Lactic Acid Bacteria: Biodiversity and Taxonomy

By Brian J.B. Wood

The lactic acid bacteria (LAB) are a group of related micro-organisms that are enormously important in the food
and beverage industries. Generally regarded as safe for human consumption (and, in the case of probiotics, positively beneficial to human health), the LAB have been used for centuries, and continue to be used worldwide on an industrial scale, in food fermentation processes, including yoghurt, cheeses, fermented meats and vegetables, where they ferment carbohydrates in the foods, producing lactic acid and creating an environment unsuitable for food spoilage organisms and pathogens to survive. The shelf life of the product is thereby extended, but of course these foods are also enjoyed around the world for their organoleptic qualities.  They are also important to the brewing and winemaking industries, where they are often undesirable intruders but can in specific cases have desirable benefits. The LAB are also used in producing silage and other agricultural animal feeds.  Clinically, they can improve the digestive health of young animals, and also have human medical applications.

This book provides a much-needed and comprehensive account of the current knowledge of the lactic acid bacteria, covering the taxonomy and relevant biochemistry, physiology and molecular biology of these scientifically and commercially important micro-organisms. It is directed to bringing together the current understanding concerning the organisms’ remarkable diversity within a seemingly rather constrained
compass. The genera now identified as proper members of the LAB are treated in dedicated chapters,
and the species properly recognized as members of each genus are listed with detailed descriptions of their principal characteristics.  Each genus and species is described using a standardized format, and the relative importance of each species in food, agricultural and medical applications is assessed.  In addition, certain other bacterial groups (such as Bifidobacterium) often associated with the LAB are given in-depth coverage. The book will also contribute to a better understanding and appreciation of the role of LAB in the various ecological ecosystems and niches that they occupy.  In summary, this volume gathers together information designed to enable the organisms’ fullest industrial, nutritional and medical applications.

Lactic Acid Bacteria: Biodiversity and Taxonomy is an essential reference for research scientists, biochemists and microbiologists working in the food and fermentation industries and in research institutions. Advanced students of food science and technology will also find it an indispensable guide to the subject.

• Language: English
• Publisher: Wiley
• Release date: Apr 29, 2014
• ISBN: 9781118655276

Book preview

Lactic Acid Bacteria

Biodiversity and Taxonomy

Edited by

Wilhelm H. Holzapfel

School of Life Sciences, Handong Global University, Pohang, Gyeongbuk, South Korea; Insheimer Strasse 27, Rohrbach, Germany

Brian J.B. Wood

Formerly Reader in Applied Microbiology, Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, Scotland, UK

Title Page

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Library of Congress Cataloging-in-Publication Data

Lactic acid bacteria : biodiversity and taxonomy / edited by Wilhelm Holzapfel.

pages cm

Includes bibliographical references and index.

ISBN 978-1-4443-3383-1 (cloth)

1. Lactic acid bacteria. 2. Biodiversity. 3. Microbial diversity. 4. Lactic acid bacteria— Classification. 5. Lactic acid bacteria— Physiology. 6. Microbiological chemistry. 7. Lactic acid bacteria— Molecular aspects. I. Holzapfel, W. H.

QR121.L3335 2014

579.3′5— dc23

2013028930

A catalogue record for this book is available from the British Library.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books.

Cover image: Salted Peppers © Drbouz/ Istock

food industry manufacturing interior © picsfive/ Istock

Sourdough © karma_pema/ Istock

bacillus bacteria © sgame/ Istock

Cheese © IvonneW/ Istock

Meat-Salami © Floortje/ Istock

Yoghurt Jar © AndreaAstes/ Istock

Cover design by www.hisandhersdesign.co.uk

List of contributors

Hikmate Abriouel Departamento de Ciencias de la Salud, Paraje Las Lagunillas, s/n Edificio B-3, 23071-Jaén, Spain.

Nabil Benomar Universidad de Jaén, Departamento de Ciencias de la Salud, Campus Las Lagunillas, s/n, E-23071-Jaén, Spain.

Bruno Biavati Department of Agricultural Sciences, University of Bologna, via Fanin 42, 40127 Bologna, Italy.

Johanna Björkroth Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, FIN-00014 Helsinki University, Finland.

Gyu-Sung Cho Max Rubner-Institut, Federal Research Institute for Nutrition and Food, Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany.

Katrien De Bruyne Applied Maths NV, Keistraat 120, B-9830 Sint-Martens-Latem, Belgium.

Leon M.T. Dicks Department of Microbiology, University of Stellenbosch, ZA-7600 Stellenbosch, South Africa.

Stephanie Doores Department of Food Science, Penn State University, 432 Food Science Building, University Park, 16802, USA.

Harold L. Drake Department of Ecological Microbiology, University of Bayreuth, D-95440 Bayreuth, Germany.

Maret du Toit Institute for Wine Biotechnology, Stellenbosch University, Private Bag X1, Matieland, ZA-7602 Matieland, South Africa.

Akihito Endo Department of Microbiology, University of Stellenbosch, 7600 Stellenbosch, South Africa; Functional Foods Forum, University of Turku, 20014 Turku, Finland.

Giovanna E. Felis Department of Biotechnology, University of Verona, Strada le Grazie 15, I- 37134 Verona, Italy.

Charles M.A.P. Franz Max Rubner-Institut, Haid- und Neu-Strasse 9, D-76131 Karlsruhe, Germany.

