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K11662
Microbiolog y
“ … recommended … extremely useful … a welcome addition to many libraries.”
—International Dairy Journal
“ … an invaluable reference text … written in an easy-to-read style, making it eminently
suitable for both postgraduate students and general food scientists.”
—Food Australia
While lactic acid–producing fermentation has long been used to improve
the storability, palatability, and nutritive value of perishable foods, only recently
have we begun to understand just why it works. Since the publication of the
third edition of Lactic Acid Bacteria: Microbiological and Functional
Aspects, substantial progress has been made in a number of areas of
research. Completely updated, the Fourth Edition covers all the basic and
applied aspects of lactic acid bacteria and bifidobacteria, from the gastrointesti-
nal tract to the supermarket shelf.
Topics discussed in the new edition include:
• Revised taxonomy due to improved insights in genetics and new
molecular biological techniques
• New discoveries related to the mechanisms of lactic acid bacterial
metabolism and function
• An improved mechanistic understanding of probiotic functioning
• Applications in food and feed preparation
• Health properties of lactic acid bacteria
• The regulatory framework related to safety and efficacy
Maintaining the accessible style that made previous editions so popular, this
book is ideal as an introduction to the field and as a handbook for microbiolo-
gists, food scientists, nutritionists, clinicians, and regulatory experts.
LACTIC ACID BACTERIA

MICROBIOLOGICAL AND FUNCTIONAL ASPECTS
LACTIC ACID BACTERIA

MICROBIOLOGICAL AND FUNCTIONAL ASPECTS
ISBN: 978-1-4398-3677-4
9 781439 836774
9 00 0 0
FOURTH EDITION
L. acidophilus
ATCC 700396

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S A M P O L A H T I N E N • ART H U R C . O U W E H A N D
S E P P O S A L M I N E N • AT T E V O N W R I G H T
MICROBIOLOGICAL AND FUNCTIONAL ASPECTS
F o u r t h E d i t i o n
LACTIC ACID BACTERIA

L A H T I N E N
O U W E H A N D
S A L M I N E N
V O N W R I G H T
MICROBIOLOGICAL AND FUNCTIONAL ASPECTS
F ou rt h
E di ti on
MICROBIOLOGICAL AND FUNCTIONAL ASPECTS
F o u r t h E d i t i o n
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CRC Press is an imprint of the
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S E P P O S A L M I N E N
•
AT T E V O N W R I G H T
MICROBIOLOGICAL AND FUNCTIONAL ASPECTS
F o u r t h E d i t i o n
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v
Contents
Preface ix
Editors xi
Contributors xiii
1 Lactic Acid Bacteria: An Introduction 1
ATTE VON WRIGHT AND LARS AXELSSON
2 Genetics of Lactic Acid Bacteria 17
LORENZO MORELLI, MARIA LUISA CALLEAGRI, FINN KVIST VOGENSEN,
AND ATTE VON WRIGHT
3 Potential Applications of Probiotic, Bacteriocin-Producing Enterococci and
eir Bacteriocins 39
ANDREA LAUKOVÁ
4 Genus Lactococcus 63
ATTE VON WRIGHT
5 Genus Lactobacillus 77
RODOLPHE BARRANGOU, SAMPO J. LAHTINEN, FANDI IBRAHIM, AND
ARTHUR C. OUWEHAND
6 e Lesser LAB Gods: Pediococcus, Leuconostoc, Weissella, Carnobacterium,
and Affiliated Genera 93
GEERT HUYS, JØRGEN LEISNER, AND JOHANNA BJÖRKROTH
7 Streptococcus: A Brief Update on the Current Taxonomic Status of the Genus 123
JOHN R. TAGG, PHILIP A. WESCOMBE, AND JEREMY P. BURTON
8 Bifidobacteria: General Overview on Ecology, Taxonomy, and Genomics 147

