Brewing Yeast
Fermentation Performance
Brewing Yeast
Fermentation Performance
Second edition
Edited by
KATHERINE SMART
Oxford Brookes University
Oxford, UK
© Blackwell Science 2003
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Contributors
A. Aitchison
Scottish Courage Brewing Ltd, Technical Centre, 160 Canongate, Edinburgh
EH8 8DD, UK
P. Attfield
Centre for Fluorimetric Applications in Biotechnology, Department of Biological
Sciences, Macquarie University, North Ryde, Sydney, NSW 2109, Australia
B. Axcell

The South African Breweries Ltd, Corporate Technical Centre, PO Box 782178,
Sandton 2146, South Africa
C.W. Bamforth
Department of Food Science & Technology, University of California, Davis,
CA 95616-8598, USA
F.F. Bauer
Department of Microbiology and Institute for Wine Biotechnology, University of
Stellenbosch, Stellenbosch 7600, South Africa
H. Berg
Oy Sinebrychoff Ab, PO Box 87, FI-04201 Kerava, Finland
K. Berghof
BIOTECON Diagnostics GmbH, Hermannswerder Haus 17, 14473 Potsdam,
Germany
C. Boulton
Bass Brewers Ltd, Technical Centre, PO Box 12, Cross Street, Burton upon Trent
DE14 1XH, UK
W. Box
Bass Brewers Ltd, Technical Centre, PO Box 12, Cross Street, Burton upon Trent
DE14 1XH, UK
A. Boyd
Centre for Fluorimetric Applications in Biotechnology, Department of Biological
Sciences, Macquarie University, North Ryde, Sydney, NSW 2109, Australia
P. Chambers
School of Food Science and Technology, Victoria University, Werribee Campus,
PO Box 14428, Melbourne City, Victoria 8001, Australia
M. Chandler
School of Food Science and Technology, Victoria University, Werribee Campus,
PO Box 14428, Melbourne City, Victoria 8001, Australia
S. Collin
Université Catholique de Louvain, Unité de Brasserie et des Industries Alimentaires,

Croix du Sud 2/7, B-1348 Louvain-la-Neuve, Belgium
F.R. Dalvaux
Centre for Malting and Brewing Science, Faculty of Agricultural and Applied
Biological Sciences, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22,
3001 Heverlee, Belgium
I. Dawes
School of Biochemistry and Molecular Genetics, University of New South Wales,
Sydney, NSW 2052, Australia
A. Debourg
Department of Brewing Sciences and Fermentation Technologies, Institut Meurice,
1 Avenue E. Gryson, B-1070 Brussels, Belgium
F.R. Delvaux
Centre for Malting and Brewing Science, Faculty of Agricultural and Applied
Biological Sciences, Kathalieke Universiteit Leuven, Kasteelpark Arenberg 22,
3001 Heverlee, Belgium
G. Derdelinckx
Centre for Malting and Brewing Science, Faculty of Agricultural and Applied
Biological Sciences, Kathalieke Universiteit Leuven, Kasteelpark Arenberg 22,
3001 Heverlee, Belgium
J. R. Dickinson
Cardiff School of Biosciences, Cardiff University, PO Box 915, Cardiff CF10 3TL, UK
M. Dillemans
Department of Brewing Sciences and Fermentation Technologies, Institut Meurice,
1 Avenue E. Gryson, B-1070 Brussels, Belgium
J P. Dufour
Department of Food Science, University of Otago, PO Box 56, Dunedin, New
Zealand
M. Fandke
BIOTECON Diagnostics GmbH, Hermannswerder Haus 17, 14473 Potsdam,
Germany