Antonio Gálvez Departamento de Ciencias de la Salud, Paraje Las Lagunillas, Edificio B-3, E-23071-Jaén, Spain.

Giorgio Giraffa Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie (CRA-FLC), 26900 Lodi, Italy.

Cho Gyu-Sung Max Rubner-Institut, Haid- und Neu-Strasse 9, D-76131 Karlsruhe, Germany.

Walter Hammes Talstr. 60/1, D-70794 Filderstadt, Germany.

Kikue Hirota Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.

Wilhelm H. Holzapfel School of Life Sciences, Handong Global University, Pohang, Gyeongbuk, 7891-798, South Korea; Insheimer Strasse 27, D-76865 Rohrbach, Germany.

Richard B. Hoover Athens State University, 300 North Beaty Street, Athens, Alabama 35611, USA.

Lesley Hoyles Department of Microbiology, University College Cork, Cork, Ireland.

Melanie Huch Max Rubner-Institut, Haid- und Neu-Strasse 9, D-76131 Karlsruhe, Germany.

Morio Ishikawa Department of Fermentation Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture, 1-1, Sakuragaoka 1-chome, Setagaya-ku, Tokyo 156-8502, Japan.

Annelies Justé Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), Cluster for Bioengineering Technology (CBeT), Department of Microbial and Molecular Systems (M²S), KU Leuven Association, Thomas More Mechelen, Sint-Katelijne-Waver, Belgium; Scientia Terrae Research Institute, B-2860 Sint-Katelijne-Waver, Belgium; Leuven Food Science and Nutrition Research Centre (LFoRCe), B-3001 Leuven, Belgium.

Wonyong Kim Department of Microbiology, Chung-Ang University, 156-756 Seoul, Republic of Korea.

Paul A. Lawson Department of Microbiology and Plant Biology, and Graduate Program in Ecology and Evolutionary Biology, University of Oklahoma, Norman, Oklahoma 73019, USA.

Jørgen J. Leisner Department of Veterinary Disease Biology, Faculty of Health Sciences, University of Copenhagen, Grønnegårdsvej 15, DK-1870 Frederiksberg C, Denmark.

Bart Lievens Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), Cluster for Bioengineering Technology (CBeT), Department of Microbial and Molecular Systems (M²S), KU Leuven Association, Thomas More Mechelen, Sint-Katelijne-Waver, Belgium; Scientia Terrae Research Institute, B-2860 Sint-Katelijne-Waver, Belgium; Leuven Food Science and Nutrition Research Centre (LFoRCe), B-3001 Leuven, Belgium.

Oksana Lukjancenko Center for Biological Sequence Analysis, Department of Systems Biology, The Technical University of Denmark, Building 208, DK-2800 Kgs. Lyngby, Denmark.

Paola Mattarelli Department of Agricultural Sciences, University of Bologna, via Fanin 42, 40127 Bologna, Italy.

Kenji Nakajima Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.

Konstantinos Papadimitriou Laboratory of Dairy Research, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece; Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Panepistimioupolis-Zographou, 157 84 Athens, Greece.

Elena V. Pikuta Department of Mathematical, Computer and Natural Sciences, Waters Hall, N204, Athens State University, 300 North Beaty Street, Athens, Alabama 35611, USA.

Bruno Pot Lactic Acid Bacteria and Mucosal Immunology, Center for Infection and Immunity Lille, Institut Pasteur de Lille, Université Lille Nord de France, CNRS, UMR 8204; Institut National de la Santé et de la Recherche Médicale, U1019, 1, Rue du Professeur Calmette, F-59019 Lille, France.

Hans Rediers Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), Cluster for Bioengineering Technology (CBeT), Department of Microbial and Molecular Systems (M²S), KU Leuven Association, Thomas More Mechelen, Sint-Katelijne-Waver, Belgium; Scientia Terrae Research Institute, B-2860 Sint-Katelijne-Waver, Belgium; Leuven Food Science and Nutrition Research Centre (LFoRCe), B-3001 Leuven, Belgium.

Ulrich Schillinger Institute for Microbiology and Biotechnology, Max Rubner-Institut (MRI), Haid- und Neu-Str. 9, D-76131 Karlsruhe, Germany.

Pavel Švec Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Tvrdého 14, 602 00 Brno, Czech Republic.

Effie Tsakalidou Laboratory of Dairy Research, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece.

Peter Vandamme Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium.

Carol A. Van Reenen Department of Microbiology, University of Stellenbosch, ZA-7600 Stellenbosch, South Africa.

Trudy M. Wassenaar Molecular Microbiology and Genomics Consultants, Tannenstrasse 7, D-55576 Zotzenheim, Germany.

Kris A. Willems Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), Cluster for Bioengineering Technology (CBeT), Department of Microbial and Molecular Systems (M²S), KU Leuven Association, Thomas More Mechelen, Sint-Katelijne-Waver, Belgium; Scientia Terrae Research Institute, B-2860 Sint-Katelijne-Waver, Belgium; Leuven Food Science and Nutrition Research Centre (LFoRCe), B-3001 Leuven, Belgium.

Brian J.B. Wood Formerly Reader in Applied Microbiology, Strathclyde Institute for Pharmacy and Biomedical Sciences, Arbuthnott Building, University of Strathclyde, Cathedral Street, Glasgow, Scotland, G4 0RE, UK.

Kazuhide Yamasato Department of Fermentation Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture, 1-1, Sakuragaoka 1-chome, Setagaya-ku, Tokyo 156-8502, Japan.

