in the gastrointestinal tract contributes to this controversy.[186] If enhanced fecal loss of bile acids occurs as a result of bacterial BSH activity. accounting for approximately 12% of nosocomial infections in the United States. faecium strains as the causative agent in enterococcal bacteraemia was noted. 3 E. In addition to its suggested effects of lowering cholesterol levels in probiotic strains.[191.

204] Enterococci may cause or contribute to abdominal and pelvic abscess formation and sepsis.197] Compared with a steady reduction in community-acquired cases of enterococcal bacteremia.194.205] B.196] Mortality from enterococcal bacteremia is generally high.[192.203] Infections of the central nervous system by enterococci are rare and are seen primarily in neonates and persons who have undergone complicated neurological procedures.[43.207] Enterococci are either intrinsically resistant and resistance genes are located on the chromosome. but the gastrointestinal and hepatobiliary tracts have also been implicated. faecium. had structural abnormalities. Antibiotic Resistance A specific cause for concern and a contributing factor to pathogenesis of enterococci is their resistance to a wide variety of antibiotics.190. although infections of older children and adults have also been reported.Hoang-Dung TRAN and friends Bacteremia is a common form of opportunistic enterococcal infection..[192.203] Urinary tract infections are commonly caused by enterococci.[191.[192. and strains resistant to penicillin-streptomycin or penicillin-gentamicin Copyright © 2004 by Marcel Dekker. but it is not a prerequisite for development of this infection.192.[43. All Rights Reserved.208] Examples of intrinsic antibiotic resistance include resistance to cephalosporins. tetracycline. because they are detected in the vaginal microflora in 25% of healthy women.[43.198 – 200] Risk factors associated with enterococcal bacteremia include underlying disease. . abortion. high levels of clindamycin and aminoglycosides. or urinary tract instrumentation.[191.[43. a high level of streptomycin and gentamicin resistance was reported.[19.206. faecalis more commonly involved than E.[43. kanamycin. Inc.[192. combinations of cell-wall– active antibiotics such as penicillin or ampicillin with aminoglycosides (e. nosocomial cases may have increased threefold and account for up to 77% of cases. multiple trauma. or had recurrent enterococcal infections.[192. and glycopeptides such as vancomycin.209 – 211] Since the early 1970s.196. or prior antibiotic therapy. However. presence of urethral or intravascular catheters. b-lactams. surgery.[191. fluoroquinolones. while examples of acquired resistance include resistance to chloramphenicol.200] and underlying heart disease is often present. or they possess acquired resistance determinants which are located on plasmids or transposons. These infections occur especially in persons who had surgery. and gentamicin) act synergistically and have been used successfully in treatment of enterococcal infection.[196] E.g.192] Endocarditis often occurs in patients that had preceding genitourinary instrumentation or urinary tract infection (UTI).192] Dialysis catheters and prior use of antibiotics are predisposing factors for intra-abdominal infections by enterococci.[196] Sources of enterococci causing bacteraemia without endocarditis are most commonly from the urinary tract. received antibiotics.191. high levels of b-lactams.203] Enterococci causing neonatal infection are thought to originate from the vagina. major burns. and low levels of clindamycin and aminoglycosides. faecalis have been implicated in outbreaks of neonatal central nervous system infections. most probably because of the underlying complicating factors.192. sulfonamides.192] The enterococci usually originate from the urinary tract.201] Enterococci cause an estimated 5 –15% of cases of bacterial endocarditis.207] Intrinsic resistance to many antibiotics suggests that treatment of infection could be difficult.[192.[191. especially in hospitalized patients.[43. erythromycin.[192] They were reported as a cause of spontaneous peritonitis in cirrhotics and nephrotics and may be associated with peritonitis in patients on peritoneal dialysis. with E. streptomycin. faecium and E.

[223.[211] Vancomycin resistance is of special concern because this antibiotic was considered a last resort for treatment of multiply resistant enterococcal infections. and. APH (200 ). and aminoglycoside phosphotransferases (APH).[215] Acquired high-level gentamicin resistance results from the transfer of genes encoding aminoglycoside-modifying enzymes by conjugative plasmids and transposons. tobramycin. In addition.[216 – 218] VRE have indeed been isolated from a wide variety of farm animals.[213] The gene encoding high level resistance (HLR) to gentamicin in E. two European countries (Denmark and Germany) banned the use of avoparcin.[222] Not only food.[212] The hitherto successful penicillinaminoglycoside treatment was no longer a viable option.[191] In the mid-1990s in Europe the source of vancomycin-resistant enterococci (VRE) was shown to be most likely farm animals as a result of ergotropic use of avoparcin. resulting in a major therapeutic problem. Klare et al. Moreover.[211] Ferretti et al. the incidence of VRE isolated from the feces of healthy volunteers had decreased by half.Hoang-Dung TRAN and friends combinations were also found.[191] followed by a European Union – wide ban. All Rights Reserved. a glycopeptide antibiotic.[191.215] HLR to gentamicin in E. but also contact between the farm animal and humans at avoparcin-exposed farms also resulted in human VRE colonization. and these constitute an important reservoir of VRE that could be transmitted to the hospital environment via contaminated meat. and it was suggested that enterococci received this gene from staphylococci. In contrast. and netilmicin) with the exception of streptomycin. faecalis producing a b-lactamase identical to that produced by S. resistance based on b-lactamase is more common for E. as a result. Inc. faecalis strains.225] showed that VRE were isolated from poultry and poultry farmer’s feces as well as poultry carcasses at a high incidence even 3 years after agricultural use of avoparcin was banned. faecium occurred after its appearance in E.219 – 221] These findings strongly suggest that food transmission occurred. AAD(6). AAD(40 ). amikacin. this antibiotic was given as an alternative to ampicillin or penicillin/aminoglycoside treatment to persons with allergy against penicillin or ampicillin.[209] In 1983 a strain of E. faecalis has the same nucleotide sequence as the gentamicin resistance gene of staphylococci.224] There are conflicting results on the effect of the avoparcin ban on the incidence of vancomycin resistance among enterococci.[211] HLR to aminoglycosides in enterococci is due to the synthesis of one or more of a series of aminoglycoside-modifying enzymes such as aminoglycoside acetyltransferases (AAC). The worldwide dissemination of HLR to gentamicin appears to stem from the spread of this enzyme’s (aac60 -aph200 ) gene via plasmids and transposons.[55. aureus was reported.[214] showed that the genes for the two aminoglycosidemodifying enzymes aac-60 and aph-200 had probably fused to generate an extremely powerful enzyme complex conferring resistance to all clinically useful aminoglycosides (gentamicin. One involves the production of penicillin-binding proteins (PBPs) with decreased affinity for the antibiotic. aureus. The aminoglycoside-modifying enzymes that have been identified in enterococci (and staphylococci) are APH(30 ). while the second involves hydrolysis of the penicillin molecule by b-lactamase. .217.[226] Copyright © 2004 by Marcel Dekker. faecium strains.[214.[216. and it is believed that this strain of Enterococcus received the gene from S.[226] showed that the incidence of VRE from frozen and fresh poultry meats decreased 2 years after the avoparcin ban. Borgen et al. and AAC(60 ).210] Enterococcal penicillin resistance occurs by two different mechanisms. aminoglycoside adenyltransferase (AAD). While resistance based on PBPs with reduced affinity for penicillin is more common among E. faecalis and was first reported in 1998.

C.[235.236] During this translocation process.233] Moreover. enterococci must have virulence factors that allow the infecting strains to colonize host tissue. Especially in cases of intestinal lesions. but also to extracellular matrix (ECM) proteins such as fibronectin. the ability to adhere to exposed extracellular matrix proteins is thought to promote bacterial translocation. AS was determined not only to bind to eukaryotic cells. and thus such strains pose a serious medical concern. and to cause infection. The emergence of vancomycin-resistant enterococci (VRE) in hospitals has led to infections that cannot be treated with conventional antibiotic therapy. the situation with respect to nosocomial VRE infections appears to differ considerably from that in Europe because avoparcin has not been licensed for use. faecalis. Aggregation Substance Aggregation substance (AS) (Table 4) is an adhesin that is encoded on pheromoneresponsive plasmids. . Expression of the AS gene is induced by sex pheromones that are small (7 –8 amino acids) hydrophobic peptides.[234] To cause abdominal infection and bacteremia. thrombospondin. the enterococci encounter the basal membrane and extracellular matrix proteins. This indicates that clinical use of vancomycin is responsible for development of VRE. which are excreted by plasmidless.[231] AS was shown to bind to a variety of cells via such b2-type integrins.[229. AS is also involved in binding to eukaryotic cells.Hoang-Dung TRAN and friends In the United States. and translocate through epithelial cells and evade the host’s immune response. via integrin receptors. transmission of VRE in the United States does not appear to be from the community to the hospital. Binding of the pheromones by the donor strain leads to expression of AS on the cell surface. All Rights Reserved.[222] A community prevalence survey failed to isolate VRE from healthy volunteers without hospital exposure and from environmental sources or probiotic preparations. enterococci were considered to possess subtle virulence traits that were not easily identified. and each of these may be associated with one or more of the stages of infection mentioned above. Inc. The molecule contains two RGD (Arg-Gly-Asp) amino acid motifs that promote E.[194] However. vitronectin. Furthermore. and collagen type I. Sartingen et al. but these AS-producing enterococci strains were not able to translocate enterocytes in an Copyright © 2004 by Marcel Dekker. including human macrophages and intestinal epithelial cells. AS leads to clumping of donor and recipient cells by binding to a complementary receptor termed binding substance. such virulent strains must produce pathological changes either directly by toxin production or indirectly by inflammation. Only limited data are available on translocation of enterococci through intact epithelial cell layers.[228] In the past. faecalis adhesion to eukaryotic cells. enterococci must penetrate the intestinal or genitourinary epithelium and enter the lymphatic and/or vascular system.[232] showed that AS promoted internalization of enterococci by enterocytes derived from the colon and the duodenum. considerable progress has been made in determining virulence traits from clinical isolates.[232. and food has not been implicated as a possible vehicle for transmission.[227] In contrast to Europe. invade host tissue. 1. recipient strains of E. such as pig renal tubular cells.230] However. Virulence Factors Antibiotic resistance alone cannot explain the virulence of enterococci. The clumping of cells leads to a highly efficient transfer of the pheromone plasmid on which the AS gene is encoded.

Sartingen et al.[233. e.241] AS also promoted adherence to and increased intracellular survival of enterococci in host immune cells. collagens and fibrin (role in translocation?) Can hydrolyze antibacterial peptides (evasion of host innate immune response) Adhesin. laminin. faecalis was induced. and spleen when intestinal overgrowth with E. .[231. and vitronectin are exposed. faecium (Acm) Endocarditis antigen from E. mouse in vivo studies demonstrated that enterococci migrated across the intact intestinal mucosa and spread to the mesenteric lymph nodes.232.240] It promoted adherence to human neutrophils (PMNs) as well as adherence to and phagocytosis by human macrophages. Thus. Inc. such as macrophages and neutrophils (PMNs).[233. collagen.. but it may also be associated with translocation of enterococci (Table 4). All Rights Reserved.[232] concluded that the incubation time (8 h) to allow for translocation studies may have been too short in their in vitro study. To date. there is no knowledge of virulence factors other than AS which play a role in invasion or translocation of enterococci. faecalis (Ace) or E. Adherence to ECM proteins is also thought to play a major role in wound infections and in bacterial endocarditis.242] In activated human neutrophils. a major extracellular matrix constituent—role in translocation? Evasion of host immune response Cytolysin (Cyl) Gelatinase (Gel) Enterococcal surface protein (Espfs and Espfm) Adhesin to collagen of E.g. it was shown that the phagosomes containing AS-bearing enterococci were markedly larger Copyright © 2004 by Marcel Dekker. but also invasion of eukaryotic cells in tissue culture.239.[234] AS was shown to facilitate not only adherence.[237] Thus. thrombospondin.Hoang-Dung TRAN and friends Table 4 Virulence Factors Found in Some Enterocccus Strains and (Suggested) Association with Stage of Virulence Virulence determinant Aggregation substance (AS) (Suggested) association with stage of virulence Adhesion to eukaryotic cells (adhesin)/ promotes colonization Invasion of eukaryotic cells (invasin) Adhesion to extracellular matrix proteins (may promote translocation) Increases survival in immune cells (evasion of host immune response) Eukaryotic cell toxin Lyses immune cells (evasion of host immune response) Can hydrolyze various biological peptides. promotes colonization Exhibits characteristics of MSCRAMMs—role in evasion of immune response? Adhesion to extracellular matrix proteins (may promote translocation) Exhibits MSCRAMM characteristics—role in evasion of immune response? Adhesin: role in endocarditis Degrades hyaluronic acid. faecium (EfaAfs) Hyaluronidase Capsule 8-hour period. AS not only functions as an adhesin. However.[234. faecalis or E.238] About half of enterococcal endocarditis cases occur where subendothelial extracellular matrix proteins such as fibronectin. liver.

[250] determined a fivefold increased risk of death of patients within 3 weeks of bacteremia caused by b-hemolytic enterococci. faecalis at this site.[122. faecalis (Espfs) or E. are involved in causing pathological changes such as acute inflammation. AS is therefore considered an important multifunction virulence factor because it acts as an adhesin and invasin.Hoang-Dung TRAN and friends than phagosomes containing opsonized E. The incidence of Espfs was shown to be higher among clinical strains of E. faecium (Espfm) The enterococcal surface protein (Esp) produced by either E.[255] Shankar et al. 2. as Miyazaki and coworkers[252] showed that hemolytic culture supernatants of E. faecalis lysed mouse PMNs and macrophages. probably due to inhibition of the respiratory burst. but absent in food and commensal isolates. in a European study. faecalis than isolates from healthy individuals. as well as their surface exclusion proteins. Cytolysin The b-hemolysin/bacteriocin or cytolysin is a cellular toxin that enhances virulence in animal models. the pH of PMN phagosomes was significantly higher after ingestion of nonopsonized. it is involved in translocation as well as evasion of the immune response by intracellular survival in immune cells (Table 4). faecalis bloodstream isolates in the United States. it has been shown that 60% of clinical strains involved in parenteral infection had a hemolytic phenotype.[250] However. All Rights Reserved.246 – 248] In Japan. They are cleavage products of 21. in addition. the Espþ strain did not influence histopathofs logical changes in the animal model.[256] used an Espþ strain and an isogenic mutant in a mouse model of ascendfs ing urinary tract infection to show that Espfs contributed to colonisation and persistence of E.245] 3.[250] indicating a role in pathogenicity.[249] Similar trends were observed in a study of E. only 16% of E.244.[228] They are chemotactic for human and rat PMNs in vitro and induce superoxide production and secretion of lysosomal enzymes. However.[256] The presence of Espfs also increased cell Copyright © 2004 by Marcel Dekker. . suggesting that some modification of phagosomal maturation may be involved in AS-induced resistance to killing. which led them also to suggest a role in pathogenicity. faecalis. cytolysin can evade a host immune response by destroying cells of the immune system. Production of cytolysin appears to be a major risk factor associated with pathogenic enterococci as Huycke et al. Similarly. Enterococcus Surface Protein from E. Thus. compared with only 17% of isolates from the feces of healthy individuals. faecalis (Espfs) and E. Sex Pheromones Sex pheromones themselves can be considered as virulence determinants (Table 4). faecium (Espfm) is an adhesin (Table 4).[242] Sussmuth et al. In addition.to 22-amino-acid signal peptides associated with surface lipoproteins of unknown function.[233] showed that AS-bearing enterococci were significantly more resistant to killing by human macrophage during the first 3 hours postinfection.[251] Cytolysin production can be considered as a bacterial strategy to evade the host immune response. and the gene encoding this trait in both Enterococcus species appears to be chromosomally encoded. AS¨ bearing E.[250] 4. faecalis than after ingestion of opsonized cells. compared with bacteremia caused by non – b-hemolytic strains. Inc. faecalis strains isolated from blood exhibited hemolytic activity.[228. Eaton and Gasson[254] found Espfm to be highly conserved in infection-derived isolates and environmental isolates.[243] These.

