The Yeasts: Yeast Technology

By Academic Press

This classic series covers the complete biology and biochemistry of the yeasts in six volumes. Volume 5 addresses the major areas of yeast technology relevant to the food, pharmaceutical, and biotechnology industries.

* SPECIAL FEATURES:
* Final volume of a comprehensive research level edited treatise covering biochemistry physiology, technology of yeasts. The book will cover the major areas of yeast technology relevant to the food, pharmaceutical and biotechnology industries. Yeast are highly versatile organisms, particularly suitable for industrial purposes - this book will be of interest to many.

• Language: English
• Publisher: Academic Press
• Release date: Dec 2, 2012
• ISBN: 9780080925431

Book preview

Yeast Technology

The Yeasts

Second Edition

Anthony H. Rose

School of Biological Sciences, Bath University, Claverton Down, Bath, UK

J. Stuart Harrison

Ashley House, Upper Frog Street, Tenby, Dyfed, UK

ACADEMIC PRESS

Harcourt Brace & Company, Publishers

London   San Diego   New York

Boston   Sydney   Tokyo   Toronto

Table of Contents

Cover image

Title page

Copyright page

Contributors

Preface

Contents of Volume 1

Contents of Volume 2

Contents of Volume 3

Contents of Volume 4

Abbreviations

1: Introduction

2: Brewer’s Yeasts

I INTRODUCTION

II BREWING YEAST STRAINS

III TECHNOLOGY

IV NUTRITION

V YEAST AND BEER QUALITY

VI FERMENTATION IN THE FUTURE

VII CONCLUSIONS

VIII. ACKNOWLEDGEMENTS

3: Wine-making Yeasts

I INTRODUCTION

II WINE-YEAST STRAINS

A Wine yeast-strain availability

III NATURAL VERSUS INOCULATED VINIFICATIONS

IV VINIFICATION FERMENTATIONS

V ETHANOL TOXICITY AND TOLERANCE IN WINE YEASTS

VI FERMENTATION FLAVOUR COMPONENTS

VII SECONDARY WINE FERMENTATIONS BY YEASTS

VIII SPOILAGE YEASTS

IX OTHER YEASTS ASSOCIATED WITH WINE-MAKING

X APPLICATIONS OF MOLECULAR GENETICS TO OENOLOGY

4: Saké-Brewing Yeasts

I INTRODUCTION

II SAKÉ-PRODUCTION PROCESS

A Raw materials

III TAXONOMY OF SAKÉ YEAST

IV FACTORS AFFECTING SAKÉ YIELD AND QUALITY

V RECENT STUDIES OF SAKÉ YEAST AND SAKÉ BREWING

VI CONCLUSIONS

APPENDIX DEFINITION OF JAPANESE TERMS USED IN THE SAKÉ-MANUFACTURING INDUSTRY

5: Yeasts in Cider-Making

I INTRODUCTION

II THE ORCHARD

III JUICE PROCESSING

IV FERMENTATION

6: Yeasts in Distilled Alcoholic-Beverage Production

I INTRODUCTION

II TYPES OF DISTILLED ALCOHOLIC BEVERAGES

III MAJOR FERMENTATION SUBSTRATES

IV YEASTS USED IN THE DISTILLING INDUSTRY

V DESIRABLE YEAST-STRAIN PROPERTIES

VI YEAST DEVELOPMENT

VII ACKNOWLEDGEMENTS

7: Yeasts for Production of Fuel Ethanol

I INTRODUCTION

II SUBSTRATES

III PROCESS

IV YEASTS

V YIELD-REDUCING FACTORS

VI PROCESS AND QUALITY CONTROL

VII NEW TECHNOLOGY

VIII CONCLUSIONS

8: Miscellaneous Products from Yeast

I INTRODUCTION

II PHAFFIA RHODOZYMA

III YEAST CULTURE FOR LIVESTOCK FEEDS

IV SUMMARY

9: Yeast as a Vehicle for the Expression of Heterologous Genes

I INTRODUCTION

II TRANSFORMATION

III TRANSCRIPTION AND TRANSLATION

IV POST-TRANSLATIONAL EVENTS

V CONCLUSION

10: Baker’s Yeasts

I HISTORICAL PERSPECTIVE

II BREAD-MAKING

III DESIRABLE PROPERTIES IN BAKER’S YEASTS

IV METHODS USED TO ISOLATE NOVEL BAKER’S YEAST STRAINS

V MANUFACTURE OF BAKER’S YEAST

VI IDENTIFICATION OF BAKER’S YEAST STRAINS

11: Food and Fodder Yeasts

I INTRODUCTION

II HISTORICAL

III PRODUCTION SYSTEMS

IV COMPOSITION

V THEORY

VI TECHNOLOGY

VII EFFLUENT DISPOSAL

VIII NUTRITIONAL VALUE

IX CONCLUSIONS

12: Food-Spoilage Yeasts

I INTRODUCTION

II SUGAR-RICH INGREDIENTS AND PRODUCTS

III FRUITS AND VEGETABLES

IV MILK AND DAIRY PRODUCTS

V CEREAL-BASED PRODUCTS

VI SAUCES AND SALADS

VII MEAT, POULTRY AND OTHER PROTEINACEOUS FOODS

VIII SEAFOOD

IX CONCLUDING REMARKS

X ACKNOWLEDGEMENTS

13: Yeasts as Spoilage Organisms in Beverages

I INTRODUCTION

II ECOLOGICAL AND PHYSIOLOGICAL CONSIDERATIONS

III YEASTS ISOLATED FROM BEVERAGES AND THEIR SIGNIFICANCE

IV SOURCES OF INFECTION

V PREVENTION OF SPOILAGE

VI QUALITY CONTROL OF BEVERAGES WITH RESPECT TO YEAST SPOILAGE

Subject Index

Author Index

Copyright

ACADEMIC PRESS LIMITED

24/28 Oval Road

London NW1 7DX

United States Edition published by

ACADEMIC PRESS INC.

San Diego. CA 92101

Copyright © 1993 by

ACADEMIC PRESS LIMITED

All Rights Reserved

No part of this book may be reproduced in any form by photostat, microfilm, or by any other means, without written permission from the publishers

A CIP record for this book is available from the British Library

ISBN 0-12-596415-3

Filmset by Bath Typesetting Limited

and printed in Great Britain by T. J. Press Ltd, Padstow, Cornwall

Contributors

F.W. Beech     8 Fowey Close, Pine Grove, Nailsea, Bristol BS19 2UR, UK

L.F. Bisson     Department of Viticulture and Enology, University of California, Davis, CA 95616, USA

R.G. Board     School of Biological Sciences, University of Bath, Avon BA2 7AY, UK

K.A. Dawson     Department of Animal Sciences, University of Kentucky, Lexington, KY 40546, USA

J.R.M. Hammond     BRF International, Lyttel Hall, Nutfield, Redhill, Surrey RH1 4HY, UK

J.S. Harrison     Ashley House, Upper Frog Street, Tenby, Dyfed SA70 7JD, UK

E. Hinchliffe     Bass Brewers Ltd, 137 High Street, Burton-on-Trent, Staffs DE 14 1JZ, UK

W.M. Ingledew     Applied Microbiology and Food Science Departments, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0 W0

K.A. Jacques     Alltech Biological Center, 3031 Catnip Hill Pike, Nicholasville, KY 40356, USA

E. Kenny     Bioresearch Ireland, EOLAS, Glasnevin, Dublin 9, Ireland

K. Kodama     Laboratory of Kodama Brewing Co. Ltd, Iidagawa, Japan

R.E. Kunkee     Department of Viticulture and Enology, University of California, Davis, CA 95616, USA

T.P. Lyons     Alltech Biological Center, 3031 Catnip Hill Pike, Nicholasville, KY 40356, USA

A.H. Rose     School of Biological Sciences, University of Bath, Avon BA2 7AY, UK

D.S. Thomas     38 Mansfield Road, Worksop S80 3 AD, UK

E.A. Tudor     Unilever Research, P.O. Box 114, 3130 AC Vlaardgingen, The Netherlands

G. Vijayalakshmi     Microbiology and Sanitation Department, C.F.T.R.I., Mysore 570013, India

D.C. Watson     Chivas Brothers Ltd, Keith, Banffshire, Scotland, UK

Preface

Anthony H. Rose; J. Stuart Harrison

The days when a single author was able to produce a series of volumes which could satisfactorily cover the whole gamut of a general science such as chemistry, physics or biology are long past. More recently these continuously expanding disciplines, and many more including mathematics and chemical engineering, have become essential basics for the complete study of a single class of living organisms.