MARCO VENTURA, FRANCESCA TURRONI, AND DOUWE VAN SINDEREN
9 Bacteriophage and Anti- Phage Mechanisms in Lactic Acid Bacteria 165
SUSAN MILLS, R. PAUL ROSS, HORST NEVE, AND AIDAN COFFEY
10 Lactic Acid Bacteria in Vegetable Fermentations 187
KUNYOUNG PARK AND BOH KYUNG KIM
vi ◾ Contents
11 Current Challenges for Probiotics in Food 213
JEANMICHEL ANTOINE
12 Lactic Acid Bacteria in Cereal-Based Products 227
HANNU SALOVAARA AND MICHAEL GÄNZLE
13 Lactic Acid Bacteria in Meat Fermentations 247
CECILIA FONTANA, SILVINA FADDA, PIER SANDRO COCCONCELLI, AND
GRACIELA VIGNOLO
14 Examples of Lactic- Fermented Foods of the African Continent 265
CHARLES M.A.P. FRANZ AND WILHELM H. HOLZAPFEL
15 Antimicrobial Components of Lactic Acid Bacteria 285
INGOLF F. NES, MORTEN KJOS, AND DZUNG BAO DIEP
16 Atherosclerosis and Gut Microbiota: A Potential Target for Probiotics 331
CHAN YEE KWAN, PIRKKA KIRJAVAINEN, CHEN YAN, AND HANI ELNEZAMI
17 Lactic Acid Bacteria (LAB) in Grape Fermentations—An Example of LAB as
Contaminants in Food Processing 343
EVELINE BARTOWSKY
18 Stability of Lactic Acid Bacteria in Foods and Supplements 361
MIGUEL GUEIMONDE, CLARA G. DE LOS REYES GAVILÁN, AND BORJA SÁNCHEZ
19 Lactic Acid Bacteria in the Gut 385
MARIA STOLAKI, WILLEM M. DE VOS, MICHIEL KLEEREBEZEM, AND
ERWIN G. ZOETENDAL
20 Lactic Acid Bacteria in Oral Health 403
JUKKA H. MEURMAN AND IVA STAMATOVA
21 Some Considerations for the Safety of Novel Probiotic Bacteria 423

DIANA C. DONOHUE AND MIGUEL GUEIMONDE
22 Probiotics and Human Immune Function 439
HARSHARNJIT S. GILL, JAYA PRASAD, AND OSAANA DONKOR
23 Gastrointestinal Benefits of Probiotics—Clinical Evidence 509
ANNA LYRA, SAMPO LAHTINEN, AND ARTHUR C. OUWEHAND
24 Human Studies on Probiotics: Infants and Children 525
HANIA SZAJEWSKA
25 Human Studies on Probiotics and Endogenous Lactic Acid Bacteria in the
Urogenital Tract 543
WAYNE L. MILLER AND GREGOR REID
26 Lactic Acid Bacteria and Blood Pressure 561
PAULIINA EHLERS AND RIITTA KORPELA
Contents ◾ vii
27 Probiotics for Companion Animals 579
MINNA RINKINEN
28 Prevalence and Application of Lactic Acid Bacteria in Aquatic Environments 593
HÉLÈNE L. LAUZON AND EINAR RINGØ
29 Probiotics for Farm Animals 633
ALOJZ BOMBA, RADOMÍRA NEMCOVÁ, LADISLAV STROJNÝ, AND
DAGMAR MUDROŇOVÁ
30 Health Effects of Nonviable Probiotics 671
SAMPO J. LAHTINEN AND AKIHITO ENDO
31 Probiotics: Safety and Efficacy 689
SEPPO SALMINEN AND ATTE VON WRIGHT
32 Probiotics Regulation in Asian Countries 705
YUAN KUN LEE, WEI SHAO, SU JIN, YAN WEN, BARNA GANGULY,
ENDANG S. RAHAYU, OSAMU CHONAN, KOICHI WATANABE, GEUN EOG JI,
MYEONG SOO PARK, RAHA ABD RAHIM, HOOI LING FOO, JULIE D. TAN,
MINGJU CHEN, AND SUNEE NITISINPRASERT
33 Regulation of Probiotic and Probiotic Health Claims in South America 749

CÉLIA LUCIA DE LUCES FORTES FERREIRA AND MARCELO BONNET
Index 761
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ix
Preface
e previous editions of this book have never looked very much like their predecessors, and this
fourth edition is no exception. Due to the accumulation of new findings during the seven years
that has passed since the previous update, practically all chapters are either completely rewritten
or are totally new. We (the editors) and the contributors have strived to reach a proper balance
between the well-established “eternal truths” and the novel and even controversial findings. While
keeping the format of individual chapters as reviews, a certain compromise between comprehen-
siveness and readability has been aimed at in order to avoid an excessive length and too massive a
size of the volume. In addition to purely scientific aspects related to lactic acid bacteria and their
applications, the regulatory framework related to their safety and efficacy, particularly in probiotic
use, has also been reviewed. We hope that the book will find its audience both as an introduction
to the field for an advanced student and as a handbook for microbiologists, food scientists, nutri-
tionists, clinicians, and regulatory experts. e editors are indebted to Dr. Anna Lyra for skillful
and tireless help with the editorial process.
Sampo Lahtinen
Seppo Salminen
Arthur Ouwehand
Atte von Wright
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xi
Editors
Seppo Salminen is a Professor and the Director of Functional Foods Forum at the University
of Turku, Finland, and a visiting professor at the RMIT University, Melbourne, Australia, and
Universität für Bodenkultur, Vienna, Austria. He is the author of numerous journal articles and
book chapters, and the editor or a coeditor of several books. He has served in several scientific
expert committees and working groups of the European Food Safety Authority and other interna-