[258.Hoang-Dung TRAN and friends hydrophobicity. Inc. a mechanism for evading the immune system supplied by the same molecule involved in adherence may be an elegant solution for enterococci to increase their chances of survival.252. Interestingly. which is a potential target for sortase. Acm. faecium exhibited binding to collagen type I. The differences between binding capacity of clinical and community isolate strains were statistically significant. and biofilm formation in vitro. Adhesin to Collagen from E. while strains from the feces of healthy human volunteers did not bind collagen I. and Cna are similar in that they contain an N-terminal signal sequence followed by the collagen-binding A domain. all community E. a cell wall domain with a characteristic LPKTS motif.255] Such a phenomenon is thought to be related to evasion of the immune response.[259] Because these proteins also contain repeat units and the number of repeats can vary.[255] Shankar et al. Thus. All Rights Reserved. Acm. faecalis (Ace) and E. . however. the collagen-binding protein from E. indicating that binding to collagen is a virulence factor.[253. which are thought to stretch the membrane followed by a short cytoplasmic charges tail. faecium. was shown to bind collagen types I and IV.[253] for Esp.259] The structural organization of all Ace.255] Overall.[253] and Eaton and Gasson[255] showed that different Espþ Enterococcus strains may exhibit variations in these repeat units. faecium (Acm) Ace and Acm are adhesins (Table 4) that show structural similarity to MSCRAMMs of other gram-positive bacteria. They contain a signal sequence followed by an N-terminal region and a core region that consists of repeat units. particularly during translocation or when the intestinal epidermal layer is damaged and the underlying extracellular matrix proteins are exposed.[250.[257] In addition to a role in adhesion. 5. Ace binds not only to collagen (types I and IV) but also to laminin. based on the observation that the overall structure of both Espfs and Espfm are comparable to that of MSCRAMMs. but differ in the number of repeats. faecium isolates also contained the gene for Acm. As Enterococcus cells would become exposed to immune cells at this site. particularly to the collagen-binding protein Cna of S.[261] showed that Ace was expressed by enterococci during human infections. this gene was in nonfunctional form as a result of Copyright © 2004 by Marcel Dekker. faecalis endocarditis reacted with anti-Ace antibodies.[259] Nallapareddy et al. Ace may play an important role in pathogenesis of enterococci. Ninety percent of human sera collected from patients with E.[257] Espfs was suggested to promote colonization of host tissue by direct ligand-binding activity to the extracellular matrix in the human host. aureus.[253] The C-terminal regions of the Esp proteins contain a membrane-spanning hydrophobic domain and a cell wall anchor motif involved in anchoring the protein to the bacterial surface.255] The Espfs and Espfm proteins are similar in sequence and global organization. a B region that consists of repeat units.[253. the espfs and espfm genes and the Espfs and Espfm proteins share 89% identity. which may lead to the expression of variant proteins that are identical at the amino and carboxy termini. a stretch of hydrophobic residues. this protein may also be involved in evasion of the immune response (Table 4) by mechanisms similar to those suggested by Shankar et al. Esp may also have a function in evasion of the host’s immune response (Table 4).[259] showed that particularly the clinical strains of E.[257] This suggestion was based on the fact that Espfs exhibits characteristics of surface protein receptors designated microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that mediate binding to extracellular matrix proteins. adherence to abiotic surfaces.261] Nallapareddy et al.[260.

For example. .7%) among enterococci isolated from intensive care units in Germany.[266] reported that protease-producing E. It plays a role in the infection of human tissues. Low et al. B. GelE clears the bacterial cell surface of misfolded proteins and is also responsible for activation of an autolysin. SsaB ScaA. and Coque et al. and PsaA from streptococci. A) which have homology to ABC-type metal ion transport systems.4. Expression of GelE would lead to degradation of this fibrin layer surrounding the bacteria and allow further dissemination of the organism. where the Mn2þ availability may be as low as 20 nM. EfaAfs was suggested to play a role in adhesion in endocarditis. aureus.Hoang-Dung TRAN and friends nucleotide deletions or insertion of IS6770-like insertion sequence resulting in frame-shift mutations.24.[268] GelE was shown to cleave fibrin. so far only the efaAfs gene was shown to influence pathogenicity in animal models. Inc. Gelatinase Gelatinase is an extracellular Zn-metalloprotease (EC 3.[264] The first gene efaC encodes an ATP-binding protein. faecalis in serum. the authors could not determine whether GelE independently influences the outcome of this enterococcal infection. faecalis.[265] Production of ¨ gelatinase increased pathogenicity in an animal model.[259] 6.[264] suggested that EfaCBA is a manganese-regulated operon that functions as a high-affinity manganese permease in E. Waters et al. while the third (efaA) probably functions as a solute-binding protein receptor for the ABC transporter complex.[267] showed that 54% of clinical enterococci isolates from patients with endocarditis and other nosocomial infections produced protease. the second (efaB) a hydrophobic transmembrane protein. which was suggested to have important implications in virulence of E.[262] The EfaAfs antigen shows high homology to adhesins such as FimA. faecalis (EfaAfs) or E.[263.[263] Kuhnen et al.268] suggesting that both GelE and SprE are important in the infection in this animal model. faecalis were common (63. However.30) that acts on a variety of substrates such as insulin-b chain.[262] However.[263] The genetic determinant for production of EfaA has been sequenced and the efa operon consists of three genes (efaC. faecium endocarditis antigens (EfaAfs and EfaAfm.[264] 7. respectively) (Table 4) are considered to be potential virulence determinants. the vasoconstrictor endothelin-1. Acm has a far greater similarity at the primary sequence level (62%) to the collagen-binding protein (Cna) of S. Acm has similarity to both the A and B domains of Cna. faecalis as the secreted protease can damage host tissue and thus allow bacterial migration and spread (Table 4). faecium (EfaAfm) Production of the adhesin-like E. as well as sex pheromones and their inhibitor peptides.[265] suggested that enterococci in blood infections and vegetations formed during endocarditis were likely to be coated with polymerized fibrin. All Rights Reserved. The gene for gelatinase (gelE) is located in an operon together with a gene (sprE) encoding a serine protease. Enterococcus Endocarditis Antigen from E.[268] Mutants containing both defective gelE and sprE genes led to delayed time to death in a mouse peritonitis model. collagenous material in tissues.[259] While the similarity of Acm to Ace appears to be confined to the A domain. and expression of the EfaA was previously shown to be induced by growth of E. In addition to its role in virulence. This muramidase-1 autolysin functions to Copyright © 2004 by Marcel Dekker. faecalis and E. While Ace and Acm share some (47%) amino acid sequence similarity. GelE was also shown to affect a variety of important housekeeping functions.

Hyaluronidase Enterococci may produce hyaluronidase.[270] Rice et al. Furthermore.[271] Hancock and Gilmore[272] studied a different capsular polysaccharide from E.[265] postulated that overall GelE plays a crucial role for dissemination of the organism in high-cell-density environment. faecium exhibited 42% identity and 60% similarity to a hyaluronidase from S. and phosphate in a 2 : 1 : 2 ratio.Hoang-Dung TRAN and friends reduce chain length. The nature of the linkages and the structure of the capsular polysaccharide were not determined by Copyright © 2004 by Marcel Dekker. Peptide LL-37 belongs to the family of antimicrobial peptides termed cathelicidins and is activated when cathelicidin hCAP-18 is processed by proteinase 3. and probiotic strains were also investigated. faecium strain was determined. that of Hancock and Gilmore[272] contained glucose. . By raising antibodies to this capsular polysaccharide. the gene sequence for the hyaluronidase gene hylEfm from an E. degradation of fibrin would aid not only in dissemination. All Rights Reserved. and subpopulations of lymphocytes and monocytes. The hyaluronidase from E. Isolates from animals. which encodes a putative protein of 533 amino acids with a theoretical molecular weight of 65. glycerol. glycerol. Rice et al.[271] purified a capsular polysaccharide and determined that it consisted of a repeat structure of kojibiose linked 1.[271] contained glucose. they used immunogold labeling to show the presence of the capsule surrounding enterococci in electron microscopic studies.[270] 9. However. faecium strains of clinical origin possessed such capsular polysaccharides. it was suggested that it may also play a role in enterococcal pathogenesis.[265] GelE also degrades sex pheromones and their inhibitors. and both genotypes were found primarily in vancomycin-resistant E.[194. faecalis strains and 7 vancomycin-resistant E. Inc. Because production of this enzyme was linked to pathogenesis of other microorganisms. Their data suggested that specific E. faecium isolates from nonstool cultures obtained from patients hospitalized in the United States.[271] However. faecium strains for the incidence of hylEfm and espfm genes. and phosphate in a 4 : 1 : 1 : 2 ratio. faecium strains may be enriched in determinants that make them more likely to cause clinical infections. which is a major component of the extracellular matrix (Table 4). Accordingly. pyogenes. which also increases the potential for dissemination. an enzyme that degrades hyaluronic acid. once enterococcal growth reaches high densities.270] Recently. Capsule Huebner et al.[270] screened a large number of E.[265] The supernatant from a gelatinase-expressing E. the degradation of sex pheromones decreases aggregation of bacteria. neutrophils. there is no direct evidence for the role of hyaluronidase in disease caused by enterococci. faecalis. 8.[270] This gene consisted of 1659 bp.051 kDa.2 to glycerolphosphate. but also in reduction of chain length as a result of autolysin activation. These strains were form stool or nonstool origin isolated from both hospitalized and community-based persons. waste water.[269] Degradation of antimicrobial peptides which are part of the innate immune system thus is a further GelE-associated enterococcal virulence factor (Table 4).[271] They also showed that one third of a sample of 15 clinical E. faecalis strain was also shown to inactivate the antibacterial peptide LL-37.[269] The peptide LL-37 is part of the innate immune system and has been isolated from epithelial cells.[270] showed that the presence of espfm was roughly twice that of hylEfm . while the capsular carbohydrate of Huebner et al. galactose. Waters et al. of which the overall composition of the polymer showed some relation to the carbohydrate purified by Huebner et al.

as measured by size of aortic valve vegetation in a rabbit endocarditis model.[273] However. a-helical Copyright © 2004 by Marcel Dekker.[274] Production of cytolysin is also a regulated phenotype. Other Virulence Determinants Recently. which allows production of iCF10. and cylI (encodes immunity protein). Inc.[274] Induction by cCF10 contained in the cell wall is prevented by cell membrane – associated protein PrgY. the inhibitor iCF10 is secreted at an 80-fold excess to the pheromone cCF10. An interaction of plasma components with the inhibitor peptide iCF10 was proposed as affecting the mating behavior.[274] For Asc10. Induction occurs if neighboring cells tip the balance in favor of cCF10. consisting of a nonglobular.[273] This led the investigators to believe that this particular Enterococcus strain may harbor gene(s) encoding toxin(s) similar to streptococcal pyrogenic exotoxins (spe). which are arranged in a collinear fashion. cylB (encodes ABC transporter protein). the plasmid-bearing donor cell also excretes a competitive inhibitor. cylLS (encode structural cytolysin subunits). which encode regulatory proteins. . as measured by reduction in viable microorganisms from the abdominal lymph nodes that drain this site. cylA (encodes protein for extracellular cytolysin activation). faecium. REGULATION OF ENTEROCOCCUS VIRULENCE GENE EXPRESSION Production of aggregation substance (AS) is a tightly regulated phenotype. VII. the AS of the sex pheromone plasmid pCF10. Regulation is based on autoinduction and a two-component regulatory system that responds to quorum sensing. toxin structure and the genetic basis for production of this toxin have not yet been studied.Hoang-Dung TRAN and friends Hancock and Gilmore. PrgX also controls the transcription of the prgQ promoter. To counteract autoinducing activity. which is bound in the cell wall by the pheromone-binding protein PrgZ and consequently imported into the cytoplasm by an Opp (oligopeptide permease) system. Hancock and Gilmore[272] were also able to show that the mutant was more readily cleared from a resulting abscess. The pheromone then interacts with regulatory protein PrgX to allow AS expression. cylM (encodes protein for intracellular modification of cytolysin).[274] Hirt and coworkers[274] showed that the AS of pCF10 is actually induced in vivo and could increase pathogenicity.[275] The genes necessary for cytolysin production include cylLL. All Rights Reserved. In addition. an outbreak of sepsis similar to a toxic shock – like syndrome in humans involving both humans and pigs was attributed to a strain of E.[272] Using the capsular polysaccharide-producing strain and an isogenic mutant in a murine cutaneous infection model. The involvement of the pheromone-sensing system for in AS expression in plasma was confirmed by the absence of AS induction in a mutant lacking the pheromone-sensing protein prgZ. which prevents self-induction and also provides the threshold that allows recipients in the immediate environment to overcome their inhibitory activity with their secreted pheromone. This clearly indicated that the production of a capsule does offer some protection to the host’s defense mechanisms and that the production of a capsule by some enterococcal strains may play an important role in evasion of the immune response 10. they showed that the expression of AS conferred a survival advantage to cells harboring the plasmid and led to a highly efficient transfer of plasmid. because autoinduction by a plasmid-bearing donor strain must be prevented. Upstream of these biosynthesis and immunity genes on the opposite DNA strand are two ORFs (cylR2 and cylR1).

. Transcription of the efaCBA and EfaA production is repressed by Mn2þ by a Mn2þ-responsive transcriptional regulator EfaR. These genes have homology with the Staphylococcus aureus agr genes.[279] The FsrA protein has 38% similarity to the AgrA protein that encodes the response regulator in the S. faecalis.276] In this system.[268.Hoang-Dung TRAN and friends protein with a helix-turn-helix DNA-binding motif (CylR2) and an a-helical protein with three predicted transmembrane domains (CylR1). which plays such an important role in global regulation of S. is regulated by Mn2þ. Inc.[279. toxic shock syndrome toxin 1. So far. faecalis V583 obtained from The Institute of Genomic Research (TIGR). b-toxin. consists of a protein histidine kinase and a response regulator protein pair. indicating the importance of this gene in virulence. of which the EfaA component forms the endocarditis antigen. faecalis regulator) that regulate the expression of gelE and sprE. sprE. faecalis to the Agr system of S. gelE. active cytolysin subunit CylLS00 . as mentioned above. However.280] The amino acid sequence of this peptide corresponds to the C-terminal part of a 242-amino-acid protein encoded by fsrB. resulting in EfaR-Mn2þ complexes that bind the efaCBA promoter.[275] Unlike other well-known quorum-sensing systems. and serine protease and downregulates surface proteins such as protein A. derepressing efaCBA expression and hence increasing Efa permease levels and Mn2þ scavenging. aureus virulence. a cyclic peptide termed gelatinase biosynthesis-activated pheromone (GBAP) is the autoinducer. inhibiting transcription and hence reducing Mn2þ uptake. In S. rather it depends on a small helix-turn-helix DNA-binding protein and a transmembrane protein of unknown function. Together these were shown to repress the cytolysin operon. the Fsr system is only known to regulate two genes in E. Using the genome sequence information of E. there are three genes designated fsr (for E. as mentioned above for gelatinase regulation. and the serine protease gene. coagulase. Production of gelatinase is another regulated phenotype.e. The efaCBA operon encodes a putative ABC transporter (Efa permease). All Rights Reserved. the agr/hld locus contains five genes that encode a quorum-sensing system that regulates the expression of virulence factors. this two-component regulatory system did not consist of a protein histidine kinase and a response regulator.[264] suggested that when Mn2þ is abundant. i.[275] The Enterococcus faecalis endocarditis antigen EfaAfs . the gelatinase gene. d-toxin. This may increase the survival of enterococci in the human environment and thus contribute to virulence. intracellular levels rise. they Copyright © 2004 by Marcel Dekker. respectively. the EfaR apoprotein cannot bind the efaC promoter. while agrD encodes a pheromone peptide that acts as an autoinducer. It was demonstrated that an fsrB deletion mutant attenuated the virulence in Caennorhabditis elegans and a mouse peritonitis model.[264] Low et al. aureus Agr system. aureus. and fibronectin-binding protein.[268] Qin et al.[281. if bacteria encounter host tissues or human serum where Mn2þ availability is low. which shares 27% identity with the Corynebacterium diphtheria diphtheria toxin repressor DtxR. enterotoxin B. leads to the question whether the Fsr system plays a similar role in virulence regulation. . as well as a rabbit endophthalmitis model. agrA and agrC encode a response regulator and a sensor transducer. while FsrC has 36% similarity to the ArgC protein that is the sensor transducer of the Agr system. aureus.[268] demonstrated that for the Fsr system. Such a two-component regulatory system.[268] Homology of the Fsr system of E. faecalis gelE and sprE genes. Upstream of the E. by a quorum-sensing mechanism. The inducer for expression of cytolysin was shown to be the smaller. and autoinduction was shown to be dependent on celldensity.282] Teng and coworkers[283] studied virulence of enterococci by disrupting twocomponent regulatory systems in Enterococcus faecalis.[276 – 278] The Agr regulatory system upregulates the expression of secreted proteins such as a-toxin.