We have endeavoured in this multivolume treatise to provide as wide a coverage as possible of the different areas of investigation that comprise the science of zymology, the study of yeasts. Five volumes including the present one have resulted; the final volume dealing will yeast genetics will be published early in 1994. Inevitably there will be gaps when dealing in this very broad field; for such we apologize and trust that any deficiencies may not seriously detract from the value of the Second Edition as a whole. In this Second Edition, as in the First Edition (1969–1971), prominent workers in each relevant discipline present an overall account of their chosen field with particular attention to the results of the most recent advances.

The first requirement is the recognition and definition of the members of the group, that is taxonomy and classification, closely followed by such essentials as biological functions related to the external environment and intracellular mechanisms. In these fields modern research has been very active and rewarding, advances since the publication of the First Edition being quite phenomenal. Genetics also has made tremendous strides forward during the same period, the molecular aspect being especially important. The many branches of knowledge discussed in detail are too numerous to mention here, but each volume has been planned to cover a group of related subjects, and so form a useful book in its own right.

We gratefully acknowledge the debt we owe, not only to the contributors to these volumes and others concerned for their efforts, often under difficult circumstances, but also to the vast number of workers in related fields in the past whose investigations have made possible the rapid exponential advances in zymological knowledge. We hope that these volumes will provide a useful overall view of the present state of the art.

Contents of Volume 1

Contributors v

Preface vii

1 IntroductionA.H. Rose and J. S. Harrison 1

References 4

2 Classification of YeastsN. J. W. Kreger-van Rij 5

I. Introduction 5

II. Criteria used in the classification of yeasts 6

III. Principles of classification 19

IV. Classification 22

V. Acknowledgements 54

References 55

3 Molecular TaxonomyC. P. Kurtzman and H. J. Phaff 63

I. Introduction 63

II. Nucleic acid isolation and purification 64

III. Deoxyribonucleic acid base composition 66

IV. Deoxyribonucleic acid relatedness 71

V. Macromolecules other than deoxyribonucleic acid as indicators of relatedness 83

VI. Comparison of relatedness from nucleic acid studies with that determined by other methodologies 86

VII. Molecular taxonomy as an acid to genetic research 89

References 90

4 The Typological Yeast Species, and its DelimitationJ.P. van der Walt 95

I. Introduction 95

II. Delimitation of the species on the basis of phenotypic discontinuity 97

III. Delimitation of the species on the basis of reproductively isolated populations 99

IV. Delimitation of the species on the basis of nucleic acid analyses 107

V. Delimitation of the species by numerical analyses 111

VI. Delimitation of the imperfect species 112

VII. Delimitation of the yeast species in practice 114

VIII. Classification of the industrial yeasts 115

References 117

5 Yeasts Associated with Plants, Insects and SoilH. J. Phaff and W. T. Starmer 123

I. Introduction 123

II. Methods of isolation and study 128

III. Specificity of habitats 135

IV. Evolutionary ecology of yeasts 172

References 174

6 Ecology of Aquatic YeastsA.N. Hagler and D. G. Ahearn 181

I. Introduction 181

II. Distribution of yeasts in aquatic environments 185

III. Pollution 194

IV. Physico-chemical parameters affecting aquatic yeasts 196

V. Summary 199

References 199

7 Yeasts as Human and Animal PathogensR. Hurley, J. de Louvois and A. Mulhall 207

I. Introduction 208

II. Pathogens causing candidosis 212

III. Cryptococcus neoformans and cryptococcosis 239

IV. The genus Pityrosporum 244

V. The genus Trichosporon 250

VI. The genus Geotrichum 253

VII. Histopathological differentiation of yeast-like fungi in tissues 255

VIII. Antifungal chemotherapy 258

References 273

8 Biology of the Cell Cycle in YeastsE. Wheals 283

I. Introduction 284

II. Synthesis of cellular components 298

III. Integration of the cell cycle 326

IV. Acknowledgements 365

References 365

Appendix 1 378

Appendix 2 387

Subject Index 391

Author Index 407

Contents of Volume 2

Contributors v

Preface vii

Contents of Volume 1 xiii

1 IntroductionA.H. Rose and J. S. Harrison 1

References 3

2 Responses to the Chemical EnvironmentH. Rose 5

I. Introduction 5

II. Nutrients 6

III. Antimicrobial compounds 23

References 35

3 Temperature RelationsK.G. Watson 41

I. Introduction 41

II. Temperature limits for growth 42

III. Temperature and morphology 45

IV. Temperature and transport 51

V. Temperature and proteins 53

VI. Temperature and lipids 55

VII. High-temperature stress 60

VIII. Low-temperature stress 64

IX. Acknowledgements 65

References 65

4 Effects of Radiation on YeastA.P. Janies and A. Nasim 73

I. Introduction 73

II. Repair 76

III. Inactivation 78

IV. Mutation 81

V. Recombination 86

VI. Aneuploidy 87

VII. Modification of effects 88

VIII. Mitochondria and plasmids 91

References 935

5 Batch and Continuous CultureA. Fiechter, O. Käppeli and F. Meussdoerffer 99

I. Introduction 99

II. Basic aspects of microbial growth 100

III. Cultivation of yeasts 110

IV. Concluding remarks 127

References 127

6 Killer YeastsT. W. Young 131

I. Introduction 131

II. Distribution of the killer phenomenon in yeasts 133

III. Classification of killer yeasts 138

IV. Biology of the killer phenomenon 143

V. Genetics of the killer system in Saccharomyces cerevisiae 150

VI. Killer toxins 154

VII. Applications of the killer phenomenon 159

References 161

7 Cell AggregationC.B. Calleja 165

I. Introduction 166

II. Flocculation of brewer’s yeast 171

III. Sexual agglutination in Hansenula wingei 187

IV. Sex-directed flocculation in Schizosaccharomyces pombe. 196

V. Sexual agglutination in Saccharomyces cerevisiae 209

VI. Other yeast-aggregation systems 220

VII. The importance of being aggregated 223

VIII. Acknowledgement 226References 226

IX. Addendum 237

8 Adhesion to SurfacesL.J. Douglas 239

I. Introduction 239

II. Colonization of surfaces by yeasts 243

III. Measurement of yeast adhesion in vitro 246

IV. Adhesion to epithelial cells 250

V. Adhesion to other cellular surfaces 268

VI. Adhesion to inert surfaces 272

References 275

Subject Index 281

Author Index 293

Contents of Volume 3

Contributors v

Preface vii

Contents of Volume 1 xv

Contents of Volume 2 xix

1 IntroductionAnthony H. Rose and J. Stuart Harrison 1

Reference 4

2 Solute TransportCharles P Cartwright, Anthony H. Rose, Jill Calderbank andMichael H.J. Keenan 5

I. Introduction 5

II. Types of solute-transport that operate in yeasts 8

III. Methodology 8

IV. The wall as an impediment to solute transport 12

V. Diffusion 13

VI. Facilitated transported processes 1

References 49

3 Deoxyribonucleic Acid Organization and ReplicationCarol Shaw Newlon 57

I. Introduction 57

II. Genome structure 58

III. Chromosome structure 62

IV. Replication of chromosomal deoxyribonucleic acid 73

V. Replication of 2 μm circle deoxyribonucleic acid 97

VI. Replication of mitochondrial deoxyribonucleic acid 102

VII. Concluding remarks 104

VIII. Acknowledgements 104

References 105

4 TranscriptionStephen G. Oliver and John R. Warmington 117

I. Introduction 117

II. The transcriptional apparatus 118

III. Initiation and termination of transcription 124

IV. Synthesis and processing of ribosomal ribonucleic acid 136

V. Synthesis and processing of transfer ribonucleic acid (tRNA) 138

VI. Synthesis and processing of messenger ribonucleic acid (mRNA) 144

VII. General controls of yeast transcription 148

References 152

5 Protein SynthesisMichael F. Tuite 161

I. Introduction 162

II. Mechanism 162

III. In vitro translation systems 174

IV. Inhibitors of translation 176

V. Genetics of translation 179

VI. Regulation of protein synthesis 187

VII. Mitochondrial protein synthesis 194

VIII. Summary and prospects 197

References 198

6 Energy-yielding MetabolismCarlos Gancedo and Ramón Serrano 205

I. Introduction 206

II. Pathways of carbon and energy metabolism: similarities and differences between yeast species 207

III. The common glycolytic pathway 209

IV. Dynamics of glycolysis in Saccharomyces cerevisiae: rate-limiting and controlling steps 220

V. The pentose phosphate pathway 222

VI. End products of fermentation and their utilization: gluconeogenesis 224

VII. Aerobic pathways–the tricarboxylic acid and glyoxylate cycles 232

VIII. Energetics of yeast mitochondria 234

IX. Contribution of different pathways to the energy metabolism of Saccharomyces cerevisiae in yeast: adenosine triphosphate production and redox balance during growth 234