Isao Yumoto Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.

Acknowledgements

Many people have contributed to this book's production. Clearly we owe a great debt to the individuals and teams who have produced the chapters and the introductions to the sections into which those chapters are grouped. We do recognize that they are busy people with careers to develop, and, generally, with an overcrowded schedule continuously filled with commitments. Moreover, the prevailing ethos in universities and research centres does not value producing such reviews as those presented here in the same way that original research publications are measured for promotion and career development. We are thus most fortunate to have assembled an outstanding team of internationally recognized experts who share our vision for a benchmark comprising well-crafted overviews of current developments against which future development may be measured. Although such benchmarks are essential for orderly development in any scientific discipline, this may apply to the LAB and their taxonomy in a very special way. Consequently, we are most grateful to all authors for their dedication, commitment and patience with the demands that we have placed upon them. Our task has been greatly assisted by the Wiley Blackwell staff who have worked with us at various stages in the project's development, but we tender special thanks to Mr Andrew Harrison, who so expertly guided us through the last stages toward the manuscript ready to progress to actual book publication. We also wish to acknowledge the contributions made by Mr Robert Hine, who had to read through what must have seemed a very arcane text and identify errors, anomalies, miscited references and so much more, and who did so with great patience and good humour.

Wilhelm H. Holzapfel

Brian J.B. Wood

List of Abbreviations

Abbreviations for Genera and Note on Pronunciations

*Genus (phylogenetically) not a member of the LAB.

Note on Pronunciations

The etymologies of generic and specific names are in many cases supplied with a basic pronunciation of the name as used by native speakers of standard English. Syllables are separated by full points, and the primary stressed syllable is indicated by a stress mark (′) following the stressed syllable.

Chapter 1

Introduction to the LAB

Wilhelm H. Holzapfel¹* and Brian J.B. Wood²

¹School of Life Sciences, Handong Global University, Pohang, Gyeongbuk, South Korea; Insheimer Strasse 27, D-76865 Rohrbach, Germany

²Strathclyde Institute for Pharmacy and Biomedical Sciences, Strathclyde University, Glasgow, Scotland

*Corresponding author email: [e-mail]

1.1 The scope

Lactic acid bacteria (LAB) have been intimately associated with human culture and well-being throughout history. In our time, the industrialization of food biotransformations and the positive attributes of particular microbes to sensory, quality and safety features of fermented foods have become synonymous with the positive image of LAB. Yet, the economic impact and role of LAB, both beneficial and detrimental, is as diverse as the six families, 36 genera and the increasing number of species (>200 by the end of 2011) within the order Lactobacillales may suggest.

The LAB belong to the Gram-positive bacterial phylum Firmicutes with ‘low’ (≤55 mol %) G+C in the DNA. They are grouped in the third class (Class III, the Bacilli) of the Firmicutes, with the Clostridia (Class I) and the Mollicutes (Class II) as the other two members. Based on comparative sequence analysis of the 16S rRNA gene, the Firmicutes are distinguished from the other Gram-positive phylum, the Actinobacteria, with high mol % G+C (≥55 mol %) in the DNA. The two Gram-positive phyla comprise the following:

Phylum VIII: Firmicutes (Ludwig et al. 2009, modified)

Class I: ‘Bacilli’

Order I: Bacillales with 12 families, e.g.:

Family I: Bacillaceae; Family VII: ‘Sporolactobacillaceae’ (with one genus Sporolactobacillus)

Order II: ‘Lactobacillales’ with 6 families

Class II: ‘Clostridia’

Class III: ‘Erysipelotrichia’

‘Class’ Mollicutes (cell wall-less): the Mycoplasmas

Phylum Actinobacteria (Ludwig et al. 2007) comprising more than 39 families and 130 genera (Ventura et al., 2007); examples:

Coryneform and propionic acid bacteria; Bifidobacterium; Mycobacterium; Rhodococcus; Gardnerella

Filamentous representatives: Streptomyces and other Actinomycetes.

It is clear that, by phylogenetic definition, Bifidobacterium belongs to the Actinobacteria and not to the true LAB. Still we have included this and ‘related’ genera (see Chapter 29) in this book for historical and practical reasons, one being their beneficial effects on and association with the gut, and another that bifidobacteria physiologically resemble the true LAB to some degree. Similar considerations seemed to justify the inclusion of Bacillus (Chapter 31) and ‘related’ genera (Chapter 32), in addition to the genus Sporolactobacillus (Chapter 30), all of which have some physiological features similar or comparable to the LAB. Bacillus infernus (e.g.) is a strict anaerobe that grows fermentatively on glucose (Boone et al., 1995). Bacillus coagulans is a thermophilic producer of pure lactic acid (Payot et al., 1999), while ‘probiotic’ strains of this species are being marketed under the name ‘Lactobacillus sporogenes’ (De Vecchi & Drago, 2006). Most species of the genus Geobacillus are reported to form catalase (Nazina et al., 2001), yet some strains of Geobacillus stearothermophilus (formerly Bacillus stearothermophilus ) have been found to be catalase negative (Holzapfel, unpublished results). Figure 1.1.

c01f001

Figure 1.1 ‘Domestication’ of LAB in the human environment. The close relationship between human culture and the direct food environment of humans probably partly supplied the microbial population of food fermentations, and vice versa

The LAB are grouped in Order II, the ‘Lactobacillales’ (Garrity & Holt, 2001; Ludwig et al., 2009) under Class I (Bacilli) of the Firmicutes. With presently six families and 40 genera, the LAB may be considered as ‘a rapidly expanding’ group of bacteria, especially when considering the rate at which the publication of new Lactobacillus and Streptococcus species occurs, with more than 150 (see Chapter 19) and 70 (Chapter 28) species, respectively. This wide taxonomic delineation of the LAB indeed suggests a wide diversity within this group, as is indicated in the division of the six families:

‘Aerococcaceae’ (with 7 genera);

‘Carnobacteriaceae’ (with 16 genera);

‘Enterococcaceae’ (with 7 genera);

Lactobacillaceae (with 3 genera);

‘Leuconostoccaceae’ (with 4 genera);

Streptococcaceae (with 3 genera).