and the lowest incidence was observed for starter strains. .285] which gives rise to concern. faecium strains were generally clear of virulence determinants.285. establish themselves in the human gastrointestinal tract.Hoang-Dung TRAN and friends identified 11 homologues to the PhoP-PhoS global two-component regulatory system of Bacillus subtilis. Inc. EfaAfs . while E. Such studies may allow evaluation of the safety of strains intended for use as probiotics or starter cultures. a number of studies also concerned the incidence of virulence factors of enterococci isolated from foods. and vancomycin. ampicillin.[286] showed that virulence determinants occurred among food. All Rights Reserved. they showed that the mutant was more sensitive to low pH and high temperature than the wild-type strain. faecalis strain OG1RF and one mutant. the occurrence of vancomycin-resistant strains and strains with multiple antibiotic resistances was reported.3%) or Esp (2. both environment and growth phase variations were observed. indicating that etaRS may regulate different operon(s) involved in virulence and stress response.[55 – 58. in which only a few strains produced either haemolysin (8. especially the E. faecium led these authors to speculate that the occurrence of virulence determinants is a common trait in the genus Enterococcus. but that virulence determinants were significantly associated with a high virulence potential. commensal and clinical isolates of enterococci. Esp. Strains of E.[286] The incidence of antibiotic resistance among enterococci from food was also investigated. as well as strains used as starter cultures. whereas food and commensal strains harbored fewer virulence determinants.[283] Seven of these pairs were disrupted in E. EfaA.[283] Shepard and Gilmore[284] used real-time PCR to study virulence gene expression and show that AS. and EfaAfm were found also in other enterococcal species apart from E. However. Esp. However. faecalis and E. with a much higher incidence than in E. In addition. faecium. Ace. INCIDENCE OF VIRULENCE FACTORS AMONG ENTEROCOCCI FROM FOOD Much progress has been made in determining virulence factors from clinical enterococcal isolates using molecular biological techniques and model animal experiments as described above. streptomycin. faecium.57.[282] However.1%). Moreover.287] Nevertheless.[254] A similarly low incidence of virulence factors was observed among E. disrupted in the etaR gene of the gene pair designated etaRS. showed a delayed killing and a higher lethal dose in a mouse peritonitis model. at least transiently. faecalis strains also harbored multiple virulence determinants. demonstrating the occurrence of uncharacterized control mechanisms for gene expression that may play an important role in vivo. the majority of the isolates. IX. faecalis harbored multiple virulence determinants. faecium strains isolated from food in a previous study.287] These studies showed that although many strains were found resistant to one or more of the antibiotics.[54. E. Semedo et al. Eaton and Gasson[254] showed that enterococcal virulence factors were present in food and medical isolates. TRANSMISSION ROUTES AND TRANSFER OF VIRULENCE DETERMINANTS/ANTIBIOTIC RESISTANCES Evidence is gathering that enterococci from the environment or from foods can survive in and. were sensitive to the clinically relevant antibiotics such as penicillin.[284] VIII. and Gel are induced in serum or urine. The finding that virulence determinants such as cytolysin. the incidence of virulence factors was higher among the medical strains than food isolates. The study Copyright © 2004 by Marcel Dekker.[287.

veterinary. cheese. It can be speculated that such colonization may not be of a transient nature for debilitated persons and/or persons who receive antibiotic treatment. However. in the study of Klein and Pack[297] the transfer rate was considerable lower than to a clinical E.[291] investigated vancomycin-resistant E. faecium strains used in animal nutrition by Klein and Pack. however.[36] could not confirm such a host specificity. faecium control strain.[294. These investigators suggested that there was a noticeable host specificity. faecium strains from hospitalized patients. faecalis used as starter cultures in food. faecalis and two of E. faecium strains from food. faecium strain in filter mating experiments. Their results indicated a dissemination of gentamicin-resistant enterococci from food-producing animals to humans through the food supply. Sørensen et al. In all these studies. and human fecal samples. food products from animals of the same species.[35] In a variety of studies. faecium strains isolated from food which were given to human volunteers led to only transient intestinal carriage and could not be isolated after 35 days.[299] From the above Copyright © 2004 by Marcel Dekker.[299] used a new animal model. then. However. . Lund et al. it does appear that enterococci can be naturally transmitted from food animals or foods to the human gastrointestinal tract. Berchieri[292] showed that ingestion of a vancomycin-resistant strain isolated from a chicken resulted in colonization of his own gut for 20 days. faecalis strains.223. Whether host specific or not. It has been shown.Hoang-Dung TRAN and friends of Gelsomino et al. milk. Eaton and Gasson[254] studied the probability of gene transfer to starter culture strains in vitro by showing that virulence genes on a pheromone-response plasmid could be transferred to strains of E.288. Studies done so far suggest that they will be present only transiently.[35] mentioned above showed that identical three clones. The possibility of transfer of virulence factors under in vivo conditions was studied by Huycke. predominated among farm equipment. Vancanneyt et al. and human sources isolated from different geographical origins.[298] using a hamster model of enterococcal intestinal overgrowth. the human volunteers were healthy subjects.or streptogramin-resistant E. All Rights Reserved.225. for example. nonhospitalized persons.[184] showed that a probiotic E.289] Donabedian et al.[290] studied gentamicin-resistant enterococci occurring in food animals. suggesting that animal-derived enterococci may colonize the human gut. one of E. The same could be shown for the transfer to probiotic E.295] Noting that transient colonization of environmental or food strains can occur raises the question whether there can be transfer of virulence factors or antibiotic resistance genes from strains ingested with food to gastrointestinal strains.[297] However. the streptomycin-treated minipig.[293] showed that vancomycin.[254] Lund and Edlund[296] showed that vancomycin-resistant genes could be transferred to a probiotic E. genetically indistinguishable enterococci have been found in both animals and humans. to show that the pheromone-response plasmid pCF10 could be transferred in the gastrointestinal tract to other E. and various animal sources by AFLP genotyping. faecium strain could not be recovered from the feces of human volunteers 31 days after ceased intake. that enterococci may show a high colonization potential in antibiotic-treated mice. and feces from humans and found that isolates with indistinguishable PFGE patterns could be recovered from food and from human stool.[290] In contrast. faecium starter cultures. in a genotypic study on a large number of E.[298] Licht et al. casseliflavus. they were not able to transfer virulence genes into strains of E.[219. is how well these bacteria are able to establish themselves in this environment. again even when there was no selective pressure with antibiotic. Inc. They showed that pheromone-responsive plasmids carrying either antibiotic or cytolysin genes could be effectively transferred in the hamster gastrointestinal tract. even in the absence of selective pressure with antibiotics. Willems et al. One obvious question.

In a genotyping study of E. are there other.286] It may be argued that strains that possess multiple virulence determinants associated with various stages of infection (colonization/adherence. faecalis. each particular strain should be carefully evaluated for the presence of all known virulence factors in order to assess the potential risk for its use.[36] found that all human clinical isolates of E. therefore. Questions about unrecognized virulence determinants may not be answered immediately. This is especially true in the light of findings that virulence determinants such as aggregation substance. Furthermore. It would be interesting to know. For practical present-day safety investigations. Vancanneyt et al. faecalis strains. adhesins.. X.286] However. it can be suggested that if an Enterococcus strain is considered for use as starter culture or as a probiotic. All Rights Reserved. evasion of the immune response. and the incidence of such virulence determinants appears to be low.[254. suggesting that there may be a genetic basis for strains associated with human disease. such strains should harbor no virulence determinants and should be sensitive to clinically relevant antibiotics. animals. adhesion ability). Furthermore. Ideally. E. because strains of these species generally harbor fewer recognized virulence determinants than E. but there is a need for safety decisions to be made. Furthermore.Hoang-Dung TRAN and friends examples it is noticeable that for such gene transfer studies the pheromone-response plasmids were often used. and this Copyright © 2004 by Marcel Dekker. for example. faecium and other enterococci strains appear to pose a lower risk for use in foods. and induction of pathological changes) pose a higher risk than strains that possess a single virulence determinant (e. so-far unrecognized virulence genes that are regulated by the fsr locus or by other regulatory genes such as etaRS or efaR (see above)? Low et al. and food. This suggested that EfaR may be a global regulator. as well as a lack of detailed comparative studies between clinical and food isolates. which have a natural high transfer frequency. This may also be deduced from the fact that clinical enterococci strains appear to harbor more virulence factors than food or commensal strains.g. which may also be involved in regulation of as yet unidentified virulence determinants. there is still not sufficient knowledge on all virulence factors. In general.[254. faecium strains. and cytolysin appear to be common in the genus Enterococcus and are frequently encountered in food strains. faecium fell into a defined subgroup.[264] found EfaR boxes in the promoter regions of several genes. Inc. The opposite applies to E. Thus.[229] This may exaggerate the transferability rates of virulence factors or antibiotic resistance genes that are located on other type of plasmids or the transfer of nonpheromone plasmids to other enterococcal strains that generally do not harbor pheromone-response plasmids such as E.285. including two genes that encode natural resistance –associated macrophage protein (NRAMP) homologues. whether food strains may also possess capsules and whether fsr genes occur in food strains.[254.285] One problem associated with risk assessment on the basis of virulence-determinant investigations is that knowledge is limited regarding the type and combinations of virulence factor(s) that are decisive for pathogenic potential. virulent subpopulations of strains of a species may exist. clear data about the relative importance of single virulence determinants in food strains are sorely lacking. translocation. faecium strains from humans. the question as to whether food strains possess an intrinsically lower pathogenic potential than clinical isolates has still not been fully answered. . SAFETY OF ENTEROCOCCI STRAINS FOR USE AS STARTER CULTURES OR AS PROBIOTICS The incidence of virulence determinants among food isolates studied so far appears to be strain specific.

Hoang-Dung TRAN and friends may be a result of the presence of pheromone-response plasmids which encode virulence factors. Because enterococci possess different gene transfer mechanisms (e.g., pheromoneresponsive plasmids, conjugative and nonconjugative plasmids, and transposons), it is feared that enterococci may readily acquire these determinants from other enterococcal strains. This represents a possible risk related to the use of enterococci as probiotics or starter cultures. Such a risk would probably be higher for E. faecalis strains, as the gene transfer mechanism based on pheromone response plasmids is known to generally occur in this species,[229] although the presence of sex pheromone plasmids in E. faecium has been reported.[300] Knowledge on the transfer rates of plasmids to the strain intended for use as starter or probiotic culture would be of great value to assess the likelihood of in vivo transfer. Should such transfer occur, again it would be of value to know the impact of transfer of a single or more virulence determinants to the relative pathogenicity of the recipient strain. The above discussion shows that the use of enterococci in foods or as probiotic cultures clearly represents a controversial issue in the context of food safety. One should, however, not forget that enterococci are typically associated with the human environment and in particular with the human GI tract. They are also involved in most traditional food fermentations studied thus far, where they appear to play at least some positive role in the development of product-specific typical characteristics. Moreover, particular strains may be considered to be opportunistic pathogens, but it is doubtful that they would cause disease in healthy humans. This is supported by the fact that although food enterococcal strains have been described that harbor single or multiple virulence factors, the morbidity of healthy humans resulting from foodborne enterococcal infections appears to be very low. Furthermore, there are probiotic strains on the market with a long history of safe use and large-scale commercial application. Thus the host factors (i.e., physiological condition, underlying disease, immunosuppression) appear to play a key role in the establishment of an infection with enterococci, and contact of those at risk with these bacteria should be minimized.

´ 1. Thiercelin, M.E.; Jouhaud, L. Reproduction de l’enterocoque; taches centrales; granulations ´ ´ ´ ´ peripheriques et microblastes. Comptes Rendus des Seances de la Societe de Biologie 1903, 55, 686– 688. 2. Andrewes, F.W.; Horder, T.J. A study of the streptococci pathogenic for man. Lancet 1906, II, 708– 713. 3. Lancefield, R.C. A serological differentiation of human and other groups of hemolytic streptococci. J. Exp. Med. 1933, 57, 571 – 595. 4. Sherman, J.M. The streptococci. Bacteriol. Rev. 1937, 1, 3 –97. 5. Devriese, L.A.; Pot, B. The genus Enterococcus. In The Lactic Acid Bacteria, The Genera of Lactic Acid Bacteria; Wood, B.J.B., Holzapfel, W.H. (Eds.); Blackie Academic: London, 1995; Vol. 2, 327– 367. 6. Franz, C.M.A.P.; Holzapfel, W.H.; Stiles, M.E. Enterococci at the crossroads of food safety? Int. J. Food Microbiol. 1999, 47, 1 – 24. ¨ 7. Schleifer, K.H.; Kilpper-Balz, R. Transfer of Streptococcus faecalis and Streptococcus faecium to the genus Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecium comb. nov. Int. J. Syst. Bacteriol. 1984, 34, 31– 34.

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Hoang-Dung TRAN and friends
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245. 1991. Microbiol. Agents Chemother. Huycke. N. Shankar. Appl. Immun. D. Mol. Clin.B.A... All Rights Reserved. S..J. Med. Gasson. 39 – 42. Fujino.. A plasmid-encoded surface protein on Enterococcus faecalis augments its internalization by cultured intestinal epithelial cells. 1984. D..M. D. E. Claussen. 170. 2002. Enterococcus faecalis aggregation substance promotes opsonin-independent binding to human neutrophils via a complement receptor type 3-mediated mechanism.B. Hugli. 67. commensal.. 265 – 270. Moore. Flannagan. S. J. Ember. Spiegel.. Clewell. M.S. Clin. A. Chow. A. Ike...L. 255.B.B.. Eaton..L. Wells. B. 240.-A..A.Y. Dis. 2000. Jensen... 253. Snuggs. 244.B. 254. A variant enterococcal surface protein Espfm in Enterococcus faecium. 193– 200.A. G. Hashimoto. S. D.. I. Natl. Immunol. Dunny. Kobayashi. Laufs. Yamaguchi.A. A. 1524– 1528..A. 2445– 2452. H.D. M. S.A..Hoang-Dung TRAN and friends 239. M.W. S.B. high-level gentamycin-resistant Enterococcus faecalis. Sci. 35. Miyazaki.... Marasco W. C. and environmental isolates. 252. van Winkle.. H. K. Infect. 67.J. R. C. a gene encoding a novel surface protein. 1993.B. An. Bacteremia caused by hemolytic. 25.. 68. M.. M. 35. M. F. Infection-derived Enterococcus faecalis strains are enriched in esp. Microbiol. Pathol. Antimicrob.. J. Thal.S. Microbiol. Characterization of a class of nonformylated Enterococcus faecalis-derived neutrophil chemotactic peptides: the sex pheromones. J. Huycke. 241. Enterococcal sex pheromone precursors are part of signal sequences for surface lipoproteins. Gilmore. Zervos.M. 1999. Virulence factors of Enterococcus faecalis and Enterococcus faecium blood culture isolates. 1628– 1635. W. 269–275..O. Erlandsen. Hemolysin of Streptococcus faecalis subspecies zymogenes contributes to virulence in mice.M.. V.. Clewell. 1999. Infect. Inducible expression of Enterococcus faecalis aggregation substance surface protein facilitates bacterial internalization by cultured enterocytes. M. Contribution of the pAD1-encoded cytolysin to the severity of experimental Enterococcus faecalis endophthalmitis. Simon. T. J. D. Dunny. S. D. Infect. Vazquez. Jaquez-Palas.M. Sobottka.. T.G. R. 19. Eur. 1994. Immun. G. Infect. Dunny. Microbiol. Erlandsen. Jacques-Palaz. Mee. Ohno. 248. Mariscalco. R. Lett. Inc.. S.E. Acad. 1989.J. 1987.. S. J. Enterococcus faecalis bearing aggregation substance is resistant to killing by human neutrophils despite phagocystosis and neutrophil activation. Rakita. Cytotoxic effect of haemolytic culture supernatant from Enterococcus faecalis on mouse polymorphonuclear neutrophils and macrophages. T.L. J. J.. Mack. H. . 66 – 70. distribution among food. 6067– 6075. M. Infect. 246–247.. Infect. Gilmore. Clewell.E. G. Infect. 1549– 1556. M. Characterization of the human neutrophil response to sex pheromones from Streptococcus faecalis. Dunny. 26.J. R.B.. 1999.. J.I.M. Simon. M..I. Copyright © 2004 by Marcel Dekker. G.M. M. 250.. Rakita.M. Microbiol. Hoag. M. L. Jett.. Dunny. 2001. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Wirth. Till.M.. 246. Vanek. 251. Wells.. M. G. 7190– 7194. 45.. Dis. G..N. FEMS Immunol. 797 – 805. 134. N. 249.. K. 1626– 1634. Agents Chemother. Immun. H. Goto..S. Gasson. 49 – 60.. Antimicrob... Plasmid-associated hemolysin and aggregation substance production contribute to virulence in experimental enterococcal endocarditis. Y. G. 2000. 1990. I..J.. M. Gilmore.. Clewell. Y. Olmsted...S. R....A.. M. M.. Mariscalco. Antiporta. 1992.L. 247. Elsner. 37. K. 528 – 530. Craig. Proc. H. Ike.. medical.A. Environ. Nordquist.M. Immun. R. 87.A. Sannomiya. 243..E. Immun. 2474 –2477. 216. 37. 242. High incidence of hemolysin production by Enterococcus (Streptococcus) faecalis strains associated with human parenteral infections. 1993. 2000. Lindahl.M. Vanek.N... 60.M. Hirt.M. Eaton. Am. Suzuki. FEMS Microbiol. P. Perri.. S. Donabedian. C. Microbiol. Clewell. 67. Baghdayan.. Hashimoto.