X. The Pasteur and other effects 237

XI. General catabolite control or the multiple effects of glucose 241

XII. Energy reserves 246

XIII. Perspectives 250

XIV. Acknowledgements 251

References 251

7 Metabolism of n-AlkanesAtsuo Tanaka and Saburo Fukui 261

I. Introduction 261

II. Uptake of alkanes 262

III. Initial oxidation of alkanes 263

IV. Oxidation of higher alcohols to fatty acids 266

V. Appearance and physiological significance of peroxisomes 267

VI. Activation of fatty acids 268

VII. Degradation of acyl-coenzyme A 272

VIII. Synthesis of cellular fatty acids 275

IX. Synthesis of tricarboxylic-acid cycle intermediates 278

X. Miscellaneous 282

XI. Future prospects 283

References 284

8 Metabolism of One-carbon CompoundsWim Harder and Marten Veenhuis 289

I. Introduction 289

II. Isolation and properties of yeasts that utilize one-carbon compounds 291

III. One-carbon compounds as carbon and energy sources 294

IV. One-carbon compounds as nitrogen sources 309

V. Concluding remarks 312

VI. Acknowledgements 313

References 313

9 Polysaccharide MetabolismVladimir Farkaš 317

I. Introduction 317

II. Storage polysaccharides 318

III. Cell-wall polysaccharides 327

IV. Acknowledgements 356

References 356

10 Lipids and their MetabolismColin Ratledge and Christopher T. Evans 367

I. Introduction 368

II. Extraction of lipids 369

III. Total lipid contents 372

IV. Major lipid classes 385

V. Biosynthesis of fatty acids 406

VI. Biosynthesis of lipids from fatty acids 417

VII. Biosynthesis of lipids from mevalonate 428

VIII. Metabolism of lipids 434

References 444

11 Vitamin MetabolismChisae Umezawa and Takeo Kishi 457

I. Introduction 457

II. Biotin 457

III. Folic acid 459

IV. Inositol 460

V. Nicotinic acid 462

VI. Pantothenic acid 464

VII. Riboflavin 466

VIII. Thiamin 470

IX. Vitamin B6 476

X. Ubiquinone 479

References 482

12 Sporulation in Saccharomyces cerevisiaeJ. J. Miller 491

I. Introduction 492

II. Morphological changes during sporulation 493

III. Factors controlling sporulation 499

IV. Sporulation synchrony 512

V. Cell cycle, cell age and sporulation 514

VI. Factors affecting number of spores per ascus 515

VII. Major chemical changes during sporulation 521

VIII. Induction and regulation 530

IX. Properties of the yeast spore 533

X. Ecology of sporulation . 541

XI. Acknowledgement 543

References 543

Subject Index 551

Author Index 593

Contents of Volume 4

Contributors v

Preface vii

Contents of Volume 1 xv

Contents of Volume 2 xix

Contents of Volume 3 xxiii

Abbreviations xxix

1 IntroductionAnthony H. Rose and J. Stuart Harrison 1

References 6

2 Yeast Cytology: An OverviewC. F. Robinow and B. F. Johnson 7

I. Introduction 8

II. The cell wall 9

III. The nucleus 52

IV. The cytoplasm 87

V. The Golgi body 91

VI. The vacuole 91

VII. The plasmalemma 93

VIII. Mitochondria 98

IX. Peroxisomes (microbodies) 101

X. Acknowledgements 103

XI. Addendum 104

References 110

Note added in proof 120

3 Separation of Yeast OrganellesD. Lloyd and T. G. Cartledge 121

I. History 121

II. Methods of disruption 122

III. Subcellular distribution of enzymes 124

IV. Marker enzymes 126

V. Analytical subcellular fractionation, subcellular enzyme distributions and separation of organelles 127

VI. Prospects 166

References 167

4 CapsulesW. I. Golubev 175

I. Introduction 175

II. Morphology and fine structure 176

III. Culture conditions promoting capsule formation 182

IV. Decapsulation and chemical composition 185

V. Functions 189

VI. Conclusion 194

VII. Acknowledgements 195

References 195

5 Cell wallsG. H. Fleet 199

I. Introduction 200

II. Cell wall function 201

III. Preparation of cell walls and separation of components 202

IV. Composition, structure and function of wall components 206

V. Macromolecular organization of the wall 236

VI. Factors affecting cell-wall composition and structure 238

VII. Degradation of yeast walls by enzymes 245

VIII. Cell-wall biosynthesis 257

IX. Acknowledgements 264

References 264

6 Periplasmic SpaceW. N. Arnold 279

I. Introduction 279

II. Background 280

III. Plasmolysis in yeast 280

IV. Osmotic shock 282

V. Periplasmic-space enzymes 282

VI. Protein concentration in the periplasmic space 291

VII. Periplasmic bodies 292

VIII. Concluding remarks 292

IX. Acknowledgements 293

References 293

7 Plasma MembranesP. A. Henschke and A. H. Rose 297

I. Introduction 297

II. What is the plasma membrane? 298

III. Isolation procedures 298

IV. Characterization criteria 307

V. Composition 312

VI. Molecular anatomy 324

VII. Relationships between lipid composition and plasma-membrane function 330

VIII. Acknowledgements 339

References 340

8 Vacuoles, Internal Membranous Systems and VesiclesJ. Schwencke 347

I. Introduction 348

II. The vacuolar compartment 350

III. Vacuolar proteinases and their role in some intracellular processes 371

IV. Chitosomes 390

V. Endoplasmic reticulum, Golgi complex, vacuole, vesicles and endosomes: their interrelation and their role in exocytosis and endocytosis 397

VI. Acknowledgements 420

References 420

Note added in proof 755

9 Nucleus: Chromosomes and PlasmidsD. H. Williamson 433

I. Introduction 433

II. A thumbnail sketch 434

III. Mendelian chromosomes 437

IV. Plasmids and transformation 456

V. Morphology of the working nucleus 464

References 482

10 RibosomesJ. C. Lee 489

I. Introduction 489

II. Components of the yeast ribosome 490

III. Topography of yeast ribosomal components 516

IV. Functional sites of ribosomal subunits 529

V. Acknowledgements 534

References 534

Addendum 540

11 MitochondriaB. Guérin 541

I. Introduction 541

II. Ultrastructure 543

III. Isolation of mitochondria and mitochondrial membranes 545

IV. Lipids 548

V. Mitochondrial compartments 550

VI. Oxidative phosphorylation 557

VII. Different complexes in oxidative phosphorylation 566

VIII. Cyanide-insensitive alternative respiratory pathways 581

IX. Permeability properties of the inner membrane and transport systems 582

X. Concluding remarks 589

References 589

12 MicrobodiesM. Veenhuis and W. Harder 601

I. Introduction 601

II. General properties of yeast microbodies 604

III. Biogenesis of microbodies during vegetative reproduction of cells 606

IV. Substructure, volume fraction and composition of yeast microbodies in relation to environmental conditions 621

V. Biogenesis of microbodies during generative reproduction 633

VI. Selective inactivation of peroxisomal enzymes and degradation of peroxisomes 636

VII. Concluding remarks 649

VIII. Acknowledgements 649

References 650

13 Storage CarbohydratesA. D. Panek 655

I. Introduction 655

II. α,α-Trehalose 657

III. Glycogen 663

IV. Function of storage carbohydrates 669

V. Acknowledgements 675

References 675

Subject Index 679

Author Index 713

Abbreviations

For further details on current concepts in yeast taxonomy see Kreger-van Rij, N.J.W. ed. (1984), The Yeasts, a Taxonomic Study, Elsevier Biomedical Press, Amsterdam and Kreger-van Rij, N.J.W. (1987) in The Yeasts (A.H. Rose and J.S. Harrison, eds), 2nd edition, volume 1, pp. 5–61, Academic Press, London.

Genus   Abbreviation

Aciculoconidium   Ac.

Ambrosiozyma   A.

Arthroascus   Ar.

Brettanomyces   Br.

Bullera   B.

Candida   C.

Citeromyces   Cit.

Clavispora   Cl.

Cryptococcus   Cr.

Cyniclomyces   Cyn.

Debaryomyces   Deb.

Dekkera   D.

Fibulobasidium   Fib.

Filobasidiella   Fil.