Table 1.1 summarizes information on the presently recognized families and genera, and a few selected ‘classical’ phenotypic characteristics of these genera. A highly interesting indication of biodiversity is the interpeptide bridge of the cell wall peptidoglycan of the LAB. At least five different peptidoglycan types have been reported, both for the relatively ‘small’ genus Alkalibacterium (with presently eight species) and the genus Weissella (presently 14 species). By contrast, only two peptidoglycan types are known for the genus Enterococcus (representing 43 species), and four types for the genus Lactobacillus (presently >150 species). Consensus on a comprehensive definition of ‘biodiversity’ probably does not exist, as it has to be delineated according to the scope or range under consideration. In terms of their biological diversity, the LAB have to be considered on the basis of taxonomic (genus, species and even strain) diversity, genetic diversity and phenotypic diversity in relation to an ecosystem and adaptation to extreme conditions. Even in earlier geological history, their physiological diversity and adaptation to a wide range of sometimes extreme habitats clearly suggested that the LAB are by no means a homogeneous group. Present-day phylogenetic approaches are valuable but do not necessarily explain the adaptation of particular LAB to specific ecological niches, and even less so the activation of adaptive survival mechanisms including stress factors. Diversity of the LAB is reflected by their association with diverse habitats, including niches with extreme conditions ranging from relatively high temperatures (around 50°C) to low temperatures (0–2°C), and also with examples of growth at high salt concentrations (up to 25% NaCl), low pH (around 3.9) and physiological bile salt concentrations (see Table 1.2). In contrast to other Gram-positive bacteria such as Bacillus or Listeria (relying on a global stress-response regulator such as σB), the LAB respond to stress with several conserved stress proteins, including DnaK, GroEL and Clp, which are also involved in cross-protection against different stress conditions. Moreover, the type of stress will determine whether other, more specific regulators or mechanisms will be utilized for protection against harmful conditions (Franz & Holzapfel, 2011).

Table 1.1 Selected ‘classical’ characteristics as key phenotypic features, and present grouping of the LABe

a Some species/strains.

b All species arginine hydrolase positive except C. viridans.

c Exception: T. patagoniensis, which grows at up to a pH of 10 and 7% NaCl.

d Exception: Weissella beninensis.

e Other genera referred to in this book are abbreviated as follows: Bacillus (B.); Bifidobacterium (Bif.); Escherichia (E.); Geobacillus (G.); Halobacillus (Hb.); Halolactibacillus (H.); Sporolactobacillus (Sp.); Staphylococcus (Staph.).

Table 1.2 Examples of growth/tolerance and association of LAB with extreme conditions. Strain variations may occur within a species (Holzapfel, unpublished data; see also Franz & Holzapfel, 2011)

a In relation to ‘bitter hop compounds’ at concentrations ranging around 55 ppm of iso- c01-math-0001 -acids.

b Higher resistance during exponential growth than in stationary phase (Hastings et al., 1986). C., Carnobacterium; Ent., Enterococcus; Lb., Lactobacillus; Leuc. Leuconostoc; O., Oenococcus; Ped., Pediococcus; Strep., Streptococcus; Tet., Tetragenococcus; W., Weisella.

Representatives of the LAB may be found in diverse habitats and under conditions defined by extreme intrinsic and extrinsic factors. Examples of the association of LAB with extreme conditions under which either growth or tolerance have been observed are given in Table 1.2. Different mechanisms may be basic to survival or adaptation to diverse habitats. Survival traits may either be determined by constitutive (‘intrinsic’) features of a species or a strain, or may depend on stress responses. Examples of the former may be associated, for example, with the (Gram-positive) cell wall properties, and with a stronger ability to maintain homeostasis in an environment with adverse conditions of high osmotic pressure or low pH. The ability to survive or adapt to extreme conditions also depends on stress responses, including tolerance to low or high temperatures or to bile salts. Stress responses may also involve resistance to environmental stresses typical of an ecosystem, for example physiological concentrations of pancreatic juice in the small intestine, or high salt concentrations typical of Asian fermented fish products. The adaptation of Lactobacillus suebicus, Lb. acetotolerans and the Lb. acidophilus ‘group’ (comprising, e.g., Lb. acidophilus, Lb. gasseri, Lb. crispatus and Lb. johnsonii) to low pH values around 3.0 is differentiated by the habitats typical of these species. The Lb. acidophilus ‘group’ is typically associated with the small intestine and the female urogenital tract (Hammes & Hertel, 2009), and may be able to either mildly ferment milk or at least survive fermentation by well-adapted species such as Streptococcus thermophilus. Strains of Lb. suebicus, isolated from mashes stored for up to a year, were found to grow at pH 2.5 and in the presence of 14% ethanol (Kleynmans et al., 1989), while Lb. acetotolerans was reported to grow even in fermenting rice vinegar broth and to tolerate 4–5% acetic acid at pH 3.5 (Entani et al., 1986). Carnobacterium viridans, originally isolated from vacuum-packaged sliced Bologna sausage, is an alkalitolerant species surviving even in saturated brine solution (Holley et al., 2002). The tetragenoccci are characterized by their high salt tolerance; an extreme example is Tetragenococcus muriaticus, strains of which are able to grow in the presence of 1–25% NaCl (Satomi et al., 1997). Strains of Lb. sakei, isolated from radurized meat, have shown, contrary to the ‘normal’ behaviour of bacteria, higher radiation resistance during exponential growth than in the stationary phase (Hastings et al., 1986). Extreme cold tolerance and ability to grow even at 1–1.5°C was reported for Leuconostoc gelidum (Shaw & Harding, 1989) and some carnobacteria (Jones, 2004) isolated from vacuum-packed cold-stored meat. This extraordinary diversity in habitats and capacities to tolerate, and even thrive in, extreme conditions is in marked contrast with earlier impressions of the LAB as highly fastidious organisms that were very restricted in their environmental tolerances and possessing very exacting nutritional requirements.

