The Yeasts: Yeast Technology
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* 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.
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The Yeasts - Academic Press
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