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By combining recombinant DNA techniques and other recently developed molecular biological methods in the study of LAB.Hoang-Dung TRAN and friends 7 Genetics of Lactic Acid Bacteria LORENZO MORELLI Catholic University of Sacred Heart. in contrast to Escherichia coli. Denmark ATTE VON WRIGHT University of Kuopio. an overview of the actual genetics of these bacteria is starting to emerge. more profound understanding about their physiology and metabolic potential is currently under development. Bacillus subtilis. “Genetic Modification of Lactic Acid Bacteria. The recombinant DNA techniques were developed for lactic acid bacteria (LAB) before a profound knowledge of the genetics of these organisms existed. Saccharomyces cerevisiae. Frederiksberg. The mass of information just becoming available makes this a challenging task. but also in the realization of the expectations regarding the eventual practical applications of genetic techniques to LAB. Italy FINN K. This situation has changed. and presentation of all the interesting 249 Copyright © 2004 by Marcel Dekker. and other microorganisms that had been subject to intense biochemical and genetic studies for decades before the advent of genetic engineering.” aptly reflected the level of knowledge about this subject in the 1990s. It is to be hoped that this understanding will result not only in more advanced basic and fundamental knowledge. and consequently a new. Inc. . All Rights Reserved. The following chapter is an attempt to give both a brief outline of the history of the field and a more detailed account of the latest developments. Piacenza. Kuopio. The first nucleotide sequences of the total genomes of LAB are becoming available. INTRODUCTION The title of this chapter in previous editions of this volume. Finland I. VOGENSEN Royal Veterinary and Agricultural University.

[3] Genome sequences resulted in sizes of 2. plantarum.[4] which has been isolated from a number of environments (plants. which belong to the same subgroup of the genus Lactobacillus (the so-called streptobacteria or facultative heterofermentative Lactobacillus) and which have been isolated from similar habitats as Lb. The amount of data accumulated in a little more than 10 years of PFGE studies has indicated that the majority of LAB have relatively small genome sizes. PFGE analysis has also provided clear evidence of a large strain-to-strain variability in the macrorestriction profiles of LAB. GENOMICS OF LAB Functional Maps of Genomes The analysis of the chromosomes of strains belonging to genera of the LAB group commenced in the early 1990s and since that time has progressed at an impressive rate. sakei. PFGE estimated a size of 2. intestinal and urogenital tracts. II. find this chapter useful as a basic introduction to the genetics of LAB. dairy products.5 Mb according to the ongoing sequencing projects. The percentage of co-migrating fragments in closely related strains of lactococci was calculated to be 80%. but physical maps have also been deduced.96 Mb.[3. respectively. Recent data obtained by genome sequencing have confirmed the accuracy of the size estimations obtained by means of PFGE. . differences in genome size. In a similar way the DICE coefficient of similarity calculated for strains of Lb. In some cases only the size of the chromosome was calculated.11 – 14] The first genetic maps were obtained for lactococci and have provided an impressive demonstration of the plasticity of the LAB genome.[9] Strain-dependent variability also includes. Oenococcus oeni. The only species with a chromosome size greatly exceeding 3 Mb is Lb. We hope that the reader will. Lb. A. differences of 2 – 3 Kb have been detected. Estimation of the genome size and physical maps were the first results achieved (reviewed in Ref. Streptococcus thermophilus. whenever closely related strains have been analyzed.42 Mb.[2] while in the case of Lactobacillus gasseri ATCC 33323.17 Mb as calculated by PFGE analysis[6] and 2.[8] in contrast to 20 –40% in nonrelated strains. it is clear is that most LAB have small chromosomes. However. before genome sequencing. Inc. as suggested by Morishita et al. and it is used as powerful strain-typing technique. This observation indicates that environmental adaptation of LAB is not only based on gene inactivation. helveticus strains ranged from 26 to 100%. the genome size estimated by PFGE was 1. All Rights Reserved. Lb. in some cases. This technique is still widely used and has yielded macrorestriction patterns of dozens of strains belonging to almost all the major species of the LAB genera. was based on pulsed-field gel electrophoresis (PFGE).84 Mb.Hoang-Dung TRAN and friends research within the scope of this chapter is not possible.36 and 1. The first approach for calculating genome size and intraspecies variability.[10] These differences could involve a sufficient number of genes to have an impact on the physiological and the technological properties of strains. plantarum. fermented foods and feeds) and which is not restricted to specific ecological niches such as milk or the intestinal tract. [1]). gasseri by hybridizing gene probes to the mapped restriction fragments. in 1981. In general. paracasei strains.[7] While it is probably too early to reach general conclusions about this matter. as they revealed the rearrangements involving large sections of the Copyright © 2004 by Marcel Dekker. however. In Lactococcus lactis IL1403.[5] but could also involve significant reduction in the size of the genome. and Lb. seem to have a smaller genome size: 2. Restriction map – based genetic maps have been obtained for Lactococcus lactis.

at the time of writing. gasseri was first obtained by Roussel et al. Genomic Sequences As a consequence of the small genome size of LAB and the availability of high-throughput sequencing facilities. III. lactis subsp. extra-chromosomal elements in strains of Lb. have an inversion of approximately 40% of the chromosome when compared with the maps of two Lc. Inc. MG1363 and FG2. The Lactococcus lactis Genome and Its Relationship to the Metabolic Capacity of the Species Lc. and IS1077 families are unevenly distributed. of two divergent clusters of the species.36 Mb of the chromosome strongly suggests that this genome may be the result of a recent recombination event between two closely related genomes: IS belonging to the IS981.[17. DL11 and IL1403. In Lb.15] This inversion has been considered related to the origin of the cremoris subspecies.5 Kb in this strain. The distribution of the IS elements through the 2. even though the two strains were representative. thermophilus A054 strain spontaneously obtained by subculturing the wild-type strain.[13] Genetic mapping of lactobacilli was achieved only recently.18] Each rrn operon contained a single copy of each of the three rrn genes-23S rRNA (rrl). one of which was inverted in orientation with respect to the others. which could be Copyright © 2004 by Marcel Dekker. have been annotated by the sequencing groups.[7] but only a few have been finished and annotated.Hoang-Dung TRAN and friends chromosome. lactis IL 1403 was the first lactic acid bacterium whose genome was completely sequenced and analyzed. one of which was due to the recombination between two adjacent rrn operons. and 5S rRNA (rrf ). This could explain the high degree of genetic plasticity encountered in LAB. 1.10. This section therefore provides general considerations on the major advances made in the functional and comparative genomics of food microorganisms. PFGE analysis confirmed the presence of a linear plasmid of 48.[14] seven rrn loci (coding for ribosomal RNA genes) were found and a total of 47 gene clusters have been mapped. This could be an element of instability in the chromosomal structure. A closer view of the cremoris strains provided further evidence of the plasticity of lactococcal chromosomes as four regions of the chromosome of the strain FG2 were found to occupy a different and inverted position with respect to the strain MG1363. All Rights Reserved. Lb. IS983. while the chromosome size was estimated to be 1. more than 20 strains (Table 1) are being sequenced. the maps of two Lc.[20] The presence of insertion sequences (IS) (see Sec. as it has been mapped[3] and also subject to a sequencing project.13] Comparative analysis revealed extensive conservation of loci order. For example.84 Mb.16] Two strains of O. sakei 23K. with a special focus on the two LAB genomes that. 16S rRNA (rrs). . as demonstrated in other LAB. Two deletions occurred in each clone.[1. microbial genomics is developing fast for this group of bacteria.[11.[1] Genomic instability has been detected by means of genetic mapping in clones of the S.[7] Mapping analysis revealed that the chromosome contained six rrn operons. oeni have been genetically mapped. A). lactis strains. B. lactis subsp. cremoris strains. and it is therefore presently impossible to provide an accurate and updated review.[12. IV) is massive: 43 IS for a total of 42 Kb belonging to six different IS groups. gasseri ATCC 33323 is another well-studied strain. on the basis of macrorestriction profiles and ribotyping. in 1993[19] by means of PFGE analysis (see Sec. The evidence of linear.

ncbi. thermophilus LMD-9 S.Hoang-Dung TRAN and friends Table 1 Finished and Ongoing Sequencing Projects: Where to Find Information Web site(s) or Acc.ad.com/GOLD ftp://ftp. sakei 23K Lb.jgi-psf. cremoris SK11 Lb. pentosaceus ATCC 25745 ftp://ftp. NCDO505. bulgaricus ATCC 11842 Lb. acidophilus NCFM/ ATCC700396 Lb. gasseri ATCC 33323 Lb.ebi.gov explained by a lateral transfer from a donor Lactocccus strain.jgi-psf.integratedgenomics.org/ http://wit.org/pub/JGI_data/Microbial/ streptococcus_thermophilus/020930/ http://wit.jgi-psf.doe.jgi.[21] Nine strains of the Lc. bulgaricus ATCC BAA-365 Lb.integratedgenomics.jgi. lactis subsp. casei BL23 Lb. carrying one type of IS. oeni PSU 1/ATCC BAA331 P. The genome plasticity of Lc. plantarum WCFS1 Bifidobacterium longum NCC2705 Bifidobacterium longum DJO10A Leuconostoc mesenteroides LA81/ ATCC8293 Lb. cremoris (NCDO712. rhamnosus HN001 Lb. . lactis subsp. lactis subsp.jgi. thermophilus CNRZ 1066 O.integratedgenomics.org/pub/JGI_data/Microbial/ Lactobacillus_casei/ http://wit. johnsonii La1 (NCC2761) Streptococcus thermophilus LMG 18311 S.org/ http://wit.jgi.uk/proteome/ http://mbgd.genoscope.tigr.gov http://www. helveticus CNRZ32 Lb.kun.gov http://wit.integratedgenomics.doe.doe.doe.gov http://www.com/GOLD http://www.integratedgenomics.integratedgenomics. Copyright © 2004 by Marcel Dekker.org/pub/JGI_data/Microbial/ Lactobacillus_delbrueckii/ http://www.de/ http://www. lactis subsp.nih.com/GOLD Species and strain Lc.integratedgenomics. Prophages are also present with either complete or defective genomes.com/GOLD ftp://ftp.nih.integratedgenomics. to a recipient strain carrying another type of IS.genome.nl/lactobacillus/ http://www. lactis IL1403 Lc.doe. paracasei ATCC 334 Lb.gsf. thermophilus ATCC BAA-491 S.nlm. delbrueckii subsp.cmbi. lactis strains has been further demonstrated by Le Bourgeois et al.gov http://www.integratedgenomics.gov http://www.com/GOLD http://www.jp/ http://pedant.tigr.brevis ATCC367 Bifidobacterium breve NCIMB8807 Lb.com/GOLD/ http://www. delbrueckii subsp.jgi. BH770319-BH771051 http://wit.gov http://www.ac.jgi-psf.com/GOLD/ http://www.nlm.org/pub/JGI_data/Microbial/ Lactobacillus_brevis/021106/ http://wit. cremoris MG1363 Lc. Inc. number http://wit.ncbi.com/GOLD/ Acc.fr/ http://wit.com/GOLD ftp://ftp. All Rights Reserved.

plantarum WCFS1. . Several macrorestriction fragment length polymorphisms (RFLP) have been found. No long stretches of continuous homology could be detected. However. Biosynthetic capabilities of lactococci.96 – 1. Comparison with 2236 predicted proteins of S. C2. lactis cannot be considered an exclusively fermentative microorganism. Inc. but 344 hypothetical proteins had no database matches.[22] It is a 3. The presence of mutation potentially leading to inactivation of the respective gene was detected in 30 of the genetic determinants involved in amino acid biosynthesis. All Rights Reserved. LM2301. MMS36. It has been possible to assign a biological function to more that 2500 predicted proteins.52 Mb region of Lb.Hoang-Dung TRAN and friends NCDO2031. The Genomic Sequence of Lactobacillus plantarum The complete genome sequence of Lb.[15] but the DNA level comparison between Lb. indicating only localized similarities. has been published. Besides the linear regions of similarity. lactis IL1403. integration of prophage DNA. This situation strongly resembles what has been previously described in the chromosomes of the two subspecies of Lc. These strains were considered adequate for the investigation of genome plasticity because they were described as belonging to the same genetic lineage. three large-scale genome inversions were detected. it is well known that this bacterium requires at least 6 amino acids to grow in a synthetic medium. lactis.[20] De novo biosynthesis of purine compounds has been suggested by the presence of 57 genes involved in this metabolism. 2. as revealed by genome analysis. which is of paramount relevance for a bacterium adapted to ferment milk. LM0230. another gram-positive bacterium with low guanine and cytosine (GC) content.[23] Analysis of Lb. Lc. gasseri ATCC 33323 genomes showed some regions of extended homologies. a derivative of the human isolate NCIMB88226. On the other hand. the extracellular protease that has been found in Lc. One of these inversions causes a strong asymmetrical GC skew distribution. lactis. the first example of such a system in LAB.3 Mb circular chromosome with 3052 potential protein-encoding genes. acidophilus NCFM and Lc. and Southern hybridization analysis correlated the polymorphic regions with genetic events such as chromosomal inversion. a gene cluster encoding for nonribosomal peptide synthesis has been found. NCDO763. 3. this strain seems to lack the primary enzyme for protein breakdown. acidophilus NCFM and the Lb. include genes for the biosynthesis of 20 amino acids. and MG1363) were studied by pulsed-field gel electrophoresis. a substrate containing low levels of these DNA building blocks.99 Mb region of Lb. acidophilus. Quite surprisingly. and location of the transposon-like structures. revealed that 905 are highly similar to those from Lc. Comparative Sequence Data Available from Other LAB A comparative analysis of the genomes of two strains of lactobacilli belonging to species inhabiting the intestinal tract has also been performed. pneumoniae. Surprisingly. A hypothetical reversion of this region would not only restore the GC skew but also reestablish a direct similarity between the 0.48– 0. lactis IL1403 showed no extended region of simi- Copyright © 2004 by Marcel Dekker. gasseri and the 1. genome analysis of this facultatively anaerobic bacterium has led to the discovery that functions required for aerobic respiration are also present among the chromosomal genes. These regions appear to be fragmented internally. therefore. The most striking feature resulting from the sequence analysis was the high potential of this bacterium to import and metabolize a range of carbon sources.

plantarum. thermophilus CNRZ 1066[25] has shown the presence of a high number of truncated ORFs. in this case we have to be prudent and wait for more data before concluding a tight linkage between catabolic potential and gut colonization ability. The ongoing sequencing projects (Table 1) will provide evidence for or against this hypothesis. plantarum belongs to the so-called facultative heterofermentative (streptobacteria) subgroup of Lactobacillus. Proteomics Proteomics was originally based on two-dimensional electrophoresis of proteins. As noted earlier. LAB form a very large and diverse group of bacteria. C. in contrast to 18 and 34 such systems. lactis (mainly milk) is reflected by the presence in Lc.Hoang-Dung TRAN and friends larity. A preliminary characterization of the genome of S. and this is apparently reflected in a number of genes involved in sugar transport and utilization in its genome.13[26] has revealed the presence of 1806 putative coding sequences. All Rights Reserved.26 Mb genome sequence of an infant-derived strain of Bifidobacterium longum. Although the genetics of Bifidobacterium is not specifically addressed in this chapter. several physiological traits could. which could have adopted different strategies for surviving in the same habitats. which is strongly reflected in the genome organization.82 Mb circular genome. acidophilus NCFM[24] have been shown to have a high degree of similarity with the same genetic structure found in distant Lactobacillus species such as Lb. paracasei. distributed in a 1.[29] Among the 1730 possible coding sequences identified. Inc. Genome Plasticity Versus Environment The genomes of LAB sequenced and commented on suggest adaptation to the environment. The sequencing of the entire chromosome of O. A large number of the predicted proteins appear to be involved in the catabolism of oligosaccharides.[27] The ability to colonize a wide variety of habitats is typical for Lb. The technique has been refined in many ways and is now being applied to LAB. Lb. respectively. Quorum sensing genes have been found in Lb. Genome analysis has confirmed the presence of genes involved in oligosaccharide utilization in Lb. which has a larger genome. it is worthwhile to mention results achieved by means of bioinformatic analysis of the 2. D. At present we cannot make general or final conclusions about the initial data provided by comparative genomics. lactis of only three potential sigma factors and eight two-component regulatory systems. lactis genome. plantarum WCFS1. Both species have been isolated from the same types of environments. it turns out that the genes involved in basic information processes and gene regulation are highly similar to those found in Bacillus subtilis. the colon. this observation could indicate an adaptation of this bacterium to a nutrient-rich environment such as milk. However. subtilis. in B. explain the successful adaptation of this bacterium to one peculiar ecological niche. oeni Lo84.[28] but it is unclear if these genes have a real role in this intestinal isolate. at least partially.[23] Stress operons in Lb. the narrow range of habitats occupied by Lc. sakei. This ability to scavenge a variety of nutrients could explain the observed competitiveness and persistence of bifidobacteria in the colon. . However. If we take into consideration the Lc. Lb.[30] While Copyright © 2004 by Marcel Dekker. in which the Embden-Mayerhof pathway is active for six-carbon sugars and the phosphoketolase pathway for pentoses. plantarum has a chromosomal size approximately 1 Mb larger than the genome of Lb. acidophilus NCFM.