A rapidly increasing number of LAB genomes have been sequenced, and the information, in general, is being made publicly available. Comparative functional genomic analyses have become strong tools in support of a deeper understanding of the mechanisms behind biodiversity and adaptation of LAB to diverse habitats. The trend of extensive gene loss or ‘ongoing reduction in genome size’, called ‘reductive evolution’ (Van de Guchte et al., 2006), combined with key gene acquisitions via horizontal gene transfer, may explain the specialization of LAB to a variety of nutritionally rich environments (Makarova et al., 2006; Makarova & Koonin, 2007; Schroeter & Klaenhammer, 2009). Adaptation of LAB to food and intestinal ecosystems is explained by genomic analyses revealing species-to-species variation in the number of pseudogenes, and functional genes directing metabolic ability and nutrient uptake. Even with a general trend of genome reduction, it appears that certain niche-specific genes have been acquired with location on plasmids or adjacent to prophages (Schroeter & Klaenhammer, 2009).

An interesting example is the in silico analysis by Lebeer et al. (2008) of genome sequences reflecting differences between the cheese isolate Lb. helveticus DPC4571 (genome: 2,080,931 bp, with 1618 genes and 217 ‘pseudogenes’), and the closely ‘related’ probiotic strain Lb. acidophilus NCFM (genome: 1,993,564 bp, with 1864 genes but no ‘pseudogenes’) from infant faeces. [The term ‘pseudogenes’ has been suggested for dysfunctional ‘relatives’ of known genes that have lost their protein-coding ability; they are considered to be neutral sequences ‘shaped by random mutations and chance events’ (Vanin, 1985; Andersson & Andersson, 2001; Kuo & Ochman, 2010)]. The suggested ‘loss of genes’ of Lb. helveticus DPC4571 is considered important for adaptation to the gut environment, while half of the phosphoenolpyruvate-dependent sugar phosphotransferases (PEP-PTS), cell wall-anchoring proteins, and all the mucus-binding proteins predicted for Lb. acidophilus NCFM were absent or classified as being ‘pseudogenes’ in Lb. helveticus DPC4571. Genes considered pivotal in suggesting the niche of a strain are thought to be involved in sugar metabolism, the proteolytic system and restriction modification enzymes. Of the nine niche-specific genes identified, six were dairy-specific genes identified for Lb. helveticus DPC4571 and encoded components of the proteolytic system and restriction endonuclease genes. The three gut-specific genes of Lb. acidophilus NCFM encoded bile salt hydrolase and sugar metabolism enzymes (O'Sullivan et al., 2009).

Understanding the genomic information responsible for various phenotypes and their persistence and survival in specific ecosystems and niches is an exciting and rapidly expanding field of research in our time. Specific information on LAB genomics, with specific focus on functional (including probiotic) LAB is presented in Chapter 5.

When discussing biodiversity of a specific group of microorganisms such as the LAB, the importance of taxonomy as a basis of communication is obvious. In this context, it was envisaged that the title (‘Lactic Acid Bacteria—Biodiversity and Taxonomy’) would suggest the intricate complexity of the interplay between biodiversity and taxonomy of this exciting group of bacteria.

1.2 A little history

Pioneering contributions are frequently overlooked or even forgotten in our ‘post-modern’ era. This applies in a special way to the LAB and their key position in early microbiological studies in the 19th century. The second part of the 19th century is characterized by the advent of microbiology as a science. It is fascinating to note that some of the earliest studies on bacteria were conducted on various types of LAB, most of which were either of socio-economic or medical importance during that time. Probably the first starter cultures to be applied for industrial purposes were introduced in 1890, in Denmark, Germany and the USA, for the production of cheese and sour milk. This initiative laid the foundation for the development of the diverse branches of industrial microbiology and modern biotechnology.

The early interest in LAB as microorganisms was prompted by practical issues related to the food and fermentation industries. Louis Pasteur can be considered as the father of microbiology and immunology, but was a chemist. His studies on the molecular structures of tartaric acid laid the foundations of stereochemistry. These were followed in the summer of 1856 by investigations on a problem with improper fermentation, where he detected lactic acid instead of the by-product alcohol. Subjecting the mixture to high temperature (‘pasteurization’) and thereby killing the microorganisms, enabled him to achieve a predictable fermentation by introducing pure microbial cultures (http://www.famous-scientists.net/Louis-Pasteur.html).