tional committees including ILSI Europe and International Dairy Federation. Professor Salminen
received his M.S. degree (1978) in food science from Washington State University, Pullman, the
M.Sc. degree (1979) in food chemistry and technology from the University of Helsinki, Finland,
and the Ph.D. degree (1982) in biochemistry and toxicology from the University of Surrey, UK.
Professor Atte von Wright graduated from the University of Helsinki (Helsinki, Finland) in
1975 and obtained his Ph.D. in microbiology in 1981 at the University of Sussex, UK. He has
a professional background both in industry and academia with research interests spanning from
food toxicology to molecular biology and safety aspects of lactic acid bacteria. Since 1998, Atte
von Wright has been a Professor of Nutritional and Food Biotechnology at the University of
Kuopio (since 2010, the University of Eastern Finland), Kuopio, Finland. He has also served in
many expert functions of the EU (a member of the Scientific Committee of Animal Nutrition,
1997–2003; a member of the EFSA Scientific Panel on additives and products or substances used
in animal feed, 1996–2009; and a member of the EFSA Scientific Panel on Genetically Modified
Organisms from 2009 onward). He has more than 120 original scientific publications and reviews
in international refereed journals.
Dr. Sampo Lahtinen is a Health & Nutrition Group Manager at Danisco Health & Nutrition,
Kantvik, Finland. He has a professional background both from industry and academia with a
focus on probiotic and intestinal bacteria, prebiotics, and functional foods, in general. He received
his Ph.D. degree (2007) in Food Chemistry from the University of Turku, Finland, and was
nominated in 2009 as an Adjunct Professor of Applied Microbiology of the University of Turku.
He is the author of more than 50 journal articles and book chapters on probiotics and prebiotics.
Dr. Arthur Ouwehand is an R&D group manager at Danisco Health & Nutrition in Kantvik,
Finland. He has a research background in both academia and industry. His main interest is on
functional foods, in particular, probiotics and prebiotics and their influence on the intestinal
microbiota. He is active in the International Life Sciences Institute Europe, the International
xii ◾ Editors
Dairy Federation, and the International Scientific Association for Probiotics and Prebiotics. Dr.
Ouwehand received his M.S. degree (1992) in cell biology from Wageningen University (the
Netherlands) and his Ph.D. degree (1996) in microbiology from Göteborg University (Sweden).
In 1999, he was appointed as an Adjunct Professor in Applied Microbiology at the University of

Turku (Finland), and he is the author of more than 150 journal articles and book chapters.
xiii
Contributors
Jean-Michel Antoine
Danone Research
Palaiseau, France
Lars Axelsson
Nofima, e Norwegian Institute of Food,
Fisheries and Aquaculture Research
Ås, Norway
Rodolphe Barrangou
Danisco USA Inc.
Madison, Wisconsin
Eveline Bartowsky
e Australian Wine Research Institute
Adelaide, Australia
Johanna Björkroth
Department of Food Hygiene and
Environmental Health, Faculty of
Veterinary Medicine
University of Helsinki
Helsinki, Finland
Alojz Bomba
Department of Experimental Medicine,
Faculty of Medicine
Pavol Jozef Šafárik University in Košice
Košice, Slovakia
Marcelo Bonnet
Dairy Cattle Research Unit
Brazilian Agricultural Research

Corporation-EMBRAPA
Juiz de Fora, Brazil
Jeremy P. Burton
BLIS Technologies Ltd, Centre for Innovation
University of Otago
Dunedin, New Zealand
Maria Luisa Calleagri
Facoltà di Agraria
Universita Cattolica di Sacro Cuore
Piacenza, Italy
Ming-Ju Chen
Department of Animal Science and
Technology
National Taiwan University
Taipei, Taiwan
Osamu Chonan
Yakult Central Institute for Microbiological
Research
Tokyo, Japan
Pier Sandro Cocconcelli
Istituto di Microbiologia, Centro Ricerche
Biotechnologiche
Universitá Cattolica del Sacro Cuore
Piacenza-Cremona, Italy
Aidan Coffey
Department of Biological Sciences
Cork Institute of Technology
Bishopstown, Ireland
xiv ◾ Contributors
Clara G. De Los Reyes-Gavilán