Filobasidium   F.

Guilliermondella   G.

Hanseniaspora   H’spora

Hansenula   H.

Holtermannia   Holt.

Issatchenkia   I.

Kloeckera   Kl.

Kluyveromyces   K.

Leucosporidium   Leu.

Lipomyces   L.

Lodderomyces   Lod.

Malassezia   Mal.

Metschnikowia   M.

Nadsonia   N.

Nematospora   Nem.

Oosporidium   O.

Pachysolen   Pa.

Pachytichospora   P’spora

Phaffia   Ph.

Pichia   P.

Pityrosporum   Pit.

Rhodosporidium   Rhodosp.

Rhodotorula   Rh.

Saccharomyces   Sacch.

Saccharomycodes   S’codes

Saccharomycopsis   S.

Sarcinosporon   Sar.

Schizoblastosporion   Schizobl.

Schizosaccharomyces   Schiz.

Schwanniomyces   Schw.

Sirobasidium   Sir.

Sporidiobolus   Sporid.

Sporobolomyces   Sp.

Sporopachydermia   Sporop.

Stephanoascus   Steph.

Sterigmatomyces   St.

Sympodiomyces   Symp.

Torulaspora   T’spora

Torulopsis   T.

Tremella   Trem.

Trichosporon   Tr.

Trigonopsis   Trig.

Wickerhamia   W.

Wickerhamiella   Wick.

Wingea   Wi.

Zygosaccharomyces   Zygosacch.

Abbreviations used for the names of type-culture collections are:

A. T.C.C.   American Type Culture Collection, U.S.A.

C. B.S. C   entraalbureau voor Schimmelcultures, Yeast Division, Delft, The Netherlands

N.C.Y.C.   National Collection of Yeast Cultures (U.K.)

Abbreviations for chemical compounds are those recommended by the Biochemical Journal (1987, 241, 1–24). Enzymes are referred to by the trivial names recommended in Enzyme Nomenclature (1984), Academic Press, London and New York, supplemented by recommendations in the European Journal of Biochemistry, 1986, 157, 1–26. All temperatures quoted are in degrees Celsius.

1

Introduction

Anthony H. Rose*    * School of Biological Sciences, University of Bath, Bath BA2 7AY, Avon, UK

J. Stuart Harrison†    † Ashley House, Upper Frog Street, Tenby, Dyfed SA70 7JD, UK

References   5

The theme of this volume is the industrial application of the many biological properties of yeasts for practical purposes. Six of the 13 chapters are devoted to anaerobic fermentation processes employed for the manufacture of products based on ethanol, two deal with aerobic systems designed to provide yeast for baking and nutritional purposes and two review methods of detection and control of spoilage of foods and beverages. Two other chapters discuss recent developments in yeast research.

Since the volume on yeast technology in the first edition of The Yeasts was published in 1970, a whole portfolio of techniques has been developed, now usually referred to as recombinant DNA technology, and these have been brought to bear on yeasts, particularly Saccharomyces cerevisiae. As far as yeast research is concerned, a major advance was made when Fink and his colleagues (Hinnen et al., 1975) discovered that naked DNA molecules could be taken up by Sacch. cerevisiae strains after the wall had been removed. In other words, yeast cells or, more accurately, sphaeroplasts could be transformed as some bacteria had been known to be for many years. The discovery of yeast transformation was followed by development of techniques for chopping up DNA molecules in a controlled manner, using restriction endonucleases, with the result that specific domains of the yeast genome could be isolated, and used in transformation experiments. The techniques now widely used in recombinant DNA technology with yeasts are described in detail in the text edited by Guthrie and Fink (1991) and in volume 6 of this second edition of The Yeasts.

The majority of chapters in this volume review processes in which yeasts, often but not always Sacch. cerevisiae, are used to produce commercially valuable products, yeast biomass or products derived from yeast biomass. To what extent, then, have industries associated with these processes exploited the newly available techniques for inserting new information into the yeast genome? The answer to this question is Only minimally. Overall, the reason for this is that many yeast-based industries, particularly those which produce alcoholic beverages, are very traditional, reflecting perhaps the all too conservative attitude which human beings in general adopt when it comes to the nature of food and beverages which they consume. In addition, several of these industries, having been in operation for between several thousands and some hundreds of years, have discovered that, by essentially pragmatic means, their exploitation of yeast productivity has been maximized to an extent that cannot easily be extended. There are, however, exceptions, particularly where fresh demands are made on particular industrial processes.

Five of the chapters in the volume overview the role of yeasts in production of alcoholic beverages, both non-distilled and distilled. For very good reasons, conservatism in these industries remains paramount. Nevertheless, the brewing industry has faced a wide range of challenges in recent years, some of which have demanded properties of yeasts used to brew beers which are new to the industry. The move to high-gravity brewing revealed that, although yeast strains in use have some of the highest ethanol tolerances known among yeasts, these were still not sufficiently high for their use in brewing high-gravity beers. Although the biochemical basis of ethanol tolerance in Sacch. cerevisiae has to some extent been revealed, efforts to enhance tolerance in industrial strains of this yeast have met with little success (van Uden, 1989).However, rather more success has been realized in obtaining new strains of brewing yeast with the ability to utilize dextrins, a goal which was established following the commercial demand for beers with lower carbohydrate contents. Details of these developments in the brewing industry are given by Hammond in chapter 2.

The use of strains of Sacch. cerevisiae in production of wines and ciders remains little changed from that employed 20 years ago. One major development, however, is the wider use of active-dried yeast in both of these industries. Kunkee and Bisson review the role of yeasts in wine-making (chapter 3), and Beech in cider manufacture (chapter 5).

The classical Japanese procedure for producing saké is highly traditional, having been in operation unchanged for centuries, as described by Kodama (1970). However, the same author discusses in chapter 4 a number of recent innovations which, without altering the basic process, introduce the benefits of modern bioengineering technology and sophisticated use of recent genetic research on the use of killer yeasts to suppress wild contaminant strains. Introduction of accurate systems of temperature and humidity control and mechanical transfer of large quantities of materials are replacing older labour-intensive methods.

The use of yeast to produce distilled alcohol is now practised in two groups of industries. The first, in which potable distilled beverages are manufactured, is many centuries old. Its origins and developments have been recounted by Rose (1977), Simpson (1977), Lehtonen and Suomalainen (1977) and Lyons and Rose (1977). Recent years have seen limited developments in these industries particularly concerning the role of yeast. There has, however, been considerable progress in identifying flavour compounds, produced in part by yeasts, in these various distilled beverages. Watson (chapter 6) overviews the present status of these industries in so far as they employ yeasts. The second group of industries is much younger, and is concerned with the production of ethanol, by distillation of yeast-fermented substrates, for use principally as a fuel. Understandably, these industries were able to use the yeast strains and expertise acquired empirically when processes for making whisky, rum, gin and vodka were developed. According to Macher (1962), shortly after the construction in Germany of the world’s first beet-sugar factory in 1801, four Russian distilleries were operating, each of which produced 25 000 litres of spirit annually from fermented beet wort. The same author gives details of modern types of distillation and rectifying columns for manufacture of highly refined ethanol. This product has very many uses in medicine, perfumery and as a solvent, but since the advent of the internal combustion engine it has been possible to employ alcohol as an energy source to supplement or replace petroleum fractions. Ethanol was first sold on a commercial scale for this purpose in the 1930s, when it could be manufactured at economically acceptable cost from molasses and grain. Market values changed as petrol became relatively cheaper. More recently, however, interest has been revived, as discussed in chapter 7 by Ingledew. It is obvious from the evidence presented in this example of research into large-scale production of ethanol of a standard required for motor fuel and other industial purposes, at a cost competitive with chemical synthesis and alternative energy sources, that economic considerations outweigh all other matters in the viability of this project.

Consumption of beers and wines many years ago probably led to the realization that yeast, present as a sediment in these beverages, has a bitter but at the same time flavour-enhancing taste. Subsequently, this led to the production of various types of yeast extract to be used as a condiment with foods. Yeasts continue to be a source of other food and food-associated products, and in chapter 8 Lyons, Jaques and Dawson report developments on two recent exploitations of yeast properties. In the first, the yeast Phaffia rhodozyma is cultivated under conditions chosen to enhance production of astaxanthin, a red carotenoid pigment. Animal pigmentation experiments have shown that high levels of astaxanthin deposition can be achieved in trout, salmon, lobsters and chicken egg-yolk, provided the yeast cells fed are treated in such a way as to release the pigment. Feeding of such a product enhances, in particular, the commercial value of salmon and trout. A second process involves feeding a suitable yeast culture, along with a compatible yeast growth medium, to cattle and other animals. Evidence is presented which indicates that the digestive systems of various species are altered by the growing yeast in such a manner as to enhance conversion efficiency.