The history of LAB taxonomy also reflects key developments and understanding around food spoilage and food fermentations, as exemplified for the genus Leuconostoc. Cienkowski (1878) was probably the first to detect strains of the genus Leuconostoc as spoilage organisms in sugar factories, where they were shown to produce a characteristic slime from sucrose. Although these strains were named Leuconostoc by the French botanist Van Tieghem (1878), Orla-Jensen (1919) disregarded this and used the generic name ‘Betacoccus’ in his approach to separate the LAB genera by phenotypic means at that stage. Bacterium gracili was isolated from wine and described by Müller-Thurgau (1908), and can be considered as a non-slime-producing Leuconostoc. The priority of the earlier name Leuconostoc was supported by later studies of McCleskey et al. (1947) and Niven et al. (1949), who described the isolation of non-slime-producing sucrose-fermenting strains of Leuconostoc from sausages. During the late 1940s the biological and ecological diversity of representatives of the genus Leuconostoc became clear, and was underlined by further studies, including those by Pederson & Ward (1949), describing slime-producing strains from fermenting cucumbers.

Lister (1878) is recognized for several pioneering contributions to the understanding of sepsis and antisepsis in health services and the application of phenol for treating wound infections. Less well known is his discovery, regarding LAB, that milk clotting is caused by ‘Streptococci’, and, moreover, the first isolation, in 1873, of a pure culture he called ‘Bacterium lactis’ (Lactococcus lactis) (http://de.wikipedia.org/wiki/Joseph_Lister,_1._Baron_Lister).

In 1973 a Symposium was held between the 19th and 23rd of September in beautiful autumn weather at the Long Ashton Research Station of the University of Bristol. It was organized by Drs J.G. Carr, C.V. Cutting and G.C. Whiting, and titled ‘Lactic Acid Bacteria in Beverages and Food’. The resulting book of the proceedings (Carr et al., 1975) claimed on its dust jacket that it was ‘the first comprehensive review of lactic acid bacteria to be published in a single volume’. This was not strictly accurate as it focused on organisms associated with the industries delineated in the title, and thus there was little said about organisms such as most members of the genus Streptococcus. Despite this limitation, the meeting otherwise represented with reasonable accuracy the organisms comprising the Lactic Acid Bacteria (LAB), as they were known at that time . The same could be said of the Symposium participants (115 in number) as representatives of the scientific community active in studying the organisms at that time, and the list of ‘Participants in the Symposium’ reads, at least in part, like a roll call of the pioneers in modern study of the group. There were 23 presentations divided into six Sections, plus opening and closing addresses.

Preceding the 1973 Symposium was the milestone ‘Symposium on Lactic Acid Bacteria’ conducted during the 52nd annual meeting of the Society of American Bacteriologists at Boston, 28 April 1952, and convened by Ralph Tittsler (Tittsler et al., 1952). One of the valuable contributions of this symposium was to correct controversies in existing LAB nomenclature, confirming (e.g.) Betacoccus as Leuconostoc, and referring to early controversial opinions regarding ‘Lactobacillus bifidus’ (first suggested by Tissier, 1899) and its assignment to a new ‘non-butyric acid producing anaerobic genus’ (Orla-Jensen et al., 1936; Pederson, 1945; Tittsler et al., 1952). A special (and frequently underestimated) feature of the ‘early’ studies on LAB was the meticulous investigations and detailed reports on their physiology, with emphasis on growth factors, growth temperature ranges, and niche-specific physiological activities with regard to fermentation and spoilage.

Of particular interest in the context of the volume published on the 1973 Symposium is the number of LAB genera and species referred to in the index to the book. There were six genera and in total 65 species. The genus Lactobacillus dominated with 45 species, but the ambiguous position of the genus Bifidobacterium was illustrated by there being only one reference to the genus (no species mentioned) but four references to ‘Lactobacillus bifidus’. The only reference in the index to ‘Genetic Code’ leads to a 4-page section on ‘Mean Base Composition and Homology of DNA’. This conference surely represents the benchmark against which we can measure the growth in our understanding of the LAB in the intervening 36 years.

Exactly 10 years later the first of the now triennial LAB conferences organized by the Netherlands Society for Microbiology and originally centred at Wageningen University in The Netherlands (which is still pivotal to the continuing success of these important meetings) took place in the University. It was called ‘Lactic Acid Bacteria in Foods; Genetics, Metabolism and Applications’, with each of the topics in the subtitle being assigned a day, although, because of other activities, the main sessions actually had a morning each to themselves. There were over 200 people present, with 68 contributions drawn from 18 countries. The organizers chose deliberately to omit Classification as a topic for the meeting. Unfortunately there is no index to the special 1983 edition of the Antonie van Leeuwenhoek Journal of Microbiology (49: 209–352) containing the plenary papers, while the short presentations were presented as photocopies in a ring binder; fast, efficient, but we cannot present the sort of statistics given for the 1973 meeting concerning numbers of validly recognized genera and species. The most striking thing about the whole conference is probably the extent to which genetics had started to move centre-stage, with plenary papers on genetic transfer systems in LAB, functional properties of their plasmids and ‘The bacteriophages of LAB with emphasis on genetic aspects of group N lactic streptococci’.