Department of Microbiology and
Biochemistry of Dairy Products, Instituto
de Productos Lácteos de Asturias
Consejo Superior de Investigaciones
Científicas
Villaviciosa, Spain
Célia Lucia De Luces Fortes Ferreira
Universidade Federal de Viçosa
Viçosa, Brazil
Willem M. De Vos
Top Institute Food and Nutrition, Laboratory
of Microbiology
Wageningen University
Wageningen, the Netherlands
Dzung Bao Diep
Laboratory of Microbial Gene Technology
Department of Chemistry, Biotechnology
and Food Science
Norwegian University of Life Sciences
Ås, Norway
Osaana Donkor
Victoria University
Melbourne, Australia
Diana C. Donohue
School of Medical Sciences
RMIT University
Melbourne, Australia
Pauliina Ehlers
Institute of Biomedicine, Pharmacology
University of Helsinki

Helsinki, Finland
Hani El-Nezami
School of Biological Sciences
University of Hong Kong
Hong Kong Special Administrative Region,
China
Akihito Endo
Functional Foods Forum
University of Turku
Turku, Finland
Geun Eog Ji
Department of Food and Nutrition, Research
Institute of Human Ecology
Seoul National University
Seoul, Korea
Silvina Fadda
Centro de Referencia para Lactobacilos
Consejo Nacional de Investigaciones
Científicas y Técnicas
Tucumán, Argentina
Cecilia Fontana
Istituto di Microbiologia, Centro Ricerche
Biotechnologiche
Universitá Cattolica del Sacro Cuore
Piacenza-Cremona, Italy
Hooi Ling Foo
Faculty of Biotechnology and Biomolecular
Sciences, Institute of Bioscience
Universiti Putra Malaysia
Kuala Lumpur, Malaysia

Charles M.A.P. Franz
Department of Safety and Quality of Fruit
and Vegetables
Max Rubner Institute, Federal Research
Institute for Nutrition and Food
Karlsruhe, Germany
Barna Ganguly
Department of Pharmacology
P.S. Medical College
Gujarat, India
Harsharnjit S. Gill
Lactia Pty Ltd
Melbourne, Australia
Contributors ◾ xv
Miguel Gueimonde
Department of Microbiology and
Biochemistry of Dairy Products, Instituto
de Productos Lácteos de Asturias
Consejo Superior de Investigaciones
Científicas
Villaviciosa, Spain
Wilhelm H. Holzapfel
School of Life Sciences
Handong Global University
Pohang, South Korea
Geert Huys
Laboratory of Microbiology and BCCM/
LMG Bacteria Collection, Department of
Biochemistry and Microbiology, Faculty of
Sciences

Ghent University
Ghent, Belgium
Fandi Ibrahim
Health and Nutrition
Danisco
Kantvik, Finland
and
Functional Foods Forum
University of Turku
Turku, Finland
Su Jin
Danisco China Ltd.
Shanghai and Beijing, People’s Republic of
China
Boh Kyung Kim
Department of Food Science and Nutrition
Pusan National University
Busan, Korea
Pirkka Kirjavainen
Institute of Public Health and Clinical
Nutrition
University of Eastern Finland
Kuopio, Finland
Morten Kjos
Laboratory of Microbial Gene Technology
Department of Chemistry, Biotechnology
and Food Science
Norwegian University of Life Sciences
Ås, Norway
Michiel Kleerebezem

Top Institute Food and Nutrition
Wageningen, the Netherlands
and
Laboratory of Microbiology
Wageningen University
Wageningen, the Netherlands
and
NIZO Food Research
Ede, the Netherlands
Riitta Korpela
Institute of Biomedicine, Pharmacology
University of Helsinki
Helsinki, Finland
Yuan Kun Lee
Department of Microbiology, Yong Loo Lin
School of Medicine
National University of Singapore
Singapore, Singapore
Finn Kvist Vogensen
Department of Food Science, Faculty of Life
Sciences
University of Copenhagen
Frederiksberg C., Denmark
Chan Yee Kwan
School of Biological Sciences
University of Hong Kong
Hong Kong Special Administrative Region,
China
Sampo J. Lahtinen
Health and Nutrition

Danisco
Kantvik, Finland
xvi ◾ Contributors
Andrea Lauková
Institute of Animal Physiology
Slovak Academy of Sciences
Košice, Slovakia
Hélène L. Lauzon
Matís ohf
Icelandic Food and Biotech R&D
Reykjavík, Iceland
Jørgen Leisner
Department of Veterinary Pathobiology
Faculty of Life Sciences
University of Copenhagen
Frederiksberg C., Denmark
Anna Lyra
Health and Nutrition
Danisco
Kantvik, Finland
Jukka H. Meurman
Institute of Dentistry, University of Helsinki
Department of Oral and Maxillofacial
Diseases Helsinki, University Central
Hospital
Helsinki, Finland
Wayne L. Miller
Canadian Research and Development Centre
for Probiotics
Lawson Health Research Institute