PLASMID BIOLOGY OF LAB The observations made by L. Physical Structure. 1. in addition to their significance for LAB. McKay at the University of Minnesota about the spontaneous and acriflavine-induced loss of lactose fermentation ability of lactococcal strains suggested involvement of plasmids in this phenotype. These plasmids replicate by two basic mechanisms: the rolling circle type of replication or theta replication. in this type of replication a nick is first formed by the replication initiation protein (IP) in the so-called plus origin of replication. a replisome. Inc.34] and to analyze differential expression patterns of lactococci under standard conditions and during purine nucleotide starvation. a double-stranded plasmid and a circular single-stranded intermediate are formed by cutting and religating the replaced minus strand.[38] Because of their technological importance study of the metabolic plasmids dominated the early stages of genetic studies on LAB (see also Sec. A proteomic approach has been used to investigate the stress response of LAB[31. and a reference protein map has been obtained for these bacteria.Hoang-Dung TRAN and friends genomic analysis provides a static view of an organism. and a helicase. DNA-polymerase III holoenzyme. lactis showed an important protein polymorphism.[39] and the detailed molecular events by Novick in 1998. thermophilus were investigated by means of proteomics. presence of plasmids is characteristic of many species and genera of LAB. Replication Mechanisms.[37] Subsequent demonstration of extrachromosomal DNA in the lactococci soon led to the identification of several metabolic plasmids and their functions. After the nick. Comparison of the proteomes of glucose. lactis MG1363 and S. A new round of DNA Copyright © 2004 by Marcel Dekker. The Rolling Circle Mechanism. III. L. When a cycle has been completed.[35] Comparison at the proteome level of two strains of Lc. and incompatibility are apparently linked to the replication mechanism.[33] and 15 proteins were initially identified. 1). and Incompatibility Circular Plasmids The majority of LAB-associated plasmids belong to the standard type of covalently closed circular. a wider relevance in general biology.[31.B). proteomics allows a dynamic observation of cellular events of interest in food production. In general. These mechanisms are intimately linked with the plasmid replication functions. starts the synthesis of a new strand while displacing the plus strand (Fig. which is situated in the immediate vicinity of the IP gene. All Rights Reserved. autonomously replicating DNA molecules. lactis NCDO 763 has been investigated by two-dimensional gel electrophoresis. These include mechanisms to control the plasmid copy number and to prevent the presence in the same cell of two plasmid species sharing too identical replication functions (incompatibility). and their study has revealed many aspects that have. a closer look at them is justified. stability.32] Lc. Because plasmid host range. Lc. consisting of a single strand – binding protein.[36] III. Host Range. .and lactose-grown cells revealed an unexpected link between the nature of the carbon source and the metabolism of pyrimidine nucleotides.[40] Briefly. A. The basic features of this type of replication were reviewed by Gruss and Ehrlich in 1989.

The staphylococcal plasmid pT181 has been used as a model to characterize the copy number control and incompatibility functions of rolling circle replicating plasmids. However. to be typical for small plasmids of LAB.[41] The small lactococcal plasmids pVW01 and pSH71. Their plus origins closely resemble that of a small staphylococcal plasmid pE194.[43] Although these small lactococcal plasmids have a similar mode of replication and share homologies in the replication region with the small staphylococcal plasmids such as pE194 and pC194. All Rights Reserved. are typical examples of plasmids replicating by a rolling circle mechanism. . Solid line represents parental DNA and dashed line the newly synthesized strand. personal communication). into the lactococci. which have been used in cloning vector construction (see Sec. oeni plasmid Copyright © 2004 by Marcel Dekker. several of which have been characterized and totally or partially sequenced. VI. In this plasmid the synthesis of IP is controlled by an attenuation mechanism involving a leader sequence in the IP messenger RNA capable of interacting with antisense RNA coded by a specific replication region site (cop).[45] the O. A). the leader sequence forms a transcription stop signal structure leading to the cessation of transcription and IP synthesis.[44] This mutation apparently enhances the promoter controlling the transcription of the IP gene (J.42] These plasmids have a broad host range replicating in several other gram-positive hosts and in E.C. the latter usually do not replicate in lactococcal hosts.[39. a mutation in a single base pair in the replication region expands the host range of pC194. In the presence of antisense RNA. in general. Alonso. leading to the formation of another double-stranded plasmid. a chloramphenicol resistance plasmid.Hoang-Dung TRAN and friends Figure 1 The rolling circle type of plasmid replication. Examples include lactococcal plasmids such as pFX2. synthesis starts from the minus origin of this intermediate. coli. Inc. The rolling circle type of replication seems.

[54] and phage resistance plasmids. gasseri.[49] and pER371 from S. spanning from a few thousand to tens of thousands of bp. such as pVS40.[66] 2. plantarum plasmid pC30il. Theta-replicating plasmids have been characterized in other species of LAB. bovis.53] citrate permease plasmids. The replication protein gene (repB) is preceded by an AT-rich origin of replication (repA). The lactococcal family of theta-replicating plasmids seems also to be generally compatible with each other.[61] The incompatibility region could be tentatively located within the above-mentioned region of 22 bp direct repeats and the first inverted repeat. [62]).Hoang-Dung TRAN and friends pLo13.56] Other examples are cryptic plasmids.[46] the Lc. No extended single-stranded regions are formed. S.[59] and pWV02.[60] The last has an exceptionally small size (3. reuteri has been shown to depend on the presence of the functional minus origin.[47] the Lb. thermophilus. such as lactococcal lactose fermentation and proteinase plasmids. III. Their origin of replication does not have structures resembling the lactococcal repA region. Linear Plasmids in Lactobacillus In addition to circular covalently closed plasmids.8 kpb) for a theta-replicating plasmid. pFV1001 and pFV1201 and pJW565 and pFW094. one between the 210 region and the start of repB. two incompatible pairs. V. were found. lactis plasmid pCI411. are also regularly found. They are large (20 –60 kpb) conjugative erythromycin-resistance plasmids with a broad host range. B).[51] Theta-Replicating Plasmids.58] pCI305. The theta-replicating plasmids in LAB are generally of medium or large size.[55.[57. Enterococcal theta-replicating plasmids of pAMb1 family are other well known examples of theta-replicating plasmids (reviewed in Ref. The synthesis can be uni.19] showed that some bands obtained by running PFGE without any restriction digestion were able to migrate at the same relative position even when different switch times Copyright © 2004 by Marcel Dekker. especially in their repB gene[63 – 65] A small 2.[48] Lb. . and Tetragenococcus halophilus represent a group of theta-replicating plasmids sharing a high degree of homology. Enterococcus faecalis.[52. The host range of these theta-replicating plasmids is rather limited in comparison to rolling circle plasmids.or bidirectional. Their wide host range and ability to mobilize nonconjugative plasmids (see Sec.[50] The stability of plasmid pGT232 from Lb. fermentum plasmid pLEM3. because typical genetic elements of the rolling circle type of replication were absent and no single-stranded plasmid DNA could be detected. acidophilus.665 bp cryptic plasmid pTXL1 from Leuconostoc mesenteroides has been assumed to replicate via the theta-replication mechanism. Evidence obtained by means of PFGE analysis[3. These plasmids include large metabolic plasmids (see Sec. All Rights Reserved. The sequences of the lactococcal plasmids mentioned above share a remarkable degree of homology in their respective replication regions. plantarum. After screening of 12 theta-replicating plasmids. It appears that Lb.A) has made them useful in genetic studies of LAB. Two inverted repeats. Lb. the presence of linear plasmid has been suggested in strains of Lb. as well as two AT-rich short (9–10 bp) repeats further upstream of the 22 bp sequences. Inc. Theta replication is based on a progressive replication fork with simultaneous synthesis of both new strands. One striking feature is the presence of three successive complete and one incomplete 22 bp direct repeats with a general consensus sequence of TATANNNNN(A/T)NAAAAA(A/ T)C(T/G)(G/A)TC immediately before the promoter of the repB gene.

lactis subsp.[75] The plasmid encoded b-galactosidase system is also common in strains of Leuconostoc. plasmid linkage of proteinase activity has been reported in at least one strain of Lb.3 kbp) in at least three strains of Pediococcus pentosaceus. Plasmids Associated with Aroma Production Diacetyl is an important aroma compound (“butter flavor”) in dairy products. a trisaccharide. see Ref.[71] and one of the key enzymes of the system.[72] Two different plasmids. which is subsequently split into galactose-6-phosphate and glucose by phospho-b-galactosidase. but do not form an operon. casei plasmid pLY101.[77. They form an essential part of an intricate machinery producing and transporting peptides necessary for the growth of the cell (reviewed in Ref. while the permease gene is separated by a 2 kbp DNA fragment containing an IS sequence. Metabolic Plasmids Carbohydrate Fermentation and Proteinase Plasmids Plasmids coding for important technological properties have naturally attracted much attention since their discovery in the lactococci. All Rights Reserved. casei strains. Glucose is further metabolized by the Embden-MeyerhofParnas pathway. plantarum strains. acidophilus TK8912. lactis both lactose permease and b-galactosidase genes reside on the same plasmid. have been characterized in Lb. 1. This is an indication of the linear nature of the migrating DNA. . one coding for 6-phospho-b-galactosidase and the other for the genes of the tagatose diphosphate pathway.[70] The presence of carbohydrate-fermentation plasmids is not as common in lactobacilli as in lactococci. Plasmid linked PEP-dependent lactose phosphotransferase system has been associated with Lb. Plasmids coding for b-galactosidase seem to be typical for Lb. and is subsequently split by b-galactosidase.50 kbp) plasmids. the lactose permease pathway.2 –47. plantarum strains there simultaneously exists both a plasmid-associated and chromosomally coded copy of the gene. gasseri seems so far to be the only lactic acid bacterium in which this kind of extra-chromosomal DNA has been detected.[68] The lactococcal lactose fermentation is based on the phosphoenolpyruvate (PEP) –dependent phosphotransferase system. possibly reflecting adaptation of these strains to the dairy environment. B. [68]). Inc.78] As an example of a carbohydrate other than lactose. [69]).Hoang-Dung TRAN and friends were used.[79] 2. 6-phospho-b-galactosidase. Lactose enters the cell unphosphorylated. the utilization of raffinose. However. examples of lactose fermentation plasmids are known also among lactobacilli. lactis biovar. In these bacteria the lactose fermentation and proteinase activities are almost invariably associated with relatively large (from 17 to .[74] It also appears that in many Lb. Linear plasmids have been discovered in several bacteria. is located on a Lb.[76] In Le. Copyright © 2004 by Marcel Dekker. The b-galactosidase gene is coded by two partially overlapping genes (lacL and LacM). but Lb. whereas galactose-6-phosphate is first converted into tagatose diphosphate before it can be split into two triose phosphates and enter the normal glycolytic pathway (for review.[73] In some lactobacilli the plasmid-encoded lactose utilization is based on another mechanism. Except for the lactococci. helveticus. Lactose enters the cell as lactose6-phosphate. The lactococcal proteinases are cell wall –associated enzymes that are very closely related to each other. the key enzyme of the system. is linked to relatively large plasmids (36. In dairy cultures diacetyl is produced from citrate by strains of Lc.

[92.[94] 1.[82] Although the genes involved in the citrate metabolism share a high degree of homology between the two species. V). lactis to small (approximately 8. Nisin is a well-known example of class I bacteriocins.[96] is a typical example. It is particularly active against Clostridium tyrobutyricum. etc. the production of which is coded by an 81 kbp conjugative plasmid. they are differently regulated. The bacteriocins are classified into three groups:[94] lantibiotics or bacteriocins containing modified amino acids. which mediates the citrate uptake by the cells. mucoidness has been correlated with the presence of plasmids ranging in size from approximately 7 to 30 kbp. Lacticins represent plasmid-encoded class I bacteriocins.86] Several lactococcal plasmids ranging in size between 27 and 47 kbp associated with mucoid phenotype have been identified. The immunity gene is usually intimately linked to the bacteriocin gene and expressed concomitantly with it. In at least some strains of Lb. coded by a six-gene operon located on a 70 kbp plasmid in Lc. The linkage of citrate permease gene in Lc. and large heat-labile bacteriocins (class III). Clostridium. pNZ4000.[83. casei. Inc.84] 3. The key enzyme in the pathway is citrate permease. All Rights Reserved.Hoang-Dung TRAN and friends diacetylactis and members of the genus Leuconostoc. in lactococci by external pH and in leuconostocs by citrate. the bacteriocin gene clusters have certain organizational similarities. or the ability to produce extracellular polysaccharides.[95] Apparently no further genetic or molecular biological analysis has been done on this system.2 kbp plasmid has been elucidated. . such as lanthionine and b-methyllanthionine (class I—bacteriocins).[90] The complete sequence of this 42. has indicated the involvement of at least 14 genes in the exopolysaccharide production. small heat-stable nonlantibiotic peptides (class II). The resulting N-terminal leader sequence inhibits the intracellular activity of the bacteriocin while simultaneously providing the signal to the transport system. Typically the bacteriocin gene contains a leader sequence with a double function. it was found that at least some leuconostocs have a similar system. which has been completely sequenced.81] Subsequently.).7 kbp) plasmids was detected relatively early. is a property of some lactococcal strains that have traditionally been used to give body and texture to certain types of Scandinavian fermented milks. Plasmid-Encoded Lactococcal Bacteriocins Historically. Regardless of class. it is now well established that the trait is coded by a chromosomally located conjugative transposon (see Sec. Listeria. Lacticin 481. Although early evidence suggested plasmid involvement in nisin production. Mucoidness Mucoidness. Plasmids Associated with Bacteriocin Production and Immunity Bacteriocins are proteins or peptides secreted by bacteria that inhibit the growth of other bacterial strains or species.93] C. LAB produce a wide variety of bacteriocins active against other gram-positive bacteria (other species and strains of LAB.[87 – 89] Genetic analysis of one of the plasmids. lactis ADRIA 85LO30.[80.[85. one the very first plasmid-associated lactococcal bacteriocins was diplococcin.[97] Another plasmidencoded bacteriocin belonging to the class I bacteriocins is the two-component lantibiotic lacticin 3157 encoded by a 60 kb plasmid.[98] Copyright © 2004 by Marcel Dekker.[91] Mucoid variants are common among other species of LAB.