It would be tedious and probably pointless to present a discussion of each of the intervening LAB Conferences (the latest one, in 2011, was the 10th in the Dutch series) but anyone who has attended the more recent meetings will readily appreciate how the field has grown and changed. We may also note that for several years there were LAB conferences held in France, in the historic town of Caen, and organized under the auspices of Adria Normandie. These tended to focus more on applications of the bacteria, and were a nice counterpoint to the Netherlands congresses. The other great change of consequence for the present work is the massive increase in the number of both genera and species within the LAB that has taken place since 1973. The 1973 conference had reference to only four genera that we would now regard as LAB (Lactobacillus, Leuconostoc, Pediococcus, Streptococcus). When The Genera of Lactic Acid Bacteria was published (Wood & Holzapfel, 1995) we find that in contrast to the four genera and (with the species of Streptococcus excluded for the reasons noted above) 60 species in the 1973 work, there are 16 valid genera and 178 species (139 excluding Streptococcus). In the 2009 text Lactobacillus Molecular Biology; From Genomics to Probiotics (Ljungh & Wadström, 2009) the chapter on ‘Taxonomy and Metabolism of Lactobacillus’ (Pot & Tsakalidou, 2009) lists 113 species (plus several subspecies) of the genus; this may be contrasted with 45 in the 1973 and 54 in the 1995 sources cited above.

1.3 Where are the boundaries?

In the early days of microbiology, the LAB were principally organisms associated with food fermentations such as dairy products, fermented meats (e.g. continental sausages) and vegetables, and participants (normally spoilage organisms but occasionally beneficial) in wine and beer production. Again we may note that among the streptococci there were representatives that did not fit this picture, including some pathogens, particularly organisms associated with tooth decay. In contrast, some of the new genera are represented by organisms derived from environments far removed from these traditional habitats. When considering LAB in relation to public health aspects, the major focus was on Lactobacillus and Leuconostoc species, none of which was known to be pathogenic or to cause human food poisoning. Thanks to ‘modern’ sanitary and hygienic measurements, food-borne epidemics by transfer of pathogenic LAB strains by food were considered a rarity in the early 1950s, even with regard to recognized human pathogenic Streptococcus species such as Strep. pyogenes. However, it was considered that ‘one group of streptococci, namely the enterococci’, might have health-related significance in the food industries. Reference was also made to the suggested use of these enterococci as an ‘index of pollution’ by health authorities (Tittsler et al., 1952).

Another striking feature of the newer entrants is the number of genera with just a single species at this time. In considering the future for these organisms, it may be helpful to draw comparison with the human stomach in general and in particular with the genus Helicobacter. Thirty years ago everyone ‘knew’ that the acid conditions prevailing in the stomach precluded any microbial growth there; indeed, these conditions were at least a partial barrier against the ingress of harmful organisms to the lower parts of the digestive system. Then someone incubated a Petri plate for a little longer than the usual time. The sample loaded onto it was from a patient suffering from a stomach ulcer, and the extended incubation revealed the presence of a bacterium, now the infamous Helicobacter pylori, regarded as the cause of stomach and duodenal ulcers. This s despite the fact that only around 7% of people ever get such an ulcer, although it is said that more like 57% of the adult population harbour the bacterium without evident pathology. On the 20th anniversary of this organism's discovery the genus contained upwards of 20 species. A recent report (Pennisi, 2010) states that the human stomach is home to 25 bacterial genera. It is reasonable to suggest that much of this change in our understanding of the stomach's microbiology stems from the realization that Helicobacter could reside in this environment, and thus that where there is one there may be more. Similarly, now that the newest techniques in microbial identification permit those first discovering them to assign new isolates confidently to the LAB despite their deriving from harsh environments a long way from the traditional LAB homes, it is surely reasonable to hope that there will be more species discovered to populate these new genera, and that there are new genera awaiting discovery from unlikely habitats.

The authoritative taxonomic contributions by Orla-Jensen (1919, 1942) formed the basis of perhaps the earliest, well-founded definition of the LAB as ‘…Gram-positive, non-motile, non-sporeforming, rod- or coccus-shaped organisms that ferment carbohydrates and higher alcohols to form mainly lactic acid’. This classical approach was based on morphological and physiological characteristics, but, thanks to developments and advances in molecular techniques, an unequivocal definition soon became overruled (Stiles & Holzapfel 1997). In a general sense, the contemporary consensus definition considers a ‘typical’ LAB as Gram-positive, non-sporeforming, catalase-negative, devoid of cytochromes, non-aerobic but aerotolerant, nutritionally fastidious, acid-tolerant and strictly fermentative, with lactic acid as the major end-product of sugar fermentation (Klein et al., 1998; Franz & Holzapfel, 2011).

The LAB have traditionally been characterized as facultatively anaerobic, strictly fermentative bacteria, and were therefore generally considered not capable of aerobic respiration. Yet, a haem-requiring catalase has been described for some strains even in the 1960s (Whittenbury, 1964), while haem- (and menaquinone-) stimulated aerobic growth was reported for several species, including Lb. plantarum, Lb. rhamnosus, Lb. brevis, Lb. paralimentarius, Strep. entericus and Lactococcus garviae (Bryan-Jones & Whittenbury, 1969). Recent reports (Brooijmans et al., 2007; Wegmann et al., 2007) clearly confirmed a respiratory ability for Lactococcus lactis by generating a proton-motive force via a membrane integral electron shuttle involving quinones and cytochromes. Menaquinone production and the corresponding encoding genes were reported for Lactococcus lactis (Wegmann et al., 2007). The presence of a haem-dependent bd-type cytochrome in the respiratory chain confirmed the ability to generate a proton-motive force (Brooijmans et al., 2007, 2009). It has also been reported that Fructobacillus species grow well on glucose under aerobic conditions, thus suggesting that oxygen is used as an electron acceptor (see Chapter 22).