London, Canada
and
Department of Microbiology and
Immunology
e University of Western Ontario
London, Canada
Susan Mills
Teagasc Food Research Center
Moorepark, Fermoy, Co.
Cork, Ireland
and
CSK Food Enrichment
Ede, the Netherlands
Lorenzo Morelli
Facoltà di Agraria
Universita Cattolica di Sacro Cuore
Piacenza, Italy
Dagmar Mudroňová
University of Veterinary Medicine and
Pharmacy in Košice
Košice, Slovakia
Radomíra Nemcová
University of Veterinary Medicine and
Pharmacy in Košice
Košice, Slovakia
Ingolf F. Nes
Laboratory of Microbial Gene Technology
Department of Chemistry, Biotechnology
and Food Science
Norwegian University of Life Sciences

Ås, Norway
Horst Neve
Department of Microbiology and
Biotechnology
Max Rubner Institute, Federal Research
Institute for Nutrition and Food
Kiel, Germany
Sunee Nitisinprasert
Department of Biotechnology, Faculty of
Agro-Industry
Kasetsart University
Bangkok, ailand
Arthur C. Ouwehand
Health and Nutrition
Danisco
Kantvik, Finland
Kun-Young Park
Department of Food Science and Nutrition
Pusan National University
Busan, Korea
Contributors ◾ xvii
Myeong Soo Park
Department of Hotel Culinary Arts
Anyang Technical College
Anyang, Korea
Jaya Prasad
Dairy Innovation Australia Limited
Werribee, Australia
Endang S. Rahayu
Faculty of Agricultural Technology

Gadjah Mada University
Yogyakarta, Indonesia
Raha Abd Rahim
Faculty of Biotechnology and Biomolecular
Sciences, Institute of Bioscience
Universiti Putra Malaysia
Kuala Lumpur, Malaysia
Gregor Reid
Canadian Research and Development Centre
for Probiotics
Lawson Health Research Institute
London, Canada
and
Departments of Microbiology and
Immunology, and Surgery
e University of Western Ontario
London, Canada
Einar Ringø
Norwegian College of Fishery Science, Faculty
of Biosciences, Fisheries and Economics
University of Tromsø
Tromsø, Norway
Minna Rinkinen
Medical Affairs, Business Development and
Support
Orion Corporation Orion Pharma
Espoo, Finland
R. Paul Ross
Teagasc Food Research Center
Moorepark, Fermoy, Co.

Cork, Ireland
Seppo Salminen
Functional Foods Forum
University of Turku
Turku, Finland
Hannu Salovaara
Department of Food and Environmental
Sciences
University of Helsinki
Helsinki, Finland
Borja Sánchez
Department of Microbiology and
Biochemistry of Dairy Products, Instituto
de Productos Lácteos de Asturias
Consejo Superior de Investigaciones
Científicas
Villaviciosa, Asturias, Spain
Wei Shao
Chinese Institute of Food Science and
Technology
Beijing, China
Iva Stamatova
Faculty of Dental Medicine
Medical University of Plovdiv
Plovdiv, Bulgaria
Maria Stolaki
Top Institute Food and Nutrition
Wageningen, the Netherlands
and
Laboratory of Microbiology

Wageningen University
Wageningen, the Netherlands
and
TNO Quality of Life
Zeist, the Netherlands
Ladislav Strojný
Department of Experimental Medicine
Faculty of Medicine
Pavol Jozef Šafárik University in Košice
Košice, Slovakia
xviii ◾ Contributors
Hania Szajewska
Department of Paediatrics
e Medical University of Warsaw
Warsaw, Poland
John R. Tagg
Department of Microbiology and
Immunology
and
BLIS Technologies Ltd, Centre for Innovation
University of Otago
Dunedin, New Zealand
Julie D. Tan
Philippine Root Crop Research and Training
Center
Visayas State University
Baybay City, Philippines
Francesca Turroni
Laboratory of Probiogenomics, Department
of Genetics, Biology of Microorganisms,

Anthropology and Evolution
University of Parma
Parma, Italy
Douwe van Sinderen
Alimentary Pharmabiotic Centre and
Department of Microbiology, Bioscience
Institute
National University of Ireland
Cork, Ireland
Marco Ventura
Laboratory of Probiogenomics, Department
of Genetics, Biology of Microorganisms,
Anthropology and Evolution
University of Parma
Parma, Italy
Graciela Vignolo
Centro de Referencia para Lactobacilos
Consejo Nacional de Investigaciones
Científicas y Técnicas
Tucumán, Argentina
Atte Von Wright
Department of Biosciences
University of Eastern Finland
Kuopio, Finland
Koichi Watanabe
Yakult Central Institute for Microbiological
Research
Tokyo, Japan
Yan Wen
Danisco China Ltd.