pCP49 and pCP40. coded by two operons in a 50 kbp plasmid pCIM1 in Lb.[104] both produced by 14 kbp plasmids (pLA103 and pCV461. Carnobacteriocins A and B2 are coded by two separate plasmids. Inc. sakei L45.[106. lactis ssp. Loss of bacteriocin activity is connected with novobiocin-induced curing of a 49 kbp plasmid of the E. Other Plasmid-Associated Bacteriocins from LAB Pediocins and pediocin-like bacteriocins are often referred to as class IIa bacteriocins. acidophilus.[102] Other examples of plasmid involvement in bacteriocin production are acidocin 8912[103] and acidocin B. The resulting recombinant Carnobacterium strain is capable of producing both lactacin F and carnobacteriocins. among others. An illustrative example of a plasmid coding for multiple bacteriocins is p9B4-6 from Lc. lactis biovar. mesenteroides Y105 harboring a 35 kbp production plasmid. .[99] In this plasmid one bacteriocin locus consisting of two genes jointly produced the so-called lactococcin M (low antagonistic activity). Bacteriophage Defence Mechanisms Associated with the Plasmid pNP40 A 65 kbp conjugative plasmid pNP40 from Lc. and BM1. B2. the plasmid has been shown to contain Copyright © 2004 by Marcel Dekker. johnsonii. The genes have been cloned and expressed in Carnobacterium pisciciola LV17.[108] Enterocin ON-157 is a recently characterized enterococcal bacteriocin active mainly against other enterococci. faecium production strain. restriction/modification (R/M) systems. Plasmids and Phage Defense Mechanisms Plasmids associated with increased resistance against bacteriophages are common. for the food industry.[105] 3. Tens of plasmids coding for these mechanisms are known[110. Bacteriocin Plasmids from the Lactobacilli A wide range of both chromosomally encoded and plasmid-linked lactobacillar bacteriocins is known.Hoang-Dung TRAN and friends Lactococcins belonging to the class II bacteriocins are mainly active against other lactococci.[112] Subsequently.111] (see also Chapter 8). they are particularly interesting. and sakacin A. respectively) in Lb. and abortive infection (Abi) or intracellular inhibition of phage development. while the other region coded for two independent highly active lactococcins A and B. An example of a lactobacillar lantibiotic is lactocin S. All Rights Reserved.[100. associated with a 60 kbp plasmid of Lb. Three basic phage resistance mechanisms with different subdivisions are known: inhibition of phage adsorption. cremoris 9B4. The latter strain produces three class II bacteriocins: carnobacteriocins A. diactylactis DRC3 confers resistance against the bacteriocin nisin and also protects the strain from the attack of a lytic bacteriophage c2. especially among the lactococci. lactis subsp.107] Mesentericin Y105 is an example of class IIa bacteriocins produced by Le. 1.[94] Lactacin F is a plasmid-associated two-peptide class II bacteriocin from Lb. Even a cursory attempt to describe them in this context is not possible here. sakei Lb706.[109] D. while the gene for BM1 is chromosomal. Because of their strong activity against Listeria. P.101] 2. acidilactici as well as by a number of other LAB. In the following paragraphs only the best characterized plasmids associated with multiple phage defense mechanisms are presented. They are often plasmid-encoded bacteriocins produced by.

Enterococcal Antibiotic Resistance Plasmids Resistance to a wide range of antibiotics is typical for enterococci isolated from clinical. [126. aminoglycosides. In many cases the resistances are located in mobile genetic elements (see Sec. lactis ssp.[125] the former a 26. and pork. Antibiotic Resistance Plasmids in Lactobacilli Early work by Vescovo et al. of swine origin conferring resistance to tylosin and erythromycin has been completely sequenced. ampicillin. 1. lactis ME2 is an exceptionally phage-resistant strain harboring a 46 kbp plasmid pTR2030[117] encoding both an R/M system and an Abi mechanism.[121] 2. but resistance plasmids. faecium. However. which can be used as a selective marker. are classic examples of conjugative enterococcal plasmids with a wide host range.[121] An ermT carrying 4. and fecal sources. acidophilus strains originating from pig and calf feces several resistances (penicillin. Conjugative plasmids carrying genes for erythromycin resistance Copyright © 2004 by Marcel Dekker.[116] These findings suggest a possible role for genes active in DNA recombination and repair in some of the Abi mechanisms.[120] E. The plasmid also contains five IS elements. Restriction Modification and Abortive Infection Coded by the Plasmid pTR2030 Lc. . three of which apparently originate from E. and Lb. and Enterococcus has been detected in a lactococcal strain isolated from soft cheese. plantarum of meat origin represents a completely sequenced lactobacillar tetracycline resistance (tetM) plasmid.9 kbp) from Lb. 2. All Rights Reserved. and chloramphenicol resistance determinants apparently related to corresponding genes in Staphylococcus. bacitracin) were probably plasmid-associated. a 30 kbp completely sequenced theta-replicating plasmid pK214 coding resistance for streptomycin.[118.5 kbp erythromycin resistance plasmid and the latter a 30 kbp chloramphenicol-erythromycin double resistance plasmid.Hoang-Dung TRAN and friends genes for at least two Abi systems (AbiE and AbiF) and for a mechanism blocking phage DNA injection. has been both subcloned and analyzed at the molecular level. Lb. LlaI. Antibiotic Resistance Plasmids Lactococcal Antibiotic Resistance Plasmids Antibiotic resistance plasmids are relatively rare among lactococci. further emphasizes its potential usefulness in engineering dairy starter strains for enhanced phage resistance. tetracycline.127]). many of which are conjugative. Listeria. These plasmids can be regularly conjugated to other species and genera of LAB (see Refs.[115] Among the other genes characterized in this plasmids are a homolog of recA (a gene central in DNA recombination and repair) as well as a gene-sharing homology with umuC (a gene involved in the so-called SOS response to DNA damage). chloramphenicol.[63] 3. IV).114] The fact that this plasmid also codes for cadmium resistance.[123] Plasmid pMD5057 (10. reuteri. erythromycin. tetracycline. Plasmid linkage to erythromycin and chloramphenicol resistance has been detected in Lb.119] as well as transferred to heterologous hosts. are typical to this genus. plantarum.[122] demonstrated by plasmid curing experiments that in 20 Lb. food. Inc. pigs. The R/M system associated with pT2030. Plasmids pAMb1[124] and pIP501.[113. cloxacillin.2 kbp plasmid p121BS from an unidentified Lactobacillus sp. fermentum strains isolated from poultry.

cremoris (out of 24 lactococcal strains screened). All Rights Reserved.4 kbp fragment.[129] 2.5 kbp plasmid in one strain of Lc. lactis ssp. among LAB. . While the general genetic recombination in LAB as well in other bacteria requires homologous sequences between the participating DNA molecules.[130.131] In hybridization experiments the gene coding for this heat stress protein gene could also be located on a 7. and their characteristics and occurrence in various bacteria groups. Insertion sequences represent the simplest form of transposable genetic elements. is plasmid encoded. although more difficult to detect.[131] IV. INSERTION SEQUENCES. The size range of S. Introns are sequences interrupting functional genes.128] F.[121.A). the transposable genetic elements require only short (3 – 10 bp) target sequences in the recipient DNA. which may participate in protecting the cell from heat stress. understandably. has a size of 33 kbp. Inc. UV resistance and adaptation to heat stress are currently known examples. have been extensively reviewed. leuconostocs. thermophilus a heat shock protein (Hsp16. Numerous IS elements have been characterized. While introns are common in eukaryotes. Other Plasmid-Associated Phenotypes While plasmids coding for important functional properties have. their presence in bacteria was a relatively recent finding. and by cloning experiments the UV-resistance determinant has been located in a 5.Hoang-Dung TRAN and friends (ermAM) or tetracycline resistance (tetM) have been detected in enterococci isolated from cheese or meat products. lactis IL594. typical transposons contain additional genes. including lactococci. but subsequently spliced during the processing of mRNA.8 – 11. TRANSPOSONS.[133] Copyright © 2004 by Marcel Dekker. Certain defense or adaptation mechanisms in some LAB strains are plasmid-encoded.[132] In contrast to simple IS elements. such as antibiotic resistance determinants or genes involved in conjugative gene transfer (see Sec. They are transcribed to RNA along with the rest of the gene.4 is 2. While the insertions can occur either at a specific site or apparently randomly. They have a size range of 750 – 2000 bp and typically contain only the genes necessary for the transposition flanked by short inverted repeats. Ultraviolet Resistance In Lc. 1.4). attracted much attention and antibiotic resistance plasmids are of interest regarding the safety aspects of LAB. V. type II introns have been detected in lactococci. pIL7. enterococci. depending on the mobile element. and pediococci. AND INTRONS Different transposable genetic elements are important mechanisms to enhance the genetic mobility and elasticity of bacteria. the resistance to ultraviolet (UV) irradiation is plasmidencoded. thermophilus plasmids encoding Hsp16.0 kbp. So far. flanked by IS sequences. The relevant plasmid. there probably are other plasmid-associated phenotypes worth studying. the transpositions introduce mutations and genetic rearrangements. Type II introns represent another type of bacterial integrative genetic elements. Heat Shock Proteins In some strains of S. lactis ssp. lactobacilli.

IS981. that of Lc.).[140] The 34. altogether 43 copies of IS elements could be found representing six known types (IS981.[136] 2. This block is flanked by direct repeats of TTTTTG. IS elements in Lb. was identified in Lb.[134] Subsequently insertion elements have been found both in other species of lactobacilli as well as in lactococci. B. thermophilus a novel. and its role or type of eventual transposition is therefore unclear. no inverted repeats flanking the nisin-sucrose gene block have been identified. 1. casei by Shimizu-Kadota et al. IS1077). However. etc. mesenteroides. lactis strains of plant origin. lactis ssp. lactis In the Lc. IS905. IS904. plantarum Two classes of transposase-coding regions resembling IS elements. plantarum WCFS1. and IS1077 belong to a larger IS3 family of insertion elements. lactis ssp. IS982. such as dairy enterococci.[22] are illustrative examples of the frequency and occurrence of IS elements in the genome. designated as ISP1 (eight complete copies) and ISP2 (four complete copies). 2. and the transconjugants contained insertions of variable sizes (50 – 110 kb) in their chromosome. have been detected in the genome of Lb. ISL1. IS983. IS904. conjugative. Tn5307. Transposons Lactococcal Conjugative Transposons Associated with Nisin Synthesis and Sucrose Fermentation The genes for nisin biosynthesis and immunity as well as for sucrose utilization are known to reside on a 70 kbp conjugative transposon. The ICESt1 Element of Streptococcus thermophilus In S. All Rights Reserved. while many of the IS elements characterized specially in lactococcal plasmids belong to the ISS1 class of IS6 family. The copy numbers of individual IS elements range from one (IS982.[132] Formation of cointegrates where the donor and target replicons are separated by two directly repeated IS copies is typical of this family. Tn5276. Insertion Sequences The first IS element in LAB. lactis IL 1403 genome. transposable genetic element has been recently characterized. 1.[135] and they have been successfully used for insertional mutagenization of lactococcal chromosomal genes.Hoang-Dung TRAN and friends A. The conjugative nature of the nisin-sucrose transposons allows for their introduction even to heterologuous hosts. IS905) to 15 (IS983). ISS1 elements have been shown to cause spontaneous formation of cointegrates between lactococcal plasmids. IS Elements in Lactococcous lactis ssp. plantarum WCFS1.[138] Recently transposons associated with sucrose fermentation only have been detected in Lc. The ISP2 apparently does not have the terminal inverted repeats. which has resulted in different designations (Tn5301.7 kb element ICESt1 integrates site-specifically to the gene Copyright © 2004 by Marcel Dekker. Inc. lactis IL1403[20] and Lb.[22] ISP1 represents a typical IS sequence having a homology with IS1165 of ISL3 family from Le.[139] These could be conjugated to sucrose-negative recipients. most likely representing a duplication of the target sequence due to transposition. The two completely sequenced LAB genomes. .[137] The nisin-sucrose transposons vary slightly from strain to strain.

[143] Characteristic of Tn916 is a very wide host range spanning both gram-positive and gram-negative bacteria. and they are regularly associated with conjugative elements or sex factors.Hoang-Dung TRAN and friends coding for putative fructose-1. This enzyme. Tn916 is still both the best known and most widely used representative of enterococcal conjugative transposons. VI. since vancomycin-resistant enterococcal strains can cause persistent nosocomial infections. which is involved in the transesterification reactions essential to the splicing. Tn920.[149] Retrohoming is a feature of group II introns.[133] Lactococci were the first bacteria in which group II introns were found. The proteins associated with the transfer functions of this element shared homology with corresponding proteins of an enterococcal transposon Tn916 (see Sec.[133] The lactococcal intron Ll. Group II Introns Characteristic of the group II introns is the formation of a closed circular structure (“lariat”) during the splicing event. enterococcal transposons can be problematic. Domain VI contains a protruding adenosine. Tn916 is accurately excised. The term “integrative and conjugative elements” has been suggested for these types of transposons. remains associated with the lariat-like RNA structure after splicing. V. was the first conjugative transposon detected. Inc. the intron can be retargeted by modifying these sequences. (see also Sec. This property has been utilized in genetic studies of LAB for the insertional inactivation of genes in recipient strains. Domain I contains the exon-binding sites (EBS1.[142] Although several others have been subsequently characterized (Tn917 and Tn916-related Tn918. All Rights Reserved.147] C. together with some other intron-coded proteins. The reverse transcriptase coded by the intron has there a key function.[148] Group II introns have a conserved secondary structure consisting of six domains. . Because the specificity of retrohoming depends on the sequences of exon-binding sites. bridging the gap in one strand.6-diphosphate aldolase. while a complementary strand is being synthesized by reverse transcriptase. Further comparison of the sequence of ICESt1 with other elements present in various gram-positive bacteria revealed a common modular structure suggesting a family of conjugative transposons with a circular intermediate involved in the integration process. Tn925). The spliced RNA is covalently joined to the DNA.[144] From the public health point of view. In the absence of tetracycline. and VI) have a role in splicing.[146. and there is also the danger of the resistance spreading to other genera. three of which (I. This 18 kbp element has subsequently been totally sequenced and analyzed at the molecular level. IV.[145] The best characterized vancomycin resistance transposon Tn1546 is itself nonconjugative. A recent example of the Copyright © 2004 by Marcel Dekker.B3). but it can be effectively mobilized by conjugative elements residing in the same host strain.ltrB is one of the best characterized bacterial group II introns. Enterococcal Transposons Enterococcal transposon Tn916. and d). Particularly the transposons associated with vancomycin resistance have received attention. when the intron RNA is excised from the mRNA.A). such as pathogenic staphylococci. which confers resistance to tetracycline. forming a DNA endonuclease that cuts the target DNA at a site recognized by the exon-binding sites of the intron. EBS2. They were first found in the organelle genomes of lower eukaryotes and plants but have subsequently been found in several species of Eubacteria but not in Archeae.[141] 3. restoring the original gene structure and function.

and even genera of LAB can occur in vivo with the known natural mechanisms. Gene Transfer In Vivo Physiological Transformation Natural transformation. species. and most convincing. Transduction Transduction is the transfer of DNA between two strains by means of bacteriophage. reliable in vitro gene transfer mechanisms are essential and have been adapted to the most important LAB. this was not a general phenomenon of plasmid transfer. Transduction was first demonstrated the early 1960s in Lactococcus by Elliker and coworkers. respectively.[151] Later DNA was shown to be the transforming principle (152. In dairy microorganisms. Møller-Madsen later claimed that only a few strains of lactococci were able to transform by natural competence and that. 1. A. In the 1970s McKay et al. [151]).[158] These observations indicate that lactococcal natural competance has to be reconsidered as a mode of gene transfer in lactococci. transduction. personal communication). 2. lactis by transferring between lactococci the ability to produce a malty flavor. indicating that a number of competence genes need to be expressed for natural competence to take place. However. see Ref. Griffith in LAB in 1929 in S.ltrB into genes coding for malate decarboxylase and tetracycline resistance in the Lc. Finally.Hoang-Dung TRAN and friends application of this technique is the targeted insertion of Ll. Knite (155. pneumoniae. Although the significance of these mechanisms varies greatly among the different species. for the actual recombinant DNA techniques.[159. see also Ref. This observation of natural competence has not been satisfactorily confirmed. these strains were lost (A.[161. GENE TRANSFER SYSTEMS Gene transfer between strains. Transformation by natural competence has been thoroughly studied in Bacillus subtilis (for a review. [156]) reported the transfer of mannitol and streptomycin resistance in lactococci by natural transformation. Møller-Madsen. However. However.[164] Birkeland and Holo[165] showed that carrying of the cohesive ends from the temperate bacteriophage fLC3 increased the transduction efficiency of plasmids approximately Copyright © 2004 by Marcel Dekker. they contribute to the horizontal gene transfer in the natural habitats of LAB and they can be utilized in the genetic modification of LAB strains. lactis IL1403 genome sequence. see also Ref. was described by F. unfortunately. physiological transformation. is the identification of the complete competence operons in the Lc. Sanders and Nicholson[157] later showed that nonprotoplasted lactococci was able to take up plasmids as well as phage DNA if polyethylene glycol (PEG) was present. . since pAMb1 was not transduced with high frequency in secondary transductions. and conjugation. [153]).[163] in repeated transduction experiments. the first gene transfer mechanism described. showed transduction of chromosomal traits like maltose and mannose utilization as well as plasmidlinked traits like lactose utilization and proteinase activities using induced temperate phages for the transduction. Inc.[150] V.162] High-frequency transduction of lactose metabolism was shown by McKay et al. lactis genome. All Rights Reserved.160] where tryptophan independence and streptomycin resistance. Møller-Madsen and Jensen[154] first described a natural transformation system in a few strains of Lc. a few results indicate that the original observations were not artefacts. was transferred by a virulent bacteriophage c2.