The exciting developments which have recently taken place in recombinant DNA technology have brought Sacch. cerevisiae even more prominently into the limelight. The reason for this is that this yeast is an extremely safe micro-organism in which to express foreign or heterologous DNA. When synthesized in Sacch. cerevisiae, it can safely be concluded that a product will not be contaminated with compounds that are in any way harmful to humans. After all, humans have for centuries been consuming Sacch. cerevisiae, albeit unwittingly, when drinking alcoholic beverages. In chapter 9, Hinchliffe and Kenny give examples of ways in which Sacch. cerevisiae has been used as a vehicle for the expression of heterologous genes.

Toxicological safety is also an important consideration in the centuries-old use of Sacch. cerevisiae to raise or leaven doughs in making bread. One of us (A.H.R.) together with Vijayalakshmi, in chapter 10, describe briefly the history of the use of yeast in bread-making. This process exploits the production of carbon dioxide during glycolysis of sugars by yeast, the ethanol that is produced at the same time being discarded up the chimney when the raised dough is baked. As emphasized in chapter 10, manufacture of baker’s yeast represents one of the great achievements in early biotechnology, living biomass with a virtually guaranteed fermentative activity being produced daily on a huge scale.

Mass-produced yeast biomass has also found other uses. It was discovered more than 200 years ago that the solid residue from beer fermentations, now known to consist largely of yeast, could be used both for the baking of bread and as feed for animals. For the latter purpose the simplest method of preparation, namely removal of excess wort by crude flteration, was employed. As demand increased, it became a practical proposition to grow food and fodder yeasts specifically for nutritional use. Large-scale plants were constructed for this purpose, particularly during war-time, to provide protein as a supplement to the human diet. In recent years, changing values have caused such production to become increasingly uneconomic, for the same reasons as already discussed in connection with manufacture of alcohol as a fuel. The rise and fall of the industry is reviewed by one of us (J.S.H.) in chapter 11. Since the late 1960s, many efforts have been made to grow food yeast, on a commercial scale, using hydrocarbon substrates. While this is technologically possible, such attempts have floundered on the same economic grounds, complicated by doubts based on medical evidence of possible health risks. Commercial production of micro-organisms other than yeast, for instance micro-algae, as a source of single-cell protein has been researched, but again economic viability appears unlikely (Benemann et al., 1979).

Only a very small number of the hundreds of recognized yeast species are pathogenic for humans and animals, and these are virtually never encountered in foods and beverages. Nevertheless, yeasts can occur as spoilage organisms in foods and beverages, where they are more of a nuisance than a worry, rendering the product unattractive more often than not. The involvement of yeasts as contributors to the spoilage of foods is reviewed by Tudor and Board in chapter 12. Although there are yeast species which can attack all types of food, the consequences are almost entirely restricted to cosmetic and nuisance factors that can be controlled relatively easily by good housekeeping and other simple means without health risks. However, the range of spoilage effects that have been investigated is extremely wide, so requiring a considerable breadth of biological knowledge and laboratory expertise on the part of those involved in the food industry. The very complete appendices give excellent information on the particular species of yeast associated with spoilage of various types of food.

The majority of alcoholic and non-alcoholic beverages have a low pH value, in the range 3.0–5.0, added to which they often contain appreciable concentrations of sugars. Both of these properties provide an ideal environment for spoilage yeasts, which are reviewed in chapter 13 by Thomas. Fortunately, in alcoholic beverages, the toxic effect of ethanol helps to combat spoilage yeasts, while in wines, ciders and many non-alcoholic beverages growth of spoilage yeasts is restricted by the presence of sulphite.

References

Benemann JR, Weissman JC, Oswald WJ. In: Rose AH, ed. London: Academic Press; 177–205. Economic Microbiology. 1979;4.

Guthrie C, Fink GR, eds. 1–933. Methods in Enzymology. 1991;194.

Hinnen A, Hicks JB, Fink GR. Proceedings of the National Academy of Sciences of the United States of America. 1975;75:1929.

Kodama K. In: Rose AH, Harrison JS, eds. 1st edn London: Academic Press; 223–282. The Yeasts. 1970;3.

Lehtonen M, Suomalainen H. In: Rose AH, ed. London: Academic Press; 595–633. Economic Microbiology. 1977;1.

Lyons TP, Rose AH. In: Rose AH, ed. London: Academic Press; 635–692. Economic Microbiology. 1977;1.

Macher L. In: Reiff F, Kautzmann R, Lüers H, Lindemann M, eds. Nürnberg: Hans Carl; 384–437. Die Hefen. 1962;2.

Rose AH. In: Rose AH, ed. London: Academic Press; 1–41. Economic Microbiology. 1977;1.

Simpson AC. In: Rose AH, ed. London: Academic Press; 537–593. Economic Microbiology. 1977;1.

van Uden N, ed. Alcohol Toxicity in Yeasts and Bacteria. Boca Raton: CRC Press; 1989.

2

Brewer’s Yeasts

John R.M. Hammond    BRF International, Lyttel Hall, Nutfield, Redhill, Surrey RH1 4HY, UK.

I. Introduction   8

II. Brewing yeast strains   9

A. Taxonomic position   9

B. Distinguishing between strains   11

III. Technology   14

A. Top and bottom yeasts   14

B. Flocculation   16

C. Yeast storage   18

C. Yeast propagation   19

E. Batch fermentation   20

F. High-gravity fermentation   22

G. Continuous fermentation   23

H. Immobilized yeast   24

IV. Nutrition   25

A. Fermentation and carbohydrate metabolism   25

B. Nitrogen metabolism   27

C. Oxygen and lipid metabolism   28

D. Wort solids and fermentation   30

E. Requirement for trace elements and vitamins   31

F. Alcohol tolerance   31

V. Yeast and beer quality   33

A. Fusel alcohols   34

B. Acetaldehyde   36

C. Vicinal diketones   36

D. Esters   40

E. Fatty acids   45

F. Organic acids   46

G. Sulphur compounds   46

H. Origin of beer flavour   48

VI. Fermentation in the future   49

A. Yeast quality   49

B. Process control   50

C. Genetic engineering   52

VII. Conclusions   56

VIII. Acknowledgements   56

References   56

I INTRODUCTION

Production of beer by fermentation of a liquid malt extract is one of the oldest biotechnologies known to humans. A record describing brewing and baking exists in a 5th Dynasty Egyptian tomb dating from about 2400 BC. Although yeasts were as essential for those ancient processes as they are today, there was no appreciation of their role until relatively recently. Not until 1876 did Pasteur demonstrate that fermentation required the participation of living organisms, and another 12 years passed before Hansen first isolated brewing yeasts and propagated them in pure culture. In the ensuing 100 years much has been learned about the behaviour of brewing fermentations and as a result there have been many changes to the brewing process.

The basic purpose of brewery fermentations still, however, remains the conversion of sweet, viscous wort into alcoholic, flavoursome beer. This is brought about by the activity of brewer’s yeast, which metabolizes wort sugars and converts them primarily into ethanol and carbon dioxide. It is the other by-products of metabolism which are of importance to beer flavour. Different yeasts produce these flavour compounds in various proportions and only those which produce an acceptable flavour balance are of use for beer fermentation. At the time of publication of the first edition of The Yeasts, the basic carbohydrate and nitrogen metabolism of brewer’s yeast was well understood, but the mechanisms underlying production of flavour compounds were far from clear (Rainbow, 1970). In the intervening years much has been learned of the fundamental biochemistry of the fermentation process but it would be foolish to assume that there is little more to understand. We are only just beginning to comprehend the complexity of the events occurring during the first hours of fermentation. Our knowledge of the mechanisms of beer-flavour production is still based on much empirical observation, and only with the greater insight that a detailed investigation of yeast intermediary metabolism will bring can precise control of beer flavour be achieved. However, as we shall see in the remainder of this chapter, we are now able to describe in considerable detail many aspects of brewery fermentations and this, coupled with the development of modern genetic methods, is beginning to make possible developments which could not have been forseen 20 years ago.