L. Gijs
Université Catholique de Louvain, Unité de Brasserie et des Industries Alimentaires,
Croix du Sud 2/7, B-1348 Louvain-la-Neuve, Belgium
X. Green
Scottish Courage Brewing Ltd, Technical Centre, 160 Canongate, Edinburgh
EH8 8DD, UK
vi CONTRIBUTORS
S. Gualdoni
School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy
Lane, Oxford OX3 0BP, UK
T. Gunasekera
Centre for Fluorimetric Applications in Biotechnology, Department of Biological
Sciences, Macquarie University, North Ryde, Sydney, NSW 2109, Australia
V. Higgins
School of Biochemistry and Molecular Genetics, University of New South Wales,
Sydney, NSW 2052, Australia
J.A. Hodgson
Scottish Courage Brewing Ltd, Technical Centre, Sugarhouse Close, 160 Canongate,
Edinburgh EH8 8DD, UK
G.A. Hulse
The South African Breweries, Beer Division, Brewing Research & Development
Department, PO Box 782178, Sandton 2146, South Africa
K.J. Hutter
Eichbaum Brauereien AG, Käfertaler Straße170, D-68169 Mannheim, Germany
C.L. Jenkins
School of Biological and Molecular Sciences, Oxford Brookes University, Headington,
Oxford OX3 0BP, UK
A.I. Kennedy
Scottish Courage Brewing Ltd, Technical Centre, Sugarhouse Close, 160 Canongate,
Edinburgh EH8 8DD, UK

M. Kiehne
BIOTECON Diagnostics GmbH, Hermannswerder Haus 17, 14473 Potsdam,
Germany
O. Kobayashi
Kirin Brewery Co., Ltd., Central Laboratories for Key Technology, 1-13-5, Fukuura,
Kanazawa-ku, Yokohama-shi, Kanagawa 236-0004, Japan
C. Lange
Eichbaum Brauereien AG, Käfertaler Straße170, D-68169 Mannheim, Germany
A. Lentini
Carlton and United Breweries Ltd/Foster’s Group Ltd, 4-6 Southampton Crescent,
Abbotsford, Victoria 3067, Australia
P. Malcorps
Interbrew, Vaarstraat 94, B-3000 Leuven, Belgium
V. Martin
School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy
Lane Campus, Headington, Oxford OX3 0BP, UK
CONTRIBUTORS vii
D.L. Maskell
School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy
Lane, Headington, Oxford OX3 0BP, UK
N. Moonjai
Centre for Malting and Brewing Science, Faculty of Agricultural and Applied
Biological Sciences, Katholuke Universiteit Leuven, Kasteelpark Arenberg 22,
3001 Heverlee, Belgium
E. Pajunen
Oy Sinebrychoff Ab, PO Box 87, FI-04201 Kerava, Finland
A. Pardigol
BIOTECON Diagnostics GmbH, Hermannswerder Haus 17, 14473 Potsdam,
Germany
P. Perpète

Université Catholique de Louvain, Unité de Brasserie et des Industries Alimentaires,
Croix du Sud 2/7, B-1348 Louvain-la-Neuve, Belgium
C.D. Powell
School of Biological and Molecular Sciences, Oxford Brookes University,
Headington, Oxford OX3 0BP, UK
I.S. Pretorius
Department of Microbiology and Institute for Wine Biotechnology, University of
Stellenbosch, Stellenbosch 7600, South Africa
D.E. Quain
Bass Brewers, Technical Centre, PO Box 12, Cross Street, Burton-upon-Trent
DE14 1XH, UK
B. Ranta
Oy Sinebrychoff Ab, PO Box 87, FI-04201 Kerava, Finland
K.E. Richardson
White Labs, Inc., 7564 Trade Street, San Diego, CA 92121, USA
P. Rogers
Carlton and United Breweries Ltd/Foster’s Group Ltd, 4-6 Southampton Crescent,
Abbotsford, Victoria 3067, Australia
A.J. Schiewe
White Labs, Inc., 7564 Trade Street, San Diego, CA 92121, USA
P. Silcock
Department of Food Science, University of Otago, PO Box 56, Dunedin, New
Zealand
O. Simal
School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy
Lane, Oxford OX3 0BP, UK
viii CONTRIBUTORS
K. Simic
Kent Brewery, Carlton and United Breweries, Broadway, Sydney, NSW 2001, Australia
K.A. Smart

School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane
Campus, Headington, Oxford OX3 0BP, UK
P. Soininen-