Shanghai and Beijing, People’s Republic of
China
Philip A. Wescombe
BLIS Technologies Ltd, Centre for Innovation
University of Otago
Dunedin, New Zealand
Chen Yan
Department of Surgery, LKS Faculty of
Medicine
University of Hong Kong
Hong Kong Special Administrative Region,
China
Erwin G. Zoetendal
Top Institute Food and Nutrition
Wageningen, the Netherlands
and
Laboratory of Microbiology
Wageningen University
Wageningen, the Netherlands
1
Chapter 1
Lactic Acid Bacteria:
An Introduction
Atte Von Wright and Lars Axelsson
Contents
1.1 Background 2
1.2 Current Taxonomic Position of LAB 2
1.3 Carbohydrate Fermentation Patterns 2
1.3.1 Homo- and Heterolactic Fermentation 2
1.3.2 Fermentation of Disaccharides 6

1.3.3 Alternative Fates of Pyruvate 7
1.3.3.1 Diacetyl/Acetoin Pathway 7
1.3.3.2 Pyruvate–Formate Lyase System 7
1.3.3.3 Pyruvate Oxidase Pathway 8
1.3.3.4 Pyruvate Dehydrogenase Pathway 9
1.3.4 Alternative Electron Acceptors 9
1.3.4.1 Oxygen as an Electron Acceptor 9
1.3.4.2 Organic Compounds as Electron Acceptors 9
1.4 Bioenergetics, Solute Transport, and Related Phenomena 10
1.4.1 Energy Recycling and PMF 10
1.4.2 Solute Transport 11
1.4.2.1 PMF-Driven Symport of Solutes 11
1.4.2.2 Primary Transport 11
1.4.2.3 Precursor–Product Antiport 11
1.4.2.4 Group Translocation: Phosphoenolpyruvate: Sugar Phosphotransferase
System 11
1.5 Nitrogen Metabolism: Proteolytic System 13
1.6 Concluding Remarks 14
References 14
2 ◾ Lactic Acid Bacteria: Microbiological and Functional Aspects
1.1 Background
At the turn of the 20th century the term “lactic acid bacteria” (LAB) was used to refer to “milk-sour-
ing organisms.” While similarities between milk-souring organisms and other bacteria producing
lactic acid were soon observed, the monograph by Orla-Jensen (1919) formed the basis of the present
classification of LAB. e criteria used by Orla-Jensen (cellular morphology, mode of glucose fer-
mentation, temperature ranges of growth, and sugar utilization patterns) are still very important for
the classification of LAB, although the advent of more modern taxonomic tools, especially molecular
biological methods, have considerably increased the number of LAB genera from the four originally
recognized by Orla-Jensen (Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus).
LAB have traditionally been associated with food and feed fermentations, and are generally

considered beneficial microorganisms, some strains even as health-promoting (probiotic) bacte-
ria. However, some genera (Streptococcus, Lactococcus, Enterococcus, Carnobacterium) also contain
species or strains that are recognized human or animal pathogens. A thorough understanding of
taxonomy, metabolism, and molecular biology of LAB is thus necessary to fully utilize the tech-
nological, nutritional, and health-promoting aspects of LAB while avoiding the potential risks.
In the following sections a brief and concise overview of the present understanding of the
taxonomy and physiological and metabolic characteristics of LAB are presented. e important
genera and species are specifically dealt with in the other chapters of this book, and some informa-
tion will, inevitably, be redundant. However, this general introduction hopefully helps the reader
to familiarize with the subject and makes the digestion of the more specific aspects easier.
1.2 Current Taxonomic Position of LAB
LAB constitutes a group of gram-positive bacteria united by certain morphological, metabolic,
and physiological characteristics. ey are nonsporulating, nonrespiring but aerotolerant cocci or
rods, which produce lactic acid as one of the main fermentation products of carbohydrates. ey
lack genuine catalase and are devoid of cytochromes. According to the current taxonomic classifi-
cation, they belong to the phylum Firmicutes, class Bacilli, and order Lactobacillales. e different
families include Aerococcaceae, Carnobacteriacea, Enterococcaceae, Lactobacillaceae, Leuconostocaceae,
and Streptococcaceae ( e common genera and their
main characteristics are listed in Table 1.1, and more specific taxonomic information is provided in
the specific chapters devoted to these LAB groups in the subsequent sections of this book.
Phylogenetically, LAB can be clustered on the basis of molecular biological criteria, such as
rRNA sequencing, and an example of a phylogenetic tree differentiating LAB from the other
bacterial groups in the phylum Firmicutes is shown in Figure 1.1 as indicated in Chapter 2 (e
Genetics of Lactic Acid Bacteria) the ancestral LAB have apparently been Bacillus-like soil organ-
isms, which subsequently have lost several genes and the associated physiological functions while
adapting to nutritionally rich ecological niches.
1.3 Carbohydrate Fermentation Patterns
1.3.1 Homo- and Heterolactic Fermentation
Because LAB do not possess a functional respiratory system, they have to obtain their energy
by substrate-level phosphorylation. With hexoses there are two basic fermentative pathways. e