II BREWING YEAST STRAINS

Brewing yeasts all belong to the genus Saccharomyces, but they form a separate group similar to, but quite distinct from, the widely studied laboratory strains of this genus. These latter strains are a narrow group of organisms specially selected for their good genetic characteristics, which make them much more amenable to laboratory investigations. It has taken many years to develop specialist techniques for genetic analysis of brewing yeast strains, and only recently has much progress been made in this area. In the meantime, much work in brewery laboratories has been devoted to investigating such factors as yeast flocculation, the biochemistry of ester and higher-alcohol production and fermentation of maltose and maltotriose in mixed-sugar fermentations; none of these topics has been pursued in the same detail in academic laboratories. Such factors, whilst of critical importance in industrial fermentations, have little significance to the taxonomist. As a result, rather different approaches have been adopted for differentiating brewing yeasts from those more traditionally applied in yeast taxonomy. Before discussing these, however, it is appropriate to digress briefly to define the taxonomic position of brewer’s yeasts.

A Taxonomic position

Traditionally, brewers have distinguished two types of brewer’s yeasts, top-fermenting strains which are used for ale fermentations and bottom-fermenting strains which are used for lager fermentations. These distinctions are becoming less clear with the almost universal adoption of cylindroconical fermentation vessels which rely on yeast sedimentation for separation of the cells from fermented beer. As a result, much ale is now produced using bottom-fermenting yeasts. These are, however, not lager yeasts but ale yeasts with their own particular flavour characteristics which have been specially bred to sediment at the end of fermentation.

Notwithstanding technologically important factors such as production of sulphur flavours by lager yeasts and their ability to ferment brewer’s wort at lower temperatures than ale yeasts, the major taxonomic distinction between the two groups of yeasts is the inability of ale yeasts to ferment the disaccharide melibiose. This was the major distinguishing feature used to differentiate the two yeast types for many years. Melibiase-negative ale yeasts have always been classified as Saccharomyces cerevisiae, but melibiase-positive lager yeasts have found themselves moved from species to species over the years. Initially named Saccharomyces carlsbergensis after the brewery where they were first isolated, they were subsequently transferred to the species Saccharomyces uvarum (Lodder, 1970). The distinction from melibiase-negative Sacch. cerevisiae was retained. However, a clear difference between laboratory and brewing strains of Sacch. uvarum was apparent in the higher maximum growth temperature of the former (Walsh and Martin, 1977). In more recent years, differentiation of species based on sugar fermentations alone has been abandoned, and the 41 Saccharomyces spp. described by Lodder (1970) have now been reduced to only seven (Kreger-van Rij, 1984). As a result, all brewer’s yeasts have been consolidated into the one species Sacch. cerevisiae. This has not met with the unanimous approval of brewing scientists since many feel that the differences between ale and lager yeasts are too great for them all to be considered one species. Recent detailed genetic analysis of the Carlsberg lager-production yeast M244 lends some weight to this argument.

Genetic analysis of brewer’s yeasts has proved extremely difficult because they are polyploid (or aneuploid) and sporulate poorly (Lewis et al., 1976; Russell and Stewart, 1979; Aigle et al., 1984) under conditions where laboratory strains sporulate freely. The sporulating ability of brewing strains has been improved by growing cells under complete catabolite derepression (Bilinski et al., 1986) and by inducing sporulation at lower temperatures (Gjermansen and Sigsgaard, 1981) but tetrad analysis is still far from simple. To overcome these problems with the lager strain M244, a technique was used whereby single chromosomes from the lager yeast were transferred into a well-characterized haploid yeast strain (Nilsson-Tillgren et al., 1980). Once in this defined genetic environment, the structure of the transferred chromosomes could be examined in considerable depth. Chromosomes III, V, X, XII and XIII have been studied in this way using both recombination and molecular-genetic techniques (Nilsson-Tillgren et al., 1980, 1981; Wettstein et al., 1984; Pedersen, 1986b; Casey, 1986a,b). The results demonstrate that the lager yeast M244 is probably a species hybrid having at least two sets of chromosomes. One set is closely homologous to the equivalent chromosomes found in Sacch. cerevisiae; the other set, whilst carrying the same gene activities, is largely unable to recombine with the equivalent Sacch. cerevisiae chromosomes and is termed homeologous. The structural differences between the two sets of chromosomes have been confirmed by restriction mapping and sequencing studies which have demonstrated significantly different nucleotide sequences (Holmberg, 1982; Casey, 1986a,b). In addition, a LEU2 gene from M244 has been cloned and shown to be different from the Sacch. cerevisiae LEU2 gene by restriction mapping. This cloned gene hybridizes preferentially with one of the two LEU2 alleles from lager yeasts (Casey and Pederson, 1988). Chromosome X from M244 has also been examined using pulsed-field electrophoresis, and three independently migrating structures have been identified, two of which are not homologous to chromosome X of Sacch. cerevisiae (Casey, 1986b). Such a complex chromosomal structure would complicate meiosis, and may help to explain the low sporulation frequency and low spore vialibility found in brewer’s yeasts.

B Distinguishing between strains

Despite the controversy surrounding the taxonomic position of ale and lager yeasts, they can be readily distinguished by the ability of the latter to utilize melibiose, a task made much simpler by the introduction of a colorimetric test based on the hydrolysis of 5-bromo-4-chloro-3-indolyl-α-D-galactoside. On media containing this indicator, melibiase-producing lager yeasts grow as blue colonies whereas ale yeast colonies remain uncoloured (Tubb and Liljeström, 1986). To the brewer, distinguishing between different yeast strains is of much more importance. This has always been so but, with the growing practices of brewing under licence and the use of several different strains in one plant, its importance has grown.

The achievement of consistent and distinct product quality has long been recognized to depend on the use of the correct yeast strain. Many tests have been devised, based on a wide range of parameters, for separating different strains of brewer’s yeasts. The most fundamental involve mimicking, on the laboratory scale, the behaviour of yeast in a brewery. The majority of the brewer’s yeasts in the UK National Collection of Yeast Cultures have been assessed for their fermentation performance in 1.5 litre tall tube fermenters. They have been classified according to their head formation, deposit formation, the degree and rate of attenuation (the change in specific gravity brought about by fermentation) and the clarity of the beer produced (Walkey and Kirsop, 1969). These data have been regularly updated over the years and more recently have been re-evaluated using numerical taxonomic techniques (Bryant and Cowan, 1979). Principal co-ordinate analysis separated the yeasts into five major groups based largely on head and deposit formation. Good head-forming yeasts, ideal for use in traditional ale breweries, were clearly separated from bottom-fermenting yeasts suitable for use in modern cylindroconical fermenters. Over the years, laboratory fermentation tests have been extended to include flavour analysis, either by means of tasting panels (Thorne, 1975) or by gas-liquid chromatography (Ferguson et al., 1972). It is generally concluded that such tests can provide valuable information, especially in the early stages of testing new yeast strains, but are too cumbersome for routine differentiation of brewing strains in a quality-control laboratory.

A number of simpler tests have been devised for the assessment of yeast-flocculation properties and several are employed routinely. Burns (1941) developed a simple test involving assessment of the volume of sedimented cells in pH 4.6 acetate buffer. An alternative method was described by Gilliland (1951) which involved visual assessment of sedimentation in small bottles of fermented wort. Four classes of flocculation could be distinguished and, by assessing 50 single colonies from the original yeast sample, an estimate could be obtained of the degree of variability of the yeast. This test was extended by Hough (1957) to include a number of other measurements, so increasing the degree of differentiation possible. The additional tests involved estimating chain formation, head formation, aggregation by ethanol, aggregation by changes in pH value and dispersal of aggregates by maltose. Over the years, many variations of these flocculation tests have appeared (for reviews, see Stewart and Russell, 1981a; Calleja, 1987) and, despite the time required for completion of the tests, they are still very popular for differentiating brewer’s yeast strains because of their relative simplicity.

Another slow but relatively straightforward procedure is the giant-colony test (Hall, 1954; Richards, 1967). Here, about 10 yeast colonies are allowed to grow for four weeks at 18 °C on nutrient-medium–gelatine plates. The morphology of the colonies is characteristic of the yeast strain although there is apparently no relationship between morphology and fermentation performance (Richards, 1967).

The traditional carbon assimilation and antibiotic sensitivity tests can be applied to the task of distinguishing strains of brewer’s yeasts. Several authors (Wiles, 1953; Kirsop, B.E., 1974a; Quain, 1986) have demonstrated the variable growth responses of strains of brewer’s yeast to a number of carbon compounds. These included melezitose, α-methyl-D-glucoside, lactic acid, glycerol, ethanol, succinic acid, mannitol, sorbitol and trehalose. By careful selection of the appropriate compounds, differential tests for a number of yeasts used in one plant can be devised. Similarly, it is possible to differentiate brewing yeasts by means of their variable sensitivity to the protein-synthesis inhibitor cycloheximide (Harris and Watson, 1968; Lawrence, 1983). This principle has been extended recently with the availability of a commercial disc-diffusion system for identification of clinically important yeasts based on six antimicrobial compounds including cycloheximide. It has been successfully used to distinguish some strains of brewer’s yeasts and to identify the presence of contaminant yeasts (Simpson et al., 1992).