Protoplast Transformation Preparation and regeneration of lactococcal protoplasts was achieved by Gasson. Since the development in the early 1980s of transformation systems for LAB. the conjugative transfer of lactococcal lactose fermentation plasmids has been well reported (see Ref. are well known. the element contains a group II intron and a gene causing a “clumping” phenotype (cluA) associated with the high incidence of conjugation.[169] demonstrated transduction in a fermented milk environment by S.[176] who also demonstrated PEG-induced protoplast fusion and the recombination of both Copyright © 2004 by Marcel Dekker. Recently. especially among the enterococci. In addition to lactococci transduction has been demonstrated in Lb. Instead. plaque formation) is not limiting for gene transfer by phage.[169. . The sex factor is also associated with an ISS1-type insertion element enabling its change of location from chromosome to plasmids.[173.[166] showed that the cos-site from the lytic phage sk1 also increases the transduction frequencies of plasmids.E. such as the origin of DNA transfer (oriT) and the various genes involved in the actual transfer event (tra). the enterococcal plasmid pAMb1. the conjugative genetic elements must have certain highly conserved common structures. An important aspect of conjugation is the mobilization of normally nonconjugative plasmids by functional sex factors and conjugative genetic elements. sex pheromones. however.[168] Heller et al. III. there has been only limited interest in transduction. thermophilus phages. This is probably due to the expected limitations in host range of the temperate and virulent bacteriophages. Irrespective of the mechanisms of achieving cell-to-cell contact. Inc.. The importance of conjugation among LAB has been indicated in previous chapters in discussions of conjugative transposons and antibiotic resistance plasmids as well as the association between conjugation and group II introns. The first efficient methods were based on protoplast transformation. However.Hoang-Dung TRAN and friends 1000-fold. [172] for review).[171] Pheromones. thus leading to the formation of donor-recipient pairs. In Vitro Gene Transfer Techniques Despite the usefulness of in vivo methods available for genetic studies. Among the metabolic plasmids. or substances produced by recipient cells promoting the synthesis of a cell aggregation factor by the donor. but they have been replaced by electroporation during recent years. such as LAB.170] 3. Conjugation During conjugation. gasseri ADH[167] and in S. lactis 712 the sex factor causing a high frequency of recombination has been thoroughly analyzed. host range (e. which.g. In Lc. Chandry et al. 1. is able to efficiently induce the conjugative transfer of proteinase plasmids in lactococcal hosts. For example. as noted in Sec.174] In addition to genes involved in the actual DNA transfer. DNA is transferred from the donor cell to the recipient by direct cellto-cell contact. but they apparently have no role in conjugation between gram-positive bacteria. All Rights Reserved. In gram-negative bacteria this contact can be mediated with structures called sex pili. development of efficient transformation techniques has been necessary for actual gene cloning in LAB. can be conjugated to several species and genera of LAB. thermophilus.[175] B. are not universal among gram-positive bacteria with conjugative genetic elements.

PEG treatment time. All Rights Reserved. have made the application of recombinant DNA techniques possible for most important LAB.[189] C.Hoang-Dung TRAN and friends chromosomal and plasmid-linked markers in the regenerated fusion products. and bacterial growth phase before protoplasting. The mechanisms involved have mainly been studied using the lactococcal integration vectors as models (see sect. The first electroporation of Lc. protoplast concentration. Leuconostoc. Inc.[191] Amplification of integrated plasmids seems to be a regular phenomenon in these experiments when selective pressure (the presence of an antibiotic against which the inserted plasmid confers resistance) is applied. which was previously very difficult to transform. Subsequently. ionic composition of transformation buffers. allowing for the shotgun cloning of both plasmid-linked[178] and chromosomal lactococcal genes[181] in lactococcal hosts. recA. Homologous sequences for these plasmids have been derived either from lactococcal chromosome[190] or from a resident prophage. casei in the same year by Chassy. 2.[183] The electroporation techniques have since then been introduced and optimized for most of the important LAB species and genera such as lactococci. The vectors available range from simple general cloning vectors to constructs Copyright © 2004 by Marcel Dekker. and Lb. and this has allowed the polymerase chain reaction (PCR)– based cloning of the lactococcal recA gene and generation of new recA mutants by insertional inactivation. General Genetic Recombination and Recombination-Deficient Mutants In general recombination the DNA introduced into the host may be integrated either with the host chromosome or plasmid.[178 – 180] With optimized methods and using different antibiotic-resistance markers. VI). VI. bulgaricus. a short electric impulse is conducted through the cell suspension. Recombination-deficient lactococcal mutants are known. The first of these was obtained by Anderson and McKay[192] after mutagenization with methyl methanesulfonate (MMS). together with the development of cloning vectors. PEG concentration and molecular weight. but introduction of chromosomal markers is inhibited. The central gene controlling bacterial recombination. thermophilus. Electroporation In electroporation.[193.194] These mutants are sensitive to different DNA-damaging agents as well as to oxygen and heat. transformation frequencies of 104 – 106 transformants per microgram of DNA were achieved. the transformation protocols were optimized in different laboratories with respect to protoplast regeneration. delbrueckii subsp. provided that the incoming DNA is sufficiently homologous with the host sequences.[184 – 188] Recently the method has also been optimized for Lb.[195] indicating a global role for RecA in various cellular processes. . Transfer of plasmids into this strain occurs normally. is highly conserved among different genera of both gram-negative and gram-positive organisms. Actual PEG-induced protoplast transformation of lactococci was reported by Kondo and McKay[177] using lactose fermentation plasmids. TOOLS FOR THE GENETIC MODIFICATION OF LAB The efficient gene transfer mechanisms. plantarum. lactis was reported by Harlander[182] and of Lb. This is sufficient for efficient DNA transfer and often for rather high transformation frequencies. S. permeabilizing the cell wall to DNA for a few nanoseconds.

the accumulation of single-stranded DNA might lead to genetic instability when foreign DNA is inserted into the constructs.[199] 2.and pSH71-based vectors). these plasmids replicate by the rolling circle mechanisms and have a wide host range. faecalis DS16 in the late 1970s. both of Enterococcus origin. usually coding for antibiotic resistance. In addition. allowing for selection. This property makes them very attractive for research purposes.[202 – 205] Subsequently. E. By choosing and combining different replication regions functional in different hosts. Integration Vectors Integration vectors for LAB have been used for several purposes—mainly for the generation of mutations. 1. first by Clewell and coworkers for the characterization of hemolysin and conjugation in E. subtilis by first combining Tn917 with a temperature-sensitive derivative of the pE194 repli- Copyright © 2004 by Marcel Dekker.[200] The integration of Tn917 into the target is induced by the presence of small non-inhibitory amounts of erythromycin. A large number of vectors have been developed. but only a few can be mentioned within the scope of this review.g. All Rights Reserved. also outside LAB. coli. Tn917 became a powerful tool for generating mutations in B. As noted in Sec. With LAB the commonly used vectors are based either on cryptic lactococcal or lactobacillar plasmids or derived from large conjugative plasmids such as pAMb1 and pIP501. . III. Inc. Enterococcus) were derived from transposons like Tn917 or Tn916. it is possible to create families of shuttle vectors able to replicate and express selection markers in widely different hosts such as lactococci.[201] Tn917 has been a workhorse for generating mutations in a large number of gram-positive bacteria. and the genetic constructs in this background are considered more stable than the ones based on plasmids with the rolling circle type of replication.A. site-specific integration vectors based on phage-integration elements allowing stabilization of genes on a specific site in the chromosome have been developed. Clewell and coworkers identified Tn917 in E..Hoang-Dung TRAN and friends designed for special purposes such as integration or efficient expression of cloned genes. It has lost the conjugative properties and does not replicate in gram-negative hosts (in contrast to pVW01. faecalis. but is a cause of concern in the genetic containment of eventual genetically modified strains in their applications.[197] Plasmid pIL253[198] is a deletion derivative of pAMb1 with a high copy number. are based on nearly identical cryptic plasmids pVW01 and pSH71. Food-grade vectors devoid of antibiotic resistance markers represent a special case in respect to eventual applications. in order to mutagenize a specific gene. and B. In addition to cloning. lactobacilli.[196] The two most common lactococcal vectors. present-day genetic techniques allow for targeted mutagenesis as an alternative to traditional random mutagenesis of strains by chemical mutagens or radiation. Also. subtilis. A. either as a random process to identify genes or by site-specific integration gene disruption or gene replacement. pGK13 and pNZ12. Cloning Vectors General Cloning Vectors The general cloning vectors typically consist of a plasmid replicon and a marker gene. This plasmid replicates by the theta mechanism. Some of the first integration vectors used in LAB (e.

followed by growth at the nonpermissive temperature (and subsequent curing of pVE6007).[208] These genetic tools have been utilized for generation of mutations in Lb.[207] and finally by introducing a promoterless LacZ gene to the end of Tn917 for the identification of promoters. This approach was first applied in LAB using replicons from plasmids pBR322 or pE194 with a selectable marker. a high excision rate was obtained. either by not having functional replication (e. Russell and Copyright © 2004 by Marcel Dekker. Tn916 has also been reported not to be able to conjugate due to the limited excision and the lack of a host factor for conjugation. however. The broad host-range cloning vector pSA3. probably because of reported hot-spot integrations. pVE6007) (see above).Hoang-Dung TRAN and friends con. All Rights Reserved. including MG1363. allowing for rapid analysis of the chromosomal insertion site. This thermosensitive replicon was later developed into the so-called pGþ host vector series containing different selectable markers[222] and has been a useful tool for generation of gene-replacement mutations in a number of mesophilic and some thermophilic LAB..[209] for promoter screening in lactococci.[216] Gene disruption and gene displacement vectors dependent on homologous recombination between cloned fragments (possibly mutagenized) and the chromosome must have conditional replication functions in the strain that should be mutagenized. Such a vector could be propagated in strains that contain the repA gene in trans. coli.g.[219] By introducing pVE6007 again.[218] It was demonstrated that at the nonpermissive temperature chromosomal integration could be selected for. faecalis. only found limited use. coli replicon) or a defective or thermosensitive replicon. plantarum. However.[210. helveticus been found to be thermosensitive at 458C[224] and used to generate gene-replacement mutants in this host.[223] has in Lb.g. by introducing the library into a strain containing the plasmid pVE6007[218] coding for a thermosensitive RepA at its permissive temperature (308C).[214] In industrial LAB.[217] A drawback of these types of suicide vectors is that they require high transformation frequencies to obtain integration in sufficient numbers of sites for the purpose of randomized mutation.[221] Although suicide vectors have been used for random mutagenesis. Tn916 and related Tn918 and Tn919 have.[219] The original pORI vectors were later improved by introducing a lacZ. a sufficient number of transformed cells was obtained. containing the replication origins of the pIP501 and pBR322. Thermosensitive vectors have been derived from pWV01 by mutagenizing the plasmid and screening for thermosensitive plasmid derivatives (e. it has been used as a genetic tool to generate mutations in both gram-positive and gram-negative bacteria.[206] then introducing a pBR322 replication origin allowing for fast identification of flanking regions of the Tn917 insertion site.211] and recently for the identification of secretion signals in lactococci.[190. vectors with an E.. Inc.[220] A food-grade version using the sucrose gene from P. In many thermophilic LAB the pGþ host vectors and the pINT/pORI two-plasmid system cannot be used because the permissive temperature is too low for sufficient growth of the transformants.[142] and it has been shown that cloned fragments containing integrated Tn916 can be precisely excised in E. which allows the detection of excision in the absence of an easy screening phenotype.[213] Due to its conjugative nature.[212] Clewell and coworkers also identified Tn916 from E. pentosaceus as a screening marker has been developed.[215] In certain Lactococcus strains. . their main use has been to generate specific gene disruptions or gene-replacement mutations.191] Later the lactococcal broad host-range plasmid pWV01 was developed to pINT/ pORI vector series by eliminating the replication initiation gene repA but leaving the replication origin intact.

Site-specific integration vectors have also been developed based on the temperate phage integration systems from LAB.[136] This is probably due to the wide host range of the temperature-sensitive pWV01-derived replicon.[230] However. gasseri the IS1223 was rescued on pSA3.g. This technology can therefore generate (random) food-grade mutants in a two-step process. . a new thermosensitive integration vector for Lb.[219] to Lb. which allowed for selection of integrant by chloramphenicol.[231] Lillehaug and Birkeland[232] described the site-specific integration in Lactococcus using fLC3 integrase and attP site. suicide thermosensitive vectors. Also. Their main use so far has been to study regulation of promoters using reporter genes. gasseri.219] However. these vectors should have restricted replication in the host.[235] Transcriptional fusion integration vectors derived from the TP901-1 integration system were developed with the gusA and lacLM as reporter genes. where their single copy mimics the chromosomal situation. Subsequently the gram-positive replication origin was deleted. coli and even to mammalian cells. A non-replicating vector containing an the ISS1 element containing an ery gene as selection marker was used to generate a physical and genetic markers in Lactococcus. based on a replicon from a cryptic Lb. delbrueckii phage mv4 may find special application in LAB because the mv4 site specifically integrates into the end of a tRNAser without Copyright © 2004 by Marcel Dekker. have been used for random mutagenesis.[234] It was later reported that the integration frequency of this resolvase-like integration system was unusually high. a pSA3 derivative without the gram-positive replication origin. dependent on homologous recombination between randomly cloned small chromosomal fragments and the chromosome. Recently. vectors used for random integration mutagenesis in industrial LAB have in most cases been based on the random integration of IS elements. curvatus plasmid pLC2. 438C) and adapted the pINT/pORI two-plasmid integration system described by Law et al. These vectors will integrate cloned genes in one copy on a specific location in the chromosome. allowing for integration into strains with low transformability.Hoang-Dung TRAN and friends Klaenhammer[225] observed that the pWV01 wild-type replicon is thermosensitive at elevated temperatures (e.[191. which can be applied in foods or in food models. personal communication). Stoll et al.[226] As mentioned above. which proved to be thermosensitive. Romero and Klaenhammer[227] constructed a composite transposon on a suicide vector based on two IS946 elements flanking the cat gene from pC194. and the derived vector pTRK327 was used for generating random mutations in Lb. This was later developed into an integration vector pINT2 generating stable one-copy integration. Varmanen.. gasseri has been developed. gasseri. The first of these integration vectors was based on the adh phage integrase gene and the corresponding attP site cloned onto pSA34. The frequently reported IS vectors used for random mutagenesis are derivatives of the pGþ host vectors carrying the ISS1 element. acidophilus and Lb.[237] showed that the TP901-1 integration system under the right conditions could be applied to E. One advantage of the last-mentioned system is that vector integrants can be excised.[236] Later a version with the pipI gene from Lb. leaving behind only an extra copy of the IS element. site-specific integration was demonstrated. Inc. Polzin and McKay[228] utilized a natural thermosensitive plasmid replicon from pSK11L from Lactococcus to develop an IS981-based integration system and a selectable erythromycin marker.[233] From the lactococcal phage TP901-1. The integrase system of the Lb. allowing for stabilization of heterologous or plasmid-encoded genes.[229] In Lb. neither of the above-mentioned vectors have gained wide use. As in the case of gene disruption and displacement vectors. All Rights Reserved. helveticus was obtained (P. indicating that the TP901-1 integrase system does not require any specific host factors except the attB site.