Lactic Acid Bacteria: An Introduction ◾ 3
Table 1.1 Common Genera of LAB and Their Differential Characteristics
Family Genera
Characteristics
Shape
CO
2

from
Glucose
Growth
at 10°C
Growth
at 45°C
Growth
in 6.5%
NaCl
Growth
in 18%
NaCl
Growth
at pH 4.4
Growth
at pH
9.6
Type
of
Lactic
Acid
Aerococcaceae Aerococcus Cocci

(tetrads)
–
+
–
+
– –
+
l
Carnobacteriaceae Carnobacterium Rods
–
+
–
ND
–
ND
–
l
Enterococcaceae Enterococcus Cocci
–
+ + +
–
+ + l
Tetrageonococcus Cocci
(tetrads)
+
–
+ +
–Variable
+
Vagococcus Cocci +

– – – –
Lactobacillaceae Lactobacillus Rods Variable Variable Variable Variable
–
Variable
–
d, l, dl
Pediococcus Cocci
(tetrads)
–
Variable Variable Variable
–
+
–
l, dl
Leuconostocaecae Leuconostoc Cocci
a
+ +
–
Variable
–
Variable
–
d
Oenococcus + +
–
Variable
–
Variable
–
d

Weissella + +
–
Variable
–
Variable
–
d, dl
Streptococcaceae Lactococcus
b
Cocci
–
+
– – –
Variable – l
Streptococcus
– –
Variable
– – – –
l
Note: ND, not determined.
a
Some Weissella strains are rod shaped.
b
In older literature lactococci are referred to as Group N streptococci.
4 ◾ Lactic Acid Bacteria: Microbiological and Functional Aspects
homofermentative pathway is based on glycolysis (or Embden–Meyerhof–Parnas pathway) and
produces virtually only lactic acid (Figure 1.2a). Heterofermentative or heterolactic fermentation
(also known as pentose phosphoketolase pathway, hexose monophosphate shunt, or 6-phospho-
gluconate pathway) produces, in addition to lactic acid, significant amounts of CO
2

and ethanol
or acetate (Figure 1.2b). As a general rule, pentoses can only be fermented heterofermentatively by
entering the pathway as either ribulose-5-phosphate or xylulose-5-phosphate (Kandler 1983), but
then (as is obvious from the fermentation scheme outlined in Figure 1.2b) CO
2
is not produced.
eoretically, homolactic fermentation produces 2 moles of ATP per mole of glucose con-
sumed. In heterolactic fermentation the corresponding yield is only 1 mole of ATP if the acetyl
phosphate formed as an intermediate is reduced to ethanol. However, if acetyl phosphate is con-
verted to acetic acid in the presence of alternative electron acceptors, an extra ATP is formed.
Hexoses other than glucose (mannose, galactose, fructose) enter the major pathways outlined
above after different isomerization and phosphorylation steps as either glucose-6-phosphate or
fructose-6-phosphate. For galactose there are two different pathways, depending on whether it
enters the cell as galactose-6-phosphate (via the so-called phosphoenolpyruvate-dependent phos-
photransferase system or PEP:PTS; see Section 1.4.2.4) or as free galactose imported by a specific
Weissella
Lb. delbrueckii group
Lb. casei–
Pediococcus
group
Lactococcus
Streptococcus
Bacillus
Staphylococcus
Carnobacterium
Enterococcus
Vagococcus
Tetragenococcus
Oenococcus
Leuconostoc