The phenotypic approach was the basis for LAB taxonomy and for characterizing the major genera until the late 1970s. Additional information for the definition of taxa was obtained by determination of the mol % G+C in the DNA, and the interpeptide bridge of the cell wall peptidoglycan. Occasionally, DNA : DNA hybridization was used to determine the similarity of an unidentified stain with an authentic strain. However, a breakthrough came in the early 1980s when DNA : DNA and DNA : rDNA hybridization studies were used to identify new genera and species within hitherto well-defined groups or taxa. Thus, the genus Streptococcus was recognized to be heterogeneous (Kilpper-Bälz et al., 1982), resulting in the separation of the genus Enterococcus (Schleifer & Kilpper-Bälz, 1984) and the genus Lactococcus (Ludwig et al., 1985; Schleifer & Kilpper-Bälz, 1987). An important pragmatic step towards the delineation and definition of the species as taxonomic unit came with the polyphasic approach (Wayne et al., 1987) and the use of a DNA reassociation value of ≥70% DNA similarity, which was shown to correlate with ≥97% sequence identity of the highly conserved 16S rRNA gene (Stackebrandt & Goebel, 1994).

Lactobacillus acidophilus is a classical example of a species that could not be reliably differentiated by phenotypic means from ‘closely related’ species such as Lb. gasseri, Lb. crispatus and Lb. amylovorus. Even in earlier studies Lb. acidophilus strains isolated from the human small intestine were shown to be heterogeneous, and a number of ‘biotypes’ were defined in terms of physiological features (Lerche & Reuter, 1962; Reuter, 1965a, 1965b, 2001). Additional tests to support species differentiation included electrophoretic analysis of soluble cellular proteins and lactate dehydrogenases, and determination of the interpeptide bridge of the cell wall. However, DNA : DNA hybridization studies by Johnson et al. (1980) showed that 78 of 89 strains of Lb. acidophilus were distributed among six distinct homology groups, which they designated Al, A2, A3, A4, B1 and B2. Group A1 of Johnson et al. (1980) was synonymous with Lb. acidophilus, and B2 with Lb. johnsonii, suggested in 1992 by Fujisawa et al. (1992), who also showed that Lb. acidophilus group A3 (Johnson et al., 1980) was synonymous with Lb. amylovorus A3 described by Nakamura (1981).

For investigations into phylogenetic relationships, comparative 16S ribosomal RNA (rRNA) sequencing analysis provided finer discrimination within taxa formerly generated on the basis of phenotypic features. Concomitant developments in molecular techniques such as methods based on the polymerase chain reaction (PCR; e.g. rep-PCR fingerprinting), restriction fragment length polymorphism (RFLP) and pulse-field gel electrophoresis (PFGE) during the late 1990s, were proven to be valuable for characterization of LAB strains from diverse origins and environments (Holzapfel et al., 2001). Moreover, culture-independent approaches such as denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) of the 16S rDNA gene and its rRNA amplicons, were found to be powerful in the analysis and monitoring of the bacterial community in complex ecosystems such as faeces and the intestinal tract (Zoetendal et al., 1998; 2002; Heilig et al., 2002).

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Chapter 2

Physiology of the LAB

Akihito Endo¹,²* and Leon M.T. Dicks¹

¹Department of Microbiology, University of Stellenbosch, Stellenbosch, South Africa

²Functional Foods Forum, University of Turku, Turku, Finland

*Corresponding author email: [e-mail]

2.1 Metabolism

2.1.1 Introduction

Lactic acid bacteria (LAB) are fastidious organisms and need rich and complex nutrients for growth, for example carbohydrates, amino acids, vitamins, and minerals. In addition, some LAB need special growth factors, including tomato juice, whey, etc. LAB degrade a number of carbohydrates and related compounds through different metabolic pathways. ATP, generated by substrate-level phosphorylation, is used for transport of solutes across the cell membrane and for biosynthetic purposes. Environmental conditions are well known to influence metabolic pathways used. This chapter discusses the growth and nutritional requirements of LAB, metabolic pathways involved, and the generation of metabolic energy.

2.1.2 Carbohydrate metabolism

2.1.2.1 Fermentation of glucose and other hexoses

Based on dissimilation of glucose, LAB are divided into two fermentation groups, homolactic and heterolactic. Homolactic LAB use the glycolysis (Embden–Meyerhof–Parnas (EMP), or Embden–Meyerhof) pathway. Heterolactic LAB use the phosphoketolase (6-phosphogluconate) pathway. Selection of pathways is determined at family level.

The glycolytic pathway (Figure 2.1A) is used by members of the families Enterococcaceae, Lactobacillaceae and Streptococcaceae, except for one group in the genus Lactobacillus. In this pathway, glucose is converted to lactic acid (2 molecules of lactic acid per molecules glucose consumed), hence the description homolactic fermentation. Fructose-1,6-diphosphatase is