Another physiological parameter which is known to vary among yeast strains is the amount of oxygen required for successful fementation (Kirsop, B. H., 1974; Jakobsen and Thorne, 1980). In addition, it has also been shown that different yeast strains have different specific oxygen-uptake rates (Daoud and Searle, 1986). These variations in oxygen requirement may permit separation of yeasts which otherwise behave similarly, although care is required to ensure that the yeasts are in a similar physiological state before the test is carried out if erroneous results are to be avoided.

Serological methods have not been widely used for differentiation of brewer’s yeasts, although they can be used to distinguish lager from ale yeasts (Campbell and Brudzynski, 1966). Among the difficulties encountered in using immunofluorescence techniques for yeast differentiation have been variations in surface antigens with physiological state of the yeast (Cowland, 1968) and changes observed on reuse of yeasts for successive brewery fermentations (Hammond and Jones, 1979). Some success has been achieved with micro-immunoelectrophoresis (Cowan and Bryant, 1981); when the electrophoretic patterns obtained were compared with fermentation properties determined in laboratory fermenters, significant interrelationships were detected between cell-surface antigens, yeast-head formation and sedimentation properties.

There has recently been a resurgence of interest in distinguishing brewing-yeast strains from one another, and a number of new analytical techniques are now being employed. The genome of brewing yeasts has been extensively studied by means of restriction-endonuclease digestion of either total DNA or mitochondrial DNA, separation of the fragments by agarose electrophoresis and then either direct examination of the banding patterns or examination of the patterns produced by hybridizing specific radioactively labelled probes to the DNA fragments. Direct examination of mitochondrial DNA can differentiate ale yeasts from lager yeasts (Lee et al., 1985; Martens et al., 1985) but the technique is unable to distinguish between strains of lager yeasts (Aigle et al., 1984; Martens et al., 1985). Direct examination of total DNA has also been used to differentiate lager and ale yeasts but, in this study, an unusual restriction enzyme was employed (Panchal et al., 1987). Reacting total DNA with a number of specific probes (RDN1, HIS4, LEU2, Ty1) has demonstrated marked differences between ale and lager yeasts with respect to genome structure (Pedersen, 1983a,b, 1985a,b, 1986a; Decock and Iserentant, 1985; Martens et al., 1985). The results obtained with lager yeasts are so homogeneous as to suggest that they are all very closely related, whereas the ale strains showed large variability, typically in their LEU2 and Ty1 banding patterns (Pedersen, 1985b). Despite similarities among lager yeasts, it has proved possible to distinguish some strains by means of variable responses to Ty1 probes. Different banding patterns are obtained from different lager strains with some Ty1 probes (Decock and Iserentant, 1985; Martens et al., 1985; Pedersen, 1985a) but not with another Ty1 probe (Aigle et al., 1984; Decock and Iserentant, 1985). Thus, with care, it seems that restriction-endonuclease fragment pattern polymorphisms can be used to distinguish different brewer’s yeast strains. The application of this approach to brewer’s yeasts has been recently reviewed (Meaden, 1990).

Another technique which has been used to investigate the genome structure of brewer’s yeasts is that of pulsed-field electrophoresis. Using this and other closely related methods it is possible to separate individual chromosomes which can then be hybridized with specific labelled probes in exactly the same way as for restriction enzyme-digested DNA. In the strains investigated to date using this technique, considerable variations in chromosomal profiles have been observed (Pedersen, 1987; Rank and Casey, 1988; Casey et al., 1988b, 1990) and it seems that this method may provide another useful way of fingerprinting industrial yeasts.

The techniques of genetic engineering may also provide a means of introducing unique selectable markers into individual yeast strains so that the strains can be distinguished by simple laboratory tests. The markers must be easily testable and yet have no adverse effects on fermentation properties of the yeasts. Suitable markers would be those for toxin resistance (e.g. heavy metals or antibiotics) or for ability to grow on a novel substrate such as lactose.

A number of other techniques have also been investigated for their ability to distinguish different yeast strains: Pyrolysis–gas chromatography, which involves controlled thermal degradation of micro-organisms followed by gas chromatography, has been successfully applied to differentiating yeast strains (Jones, 1984), as has the closely related technique of pyrolysis–mass spectroscopy (Quain, 1988), but both methods require complex and expensive equipment and will be difficult to justify in brewery laboratories if other uses cannot be found for them. Gas chromatography of long-chain fatty-acid methyl esters has been proposed as a way of distinguishing different yeast strains but, so far, this has not proved successful with strains of Sacch. cerevisiae (Oosthuizen et al., 1987). Similarly, separation of total soluble cell proteins on polyacrylamide gels followed by computer analysis of the banding patterns has demonstrated its ability to distinguish strains of yeast (Van Vuuren and Van der Meer, 1988; Dowhanick et al., 1990).

Thus, for the first time, the feasibility of distinguishing closely related brewer’s yeasts by means of a single test has been demonstrated. Further effort will, however, be required before such tests acquire the simplicity needed to supplant the flocculation and growth-based tests currently used in brewery laboratories.

III TECHNOLOGY

A Top and bottom yeasts

Until the middle of the 19th century, bottom-cropping yeasts were used only in Bavaria. At this time they began to spread to other countries, first to Czechoslovakia and Scandinavia, then to North America, until nowadays bottom fermentation is practised all over the world. In contrast, the once ubiquitous top-cropping ale yeasts are now found mainly in the UK and some of her former colonies, although they are used to a limited extent in Belgium, Germany and North America. The tendency of ale yeasts to rise to the surface of fermentations led to their use in small open fermenters from which the yeast could be skimmed. In addition, specialist fermenters, such as the Burton Union or Yorkshire stone-square systems, were developed (for a description of these fermenter types, see MacDonald et al., 1984). In contrast, lager fermenters are rather different since the yeast can be removed from the base of the fermenter once it has settled. Nowadays, much ale is produced using bottom-cropping yeasts, and the distinction between top-and bottom-cropping yeasts is beginning to disappear.

Whilst it is true that flocculation must occur before flocs settle or rise to the surface of the beer (Calleja, 1987), the final destination of the yeast seems to depend upon both the properties of the surface of the yeast and the nature of the fermentation medium. The presence of hop components in fermenting worts stimulates formation of a yeast head (Dixon, 1967). In fact, most of the hop-derived materials which are lost from wort during fermentation are found in the surface foam whether top- or bottom-fermenting yeasts are used. The presence of yeasts in the head of a fermenter increases the stability of this foam (Dixon and Kirsop, 1969). Different yeasts show a varying tendency to adhere to carbon dioxide bubbles, and so to be drawn up into the foam, and this is affected by the presence of hop components in the wort, more yeasts being found in suspension in unhopped wort than in hopped wort (Dixon, 1967).

One striking difference between top- and bottom-fermenting yeasts is that the former have a hydrophobic surface whereas bottom fermenters tend to be hydrophilic (Hinchliffe et al., 1985; Amory et al., 1988; Amory and Rouxhet, 1988). Hydrophobic yeasts adhere strongly to rising carbon dioxide bubbles, and are carried to the surface of the fermenting wort; only if they are very flocculent do they drop back into the fermenter. Proteolyticenzyme treatment of hydrophobic yeasts makes them hydrophilic and such yeasts fail to form a head (Hinchliffe et al., 1985). The hydrophobicity of bottom-cropping yeasts apparently increases under flocculating conditions, although this is not true of hydrophobic top-cropping yeasts (Kamada and Murata, 1984). In addition to hydrophobicity differences, it seems that bottom-cropping yeasts have a greater surface charge than top fermenters (Amory et al., 1988; Amory and Rouxhet, 1988), due mainly to a higher surface concentration of potassium, ammonium and phosphate ions (Amory and Rouxhet, 1988). It is clear that flocculation and cropping behaviour affect each other, but this may simply be a reflection of flocculation having to occur if rapid separation of cells from wort is to be achieved (Kirsop, B. H., 1971). The apparent independence of flocculation and cropping behaviour is further supported by the discovery that both ale and lager yeasts can be found within Gilliland’s flocculation class II (Gilliland, 1951; Windisch, 1968).