[247]) as a result of the high expression and accumulation of protein. One of the advantages is that the expression level of the PnisA promoter vectors can be fine-tuned by the amount of nisin added and the strain used. Both of these problems can be avoided by choosing vectors with natural promoters having different strengths.g. for nisin A production[259] has found widespread use.[248]). Refs.g.[260] This system. it is often necessary to add signal sequences that allow export of the expressed gene product.240 – 244]) or as a result of the analysis of specific genes (e. and a number of such vectors have been constructed with different promoters from different LAB. The PnisA promoter has been cloned on the wide host range replicon from pWV01 in different versions.246]). Although developed for a different purpose.[246] or NaCl-induced promoters. which allowed nisin-controlled expression in a number of gram-positive bacteria. The first expression vectors utilized strong constitutive promoters. plantarum. a series of synthetic constitutive promoters that allow the modulation of the expression over a 2000-fold range may be useful for fine-tuning of gene activity.. alternatively. lactis MG1363 in the pepN gene. All Rights Reserved. lactis and that this regulation is independent of nisRK. Several promoters have been suggested for this purpose. including the nisin immunity regions but with the structural nisin gene deleted..Hoang-Dung TRAN and friends disrupting the tRNA molecule. Ref.. In addition.[252] sugar-regulated promoters.[249] Another way to reduce problems with inclusion bodies or in cloning and expression of toxic gene products is to use a regulated promoter that allows for optimization of expression.g.[262. it has been reported that the PnisA promoter is also induced by galactose and lactose at least in Lc.[264] These were solved by integrating the nisRK on the chromosome using the broad-host-range integration vector pMEC1.[261] Initially the vectors were developed for Lactococcus. for vaccine production).[245..[238] and later into a number of LAB.[238] Broad host-range single-plasmid nisin-controlled vectors have recently been constructed to simplify the induction system.g. a derivative of Tn7562 containing the whole nisin operon.[266.267] Copyright © 2004 by Marcel Dekker. Many promoters have been identified from LAB either by direct screening (e.. a two-vector system was developed in which the nisRK genes are located on a separate compatible broad-host-range vector. a cell wall or membrane anchor may also be necessary.g. Expression Vectors Overexpression of homologous or heterologous genes can be achieved by a fusion of a strong or regulated promoter to the gene of interest.[250.[260] Later.[258] So far only the nisin-inducible promoter. cloning problems may occur if the gene product is toxic to the cell at the expressed concentration (e.[260] The nisRK regulator was integrated on the chromosome of Lc. including phagederived thermoinducible promoters based on genetic switches of temperate phages (e.251]). Ref. called NICE.[238] It was first shown to be able to integrate site-specifically into the corresponding tRNA in Lb.[211. was conjugated into MG1363.263] In some cases the two-vector system has been reported to cause problems.[265] Interestingly..[253 – 257] environmentally regulated promoters such as pH. Refs. PnisA.g. Common for them is that the expressed proteins are normally found in the cytoplasm as biologically inactive in inclusion bodies (e. was developed by the NIZO group in the mid-1990s and relies on the autoregulation of nisin production by nisin itself through the nisRK response regulator. . Refs. Also.[239] 3. Inc. In case the expressed protein is to be displayed on the surface of the cell (e. middle phage promoters that can be regulated by phage infection.

[273. amylovorus. particularly in Europe.g. thermophilus b-galactosidase in Lc.270 – 272] Others have been found from genes where the gene product is known to be excreted. bovis [275] and Lb. Refs. Food-grade markers can be divided into two groups: that is dominant selection and screening markers (group 1a and 1b) and complementation selection and screening (group 2a and 2b) markers. The cell-wall anchor of the S.. Similar cell-wall anchors from Staphylococcus aureus protein A[287] and lactococcal PrtP[281.[281 – 284]). while others have relied on a “clean homologous recombination” approach. it may be an additional advantage to be able to transfer the antigen to the surface of the cell. Although in most cases the antigen has been excreted to the medium. Refs.[296] The nisin and the lacticin 3147 immunity genes have been suggested as food-grade markers for vector constructions in Lactococcus. It was first found on plasmid pNP40 from Lc. lactis strain 10. the secretion signal sequences for prtP and usp45 from Lc. Food-Grade Cloning Vectors Although consumer attitudes towards genetic engineering. are skeptical. isolated from Lb.293] A cloning vector pVS40 was constructed from a related nsr gene encoded on plasmid pSF01 from Lc.088[294] and a Lactococcus plasmid origin of replication belonging to the theta group. for example. pyogenes M6 protein has been used by several groups for anchoring proteins to the surface of LAB (e. although Walker and Klaenhammer[268] described secretion of S.[279] while mutation of host genes like htrA influences the stability of the expressed and secreted proteins. lafI.298] Copyright © 2004 by Marcel Dekker. lactis by a controlled expression of a F31 holin and lysin cassette.g.[280] Within the last decade the use of LAB as live vaccines has attracted increasing interest (e. The nsr gene was identified by several groups and is unrelated to the nisin immunity gene.[212.274] the a-amylase genes from S.278] Addition of the nine-residue synthetic propeptide LEIDDTCDA after the signal sequence was shown to improve the secretion efficiency in Lactococcus significantly. lactis. a number of so-called foodgrade approaches to genetic engineering of LAB have been suggested. 4. .288] and AmcA[289] have also been used for targeting proteins on the surface.Hoang-Dung TRAN and friends Secretion signals are in most cases necessary if the expressed product has to be externalized for easier downstream processing. Among the first genes to be suggested as putative food-grade markers was a nisin-resistance gene.[297. a bacteriocin or a heavy metal ion and can therefore be directly selected for in sensitive strains. or other gene products easy to screen for.[276] and the S-layer protein gene from Lb. fermentum.[284 – 286]).[277. nisI. lactis DRC3.. All Rights Reserved. while markers in 1b and 2b must be screened.[291] and was subsequently cloned as pFM011 and sequenced. brevis.[269] A number of secretion signals have been found by screening of LAB for sequences that are able to export products of reporter genes such as a-amylase.g. b-lactamase. e. but increasingly also in the United States.[58] Both plasmids were shown to be useful for cloning in Lactococcus [57. Some of these are based on the construction of food-grade vectors using food-grade markers. Group 1a is based on genes that give resistance to. johnsonii VPI 11088 was shown to be expressed in a number of Lactobacillus species and could be selected for at least in Lb. nsr..295] The immunity gene of lactacin F.[292. Inc.[290] The 1a and 2a markers are directly selectable. peptidases were not externalized using this system. nuclease. However.

casei. lactis DN209 was complemented. lactis and Lb. the applicability in LAB in general may be questioned as two Alr activities has been observed in Lb. lacG from Lb.[305] However.[302] Data indicated that in addition to ada. One example of such a marker is the lacF gene. plantarum and Lc. The lacF marker has been used in the construction of a number of food-grade vectors using the regulated nisin promoter. acidilactici. Like the 2a markers.[309] Copyright © 2004 by Marcel Dekker.[304] as growth inhibition was seen in industrial strains when the subB suppressor was used.[306] Group 2b food-grade markers are based on complementation of some specific mutation in a carbohydrate fermentation operon. was also necessary. Inc. pentosus have beensuggested as food-grade markers[301] in a screening for transformants of Lactobacillus strains. All Rights Reserved. the D -xylose catabolism encoding genes of Lb.Hoang-Dung TRAN and friends Both these selection markers have been shown to function outside Lactococcus. Similarly. Group 2a is based on complementation of mutations in a chromosomal gene essential for growth under certain conditions. Another promising complementation system relies on the essential alanine racemase gene alr present in Lb. galA. reuteri. It is therefore only possible to conduct the complementation in strains where the corresponding mutation has previously been introduced. but screening using proper concentration of buffers. encoding enzyme III in the lactose PEP : PTS uptake system. Group 1b food-grade markers are based on the fermentation of rare or unusual carbohydrates. is that milk contains low amounts of purines and pyrimidines. The lacF gene was shown to complement a lacF deletion on a chromosomally located copy of the lactose operon in Lc. a derivative of MG5267. Recently it was shown that the nisRKFEG may also be used as a food-grade selectable marker. No direct selection for such markers is possible. Due to the variation in carbohydrate fermentation profiles. lactis was used to construct a food-grade vector pFG1 where a chromosomal auxothophic purine mutation of Lc. In the future we will undoubtedly see more food-grade markers based on bacteriocin resistance and immunity genes. plantarum heterologously complement chromosomal alr mutations in both species. Because the ability to ferment D -xylose is limited to a few species of LAB. lactis FI7794. Mutations in the alr gene make the strain auxotrophic for D -alanine. and sugar allows for isolation of transformants.[308] Also. a functional galactose permease gene. cadmium at the same time is a heavy metal. an a-galactosidase gene (ada) from Lc. . necessary for cell wall synthesis.[115. The ochre suppressor gene subB from Lc. Recently it was shown that alr genes from Lc. raffinolactis could be used as a screening marker for the utilization of melibiose in Lc.300] Although the selection for cadmium resistance is promising. indicators.[307] The advantage of this marker is its small size. Only two examples will be mentioned here. besides its small size (about 350 bp). lactis and P. although its size limits its applicability. The advantage of the suppressor system as a food-grade marker. they only function in strains where the corresponding mutation has been introduced.[299] Cadmium resistance genes encoded by natural plasmids from lactococci have also been used as a food-grade selectable markers in conjugal transfer and in vector construction. causing the vector to be stably maintained in milk fermentations. and therefore this resistance may not be considered ethical from an environmental point of view and therefore not “food-grade. lactis.[303] Later this system was improved in pFG200 using the amber suppressor gene subD in combination with amber mutation in pyrF. encoding phospho-b-galactosidase in the lactose operon. has been used in a similar approach. a number of carbohydrate catabolism genes may be utilized as screening markers in food-grade vectors.” even though the genes derive from organisms having a long history for being safe.

is the most studied antigen.[321] but inconsistent results were obtained.[318] Lactobacilli have also been used to act as vaccine delivery vectors. One of the main reasons for this is the complicated nature of food fermentations. technological advantages. and special expression and secretion vectors have been constructed.[313] The manipulated clone performed a mixed acid fermentation under anaerobic conditions. the gene for a-acetolactate decarboxylase. lactis by genetic disruption of the relevant gene.[316. etc. completely novel applications of LAB may be accomplished in the near future. Lactococci were the first LAB in which metabolic engineering studies were developed due to the large body of available data on their genomic structure and relatively well-known physiology (reviewed in Refs.[314] B. All Rights Reserved. LAB as Immunotherapeutic Agents: Genetic Aspects The possibility of introducing heterologous antigens into LAB.[319] and TTFC. Examples of Metabolic Engineering To reconstruct metabolic pathways from genome data is a formidable task made possible by the recent advances in molecular genetics. In addition to the improvement of the present starter strains and products based on them. Inc. again. in the human gut.) An especially attractive feature is the ability of some LAB to adhere to the intestinal epithelium and to persist.VII. a number of lactobacilli have been tested for antigen delivery. in which the studies conducted so far have used only the strain MG1363 or its derivatives.[315] LAB possess a number of properties that make them attractive candidates for oral vaccination (general safety. . and even reproduce themselves. The use of a surface display system based on the PrtP sequence was first used to express tetanus toxin fragment C (TTFC) in cells of Lc. Organisms to be used as oral vaccines are designed for expressing foreign epitopes on their outer surfaces. lactis.[310 – 312]). Subsequently both successful intracellular and the cell surface expression of TTFC has been achieved by means Copyright © 2004 by Marcel Dekker. no consistent picture of its final applicability has emerged. Hoang-Dung TRAN and friends APPLICATIONS OF GENETICALLY MODIFIED LAB While recombinant DNA techniques and the general developments in molecular biology have substantially increased our knowledge of the fundamental genetic aspects of LAB. Diacetyl production of up to 50% of available sugar was realized by overexpressing S. In the following paragraphs we discuss the most recently obtained results in this field. The first example was a nearly complete redirection of the metabolic flux by the inactivation of the enzyme lactate dehydrogenase in Lc. lactis (leading to less lactate and enhanced acetoin production) in combination with a genetic disruption of aldB. Genes under control of xylR or cbh promoter of Lactobacillus were initially used.[281] This organism has been widely experimentally exploited for vaccination purposes. traditions of food use.317] However. where the roles of single genes and their relative activities in the desired properties of the end products are still poorly known. A. while in the presence of oxygen it produced almost entirely acetoin. concentrating on the genetic aspects (rather than the immunological details) of this potential application. Metabolic engineering and the use of LAB as vaccines or immunotherapeutic agents are examples of new applications attracting much attention. the practical applications have been somewhat slow to materialize. lactis. mutans NADH oxidase in Lc. to be used as delivery vehicles of foreign epitopes is an exciting and rapidly evolving application of genetic engineering.[320] In contrast to Lc.

B. secreted.Hoang-Dung TRAN and friends of specially developed expression vectors. Nevertheless. and even difficult species are becoming amenable to genetic modification. P. plantarum NCIMB 8826 based on ldh constitutive promoter and transcriptional termination signals. Kordias.. Copyright © 2004 by Marcel Dekker. J. 161 – 183. N. SUMMARY Genomic sequences of the most important LAB species are increasingly becoming available. 174. Inc. Physical and genetic map of the chromosome of Lactococcus lactis subsp. M. Immunological results obtained using animal models have shown the relevance of the genetic construction to obtain good immune response in the host. Davidson.J. johnsonii to act as vaccine-delivery vehicle. REFERENCES 1. bulgaricus. the signal molecules of the immune system have a decisive role in immunological and inflammatory reactions. or surface-exposed) were obtained. is a challenging prospect.. Genomic organization of lactic acid bacteria. based on secretion signals of amylase or peptidase enzymes. 6752– 6762. the cell wall-anchored proteinase PrtB of Lb. has also been used to evaluate the possibility of using a strain of Lb. Ritzenthaler. Different localizations of the antigen (intracellular. The prospect of engineering the metabolic pathways is also becoming more real. This information is essential if the traditional uses of LAB are to be optimized using genetic techniques. M. The development of genetically modified LAB for completely new applications. 1996. M. mutans counts and caries development. A model antigen. A. zeae of an antibody fragment recognizing a streptococcal antigen typical to cariogenic S. It is to be hoped that the rapidly accumulating genetic information will enhance our understanding both of the basic physiology of LAB and especially of their role in food fermentations at the metabolic level. Dobos.[325] This finding could open new possibilities for the development of nextgeneration probiotic strains designed to specifically treat certain diseases or disorders..[322] leading to different immunological responses.. Administration of these bacteria to a rat model of dental caries resulted in the depression of both S. At the same time recombinant DNA techniques have become even more sophisticated. Antonie Van Leeuwenhoek. Results showed that this recombinant Lactobacillus can induce both systemic and local mucosal immune responses.[324] In addition to antigens and antibodies. 70. Mata. the unanswered question of public acceptance of these types of products (as well as the position of regulatory bodies) has to be taken into account. lactis designed to express IL-10 on its surface has been successfully used to treat experimentally induced murine colitis. All Rights Reserved. such as vaccines or designed probiotics. Hillier. Lautier. Le Bourgeois. . P.[323] Another approach for providing disease protection by recombinant lactobacilli has been the cloning in Lb. A number of plasmid expression vectors have been designed for Lb. 1992. mutans. the future of genetic modification of LAB may well be in their health-related uses. while the research on proteomics is starting to take advantage of the new possibilities of the accumulating sequence data. In addition to technical and scientific problems.. VIII. delbrueckii subsp. 2.E. Bacteriol.. lactis IL1403. Lc.

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[1] Their submicroscopic sperm-like morphology remained undetected until the first electron microscopes became available during the 1940s.Hoang-Dung TRAN and friends 8 Bacteriophage and Antiphage Mechanisms of Lactic Acid Bacteria JYTTE JOSEPHSEN The Royal Veterinary and Agricultural University. when Whitehead and Cox in New Zealand observed that phages were responsible for the failure in the acid-producing activity of a cheese starter culture. INTRODUCTION Food fermentations rely on actively growing lactic acid bacteria (LAB) which either are added as starter cultures or grow spontaneously in the food matrix. Germany I. Thus. phage attacks on lactic acid bacteria during the fermentation process result in an unacceptably low production of lactic acid and flavor compounds along with reduced proteolysis.[2] In the area of food fermentation. a complete failure of starter growth may occur (“dead vats”). the permanent threat of phage infection is particularly manifested in the dairy field. All Rights Reserved. Kiel. in extreme cases. residues from antibiotic or disinfectant treatments. . Frederiksberg. These bacterial viruses ´ were identified as filter-transmissible agents by the work of Twort in 1915 and d’Herelle in 1917. Phages have also 295 Copyright © 2004 by Marcel Dekker. the major commercial problem results from bacteriophage infections. Phages attacking Lactococcus lactis have been known since the 1930s. Here. starter activity is either severely affected (“slow vats”) or. such as bacteriocins. phage control is a major area of concern in handling lactic acid bacteria as starter cultures. Denmark HORST NEVE Federal Research Centre for Nutrition and Food. The fermentation capabilities of lactic acid bacteria can be severely inhibited by a panoply of nonvir