Listeria
Aerococcus
Figure 1.1 Schematic, unrooted phylogenetic tree of the LAB, including some aerobic and fac-
ultatively anaerobic Firmicutes. Note: Evolutionary distances are approximate.
Figure 1.2 Major fermentation pathways of glucose. (a) Homolactic fermentation (glycolysis,
Embden–Meyerhof–Parnas pathway). (b) Heterolactic fermentation (6-phospho-gluconate/
phosphoketolase pathway). Selected enzymes are numbered: 1. Glucokinase; 2. Fructose-
1,6-diphosphate aldolase; 3. Glyceradehyde-3-phosphate dehydrogenase; 4. Pyruvate kinase;
5.Lactate dehydrogenase; 6. Glucose-6-phosphate dehydrogenase; 7. 6-Phospho-gluconate dehy-
drogenase; 8. Phosphoketolase; 9. Acetaldehyde dehydrogenase; 10. Alcohol dehydrogenase.
Lactic Acid Bacteria: An Introduction ◾ 5
Glucose Glucose
(a) (b)
ATP
ADP
ATP
ADP
Glucose-6-P
Glucose-6-P
6-Phospho-gluconate
Ribulose-5-phosphate
Xylulose-5-phosphate
Glyceraldehyde-3-P Acetyl phosphate
CoA
CoA
9.
10.
1,3-Diphosphoglycerate
Acetyl-CoA
Acetaldehyde

3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
Fructose-6-P
Fructose-1,6-DP
Glyceraldehyde-3-P
2
2
2
2
2
2
1,3-Disphosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
H₂O
2 NAD
+
2 NADH+2H
+
3.
4.
5.
P
i
2 ADP
2 ATP

Dihydroxy-
acetone-P
1.
ATP
ADP
1.
6.
7.
8.
2.
2 ADP
2 ATP
2 NAD
+
2 NADH+2H
+
2 Lactate
NAD
+
NAD
+
NADH+H
+
NADH+H
+
CO₂
P
i
P
i

NAD
+
3.
NADH+H
+
ADP
ATP
H₂O
ADP
4.
5.
ATP
NAD
+
NADH+H
+
Lactate
Ethanol
P
i
NAD
+
NADH+H
+
NAD
+
NADH+H
+
6 ◾ Lactic Acid Bacteria: Microbiological and Functional Aspects
permease. In the former case the tagatose phosphate pathway is employed (Figure 1.3a) (Bisset

and Andersson 1974), and the so-called Leloir pathway (Figure 1.3b) in the latter (Kandler 1983).
e fermentation type is an important taxonomic criterion. e genera Leuconostoc, Oenococcus,
and Weissella are obligate heterofermentative, as well as the so-called Group III lactobacilli (e.g.,
Lactobacillus brevis, Lb. buchneri, Lb. fermentum, and Lb. reuteri). Group I lactobacilli (Lb. aci-
dophilus, Lb. delbrueckii, Lb. helveticus, Lb. salivarius), on the other hand, are obligate homofer-
mentative (i.e., they cannot metabolize pentoses). Group II or facultatively heterofermentative
lactobacilli (Lb. casei, Lb. curvatus, Lb. plantarum, and Lb. sakei) as well as most other LAB homo-
fermentatively ferment hexoses, but also ferment pentoses. e division of lactobacilli in three
groups (ermobacterium, Streptobacterium, and Betabacterium) on the basis of their fermentation
patterns, as suggested by Orla-Jensen (1919), is still used for pragmatic reasons, although it does
not reflect the present phylogeny of the genus.
It should be noted that the outline presented in this chapter represents a generalization, for
which there are exceptions, for example, the homolactic fermentation of a pentose (Tanaka et
al. 2002) and the homolactic fermentation of fructose by obligate heterofermentative lactobacilli
(Saier et al. 1996).
1.3.2 Fermentation of Disaccharides
Due to the presence of lactose in milk, the metabolism of this disaccharide has been extensively
studied, especially in the species used in dairy applications. Lactose can enter the cell either by
the means of a specific permease or as lactose phosphate by a lactose-specific PEP:PTS system,
and in some cases both systems can coexist (ompson 1979). If the transport is permease medi-
ated, lactose is cleaved to glucose and galactose by β-galactosidase, and both of these monosac-
charides can subsequently enter the major fermentation pathways. In the case of PEP:PTS system,
another enzyme, phospho-β--galactosidase, is needed to split lactose phosphate to glucose and
(a)
Galactose
PTS
Galactose-6-P
Galactose
Galactose-1-P
Glucose-1-P

Glucose-6-P
Tagatose-6-P
Tagatose-1,6-DP
DHAP GAP
Pyruvate Pyruvate
Glycolysis Glycolysis
ATP
ADP
Galactose
(b)
Permease
ATP
ADP
Figure 1.3 Galactose metabolism in LAB. (a) Tagatose-6-phosphate pathway. (b) Leloir pathway.