B Flocculation

Flocculation is critical for successful beer fermentation since it permits separation of yeast from beer once attenuation is complete. The topic has been reviewed in detail in an earlier volume in this series (Calleja, 1987), and so comments in this section will be confined to a few general remarks and a consideration of some recent findings.

In brewing, flocculation involves aggregation of dispersed cells into flocs, and occurs towards the end of fermentation. It is characteristic of stationary-phase or late experimental-phase cells of some strains of yeast (Mill, 1964). Both the genotype of cells and fermentation conditions affect flocculation. Aggregation by some brewer’s yeasts is clearly a cell wall-mediated phenomenon; sphaeroplasts fail to flocculate whereas heat-killed cells obtained from flocs reflocculate if mixed together (Mill, 1964), as do isolated wall fragments (Eddy, 1955b). Despite much, often contradictory, analytical data, no clear correlation has been established between flocculation and the phosphate content of yeast walls (Griffin and MacWilliam, 1969; Lyons and Hough, 1971) or the antigenic structure (Campbell et al., 1968) of yeasts. This has been largely due to comparisons being made between unrelated yeast strains which might be expected to show analytical differences anyway. What is clear is that the cell-surface protein phosphomannan is intimately involved in flocculation. Proteinase treatment releases large amounts of wall protein and phosphomannan, and causes flocculent cells to become permanently non-flocculent (Lyons and Hough, 1971; Nishihara et al., 1977).

The ability of yeast cells to flocculate is under metabolic control; energy generation and protein synthesis are required for flocculation (Baker and Kirsop, 1972) and flocs can be dispersed by sugars, notably mannose (Taylor and Orton, 1978), although glucose and maltose are also effective (Eddy, 1955a; Mill, 1964). It is generally agreed that calcium ions promote flocculation (Taylor and Orton, 1973, 1975; Stratford, 1989b) and that magnesium or manganese ions may act as substitutes (Stewart and Goring, 1976; Miki et al., 1982b). The role played by calcium ions in flocculation has been very controversial. Harris (1959) first suggested the idea of calcium-salt bridging between anionic groups in the wall. These anions could be either phosphate groups in the phosphomannan or carboxyl groups in proteins. The effects of phosphate removal from the wall using hydrofluoric acid (Jayatissa and Rose, 1976) and pH–electrophoretic-mobility measurements (Beavan et al., 1979) suggest that carboxyl groups are the more important, although recent investigations have concluded that the density of phosphate groups on the yeast surface is closely related to flocculation ability (Amory et al., 1988; Amory and Rouxhet, 1988). An alternative role for calcium ions was put forward by Taylor and Orton (1973). Instead of charge neutralization or salt-bridge formation, they suggested that calcium ions could be changing the conformation of a surface protein into an active binding form. Involvement of proteins in flocculation is clear since proteolytic enzymes (Nishihara et al., 1977), protein-denaturing agents (Kamada and Murata, 1984) and various protein-modification procedures (Nishihara et al., 1977) all cause loss of flocculence. Interactions between protein, phosphomannan and calcium ions have been investigated by Miki et al. (1982a,b), who proposed that mannoproteins of flocculent yeasts act in a lectin-like manner to crosslink cells, with calcium ions acting as ligands and promoting flocculation by causing conformational changes. Lectin-like interactions are further supported by the findings of Fujino and Yoshida (1976) that an acid polysaccharide present in wort and derived from malt can cause premature flocculation. They suggested that lectins on the surface of the yeast cell interact with the polysaccharide to induce flocculation. The involvement of protein–mannan interactions in coflocculation between different cells has also been demonstrated (Nishihara and Toraya, 1987).

Despite the large number of methods for measuring flocculation (see Stewart and Russell, 1981a; Calleja, 1987) which have been described, several workers have recently reinvestigated the methodologies involved. In particular, they have pointed out the need for careful control of culture conditions, agitation and cell density during measurement of flocculation (Stratford and Keenan, 1987, 1988; Amory et al., 1988; Kihn et al., 1988b). Flocculent cells have an absolute requirement for mechanical-energy input if flocculation is to occur. In shaken cultures, there is a minimum shaking speed which will induce flocculation and, thereafter, the initial rate of flocculation increases exponentially with shaking speed. It seems that an activation energy is required to overcome the repulsion between charged yeast cells. If this charge is decreased by lowering the pH value, the energy required to induce flocculation is reduced (Stratford and Keenan, 1987). It appears that quantitative measurement of flocculation requires an estimation of four parameters; these are the minimum agitation threshold below which cells will not form flocs, the initial rate of flocculation, the extent of flocculation and the size of flocs formed (Stratford and Keenan, 1988; Stratford et al., 1988). These observations clearly affect the interpretation of any results obtained using methods involving non-specific shaking or mixing. The universal involvement of calcium ions in brewer’s yeast flocculation has also been questioned (Amory et al., 1988; Kihn et al., 1988a). The former group observed that top-fermenting yeasts flocculate in the presence of ethanol and calcium ions, or ethanol alone, but not in the presence of calcium ions alone. In contrast, bottom-fermenting yeasts flocculate in the presence of ethanol and calcium ions, or calcium ions alone, but not in the presence of ethanol alone.

Clearly, flocculation of brewer’s yeast is a complex phenomenon and requires further investigation; in particular, the genetics of flocculation is far from clear (see Stewart and Russell, 1981a; Johnston and Reader, 1983; Calleja, 1987). Two distinct types of flocculation gene appear to be present in brewer’s yeasts, a FLO1 type in lager yeasts, involving a mannose-specific lectin, and NewFLO in ale yeasts, involving a broad-specificity lectin (Stratford, 1989a). A detailed review of yeast flocculation with particular emphasis on genetic aspects has recently been published (Stratford, 1992).

Brewer’s yeasts are relatively unstable with respect to flocculation character and, depending upon the type of collection system used, can gradually become less (Chester, 1963a) or more flocculent (Gyllang and Martinson, 1971). This, of course, has serious implications for the brewer, and is one of the reasons why flocculation characteristics are so carefully monitored.

C Yeast storage

Beers of consistent quality can only be produced if the fermentations used in their manufacture proceed in a controlled and reproducible manner. Good-quality yeasts of known fermentation characteristics are required. Larger brewing companies ensure their yeast supply by keeping pure stock cultures within their laboratories which are periodically used to provide a seed culture for a brewery-yeast propagation system (see Section III.D, p. 19). Smaller companies often rely on culture collections, such as the National Collection of Yeast Cultures in the UK, for storage of their yeasts. A number of techniques are employed, including regular subculturing on growth media followed by storage at 4 °C, lyophilization or storage under liquid nitrogen. In general, it has been found that yeast variation is often encountered with subculturing (Kirsop, B.E., 1974b), which is, in addition, very labour intensive. Lyophilization, on the other hand, can result in low viabilities although stability is much improved (Richards, 1975). The survival rate is very dependent upon the medium used to suspend the yeasts prior to freeze drying (Hall and Webb, 1975). The method of choice, where available, involves storage of cultures in liquid nitrogen. Survival rates are very high and strain variation is almost non-existent (Russell and Stewart, 1981).

Brewer’s yeasts are reused several times before being discarded in favour of a newly propagated population derived from laboratory stocks. In some breweries, the yeast may be used for many years before being replaced. Between fermentations, storage conditions play a vital role in maintaining the fermentative activity of the yeast which is to be reused. In most breweries, yeast is stored static under beer or water at low temperatures (Kirsop, B.H., 1974; Quain and Tubb, 1982) although agitation during storage is often used to increase yeast homogeneity (Gilliland, 1981). Under these conditions, yeast cells need to maintain their activity in an environment which contains only limited amounts of nutrients and relatively large quantities of potentially toxic ethanol. The internal storage carbohydrate glycogen appears to be important for survival of yeasts during storage, where it is used to maintain essential cellular metabolic functions (Quain and Tubb, 1982). The suggestion has been put forward that aeration of yeast during storage could be used as a way of avoiding the need for wort oxygenation (Jakobsen and Thorne, 1980). However, it appears that under these conditions stored glycogen is rapidly depleted with consequent effects on cell viability (Quain and Tubb, 1982; McCaig and Bendiak, 1985a). Moreover, sufficient lipids and sterols are not synthesized by the yeast to permit the pitching of worts without further oxygenation (Murray et al., 1984; McCaig and Bendiak, 1985a). The temperature of storage is also important. Autolysis begins at moderate temperatures (Quain and Tubb, 1982) whilst at high temperatures the yeast cells rapidly undergo autofermentation to produce ethanol and carbon dioxide (Gilliland, 1969). Temperatures above 10 °C are unsuitable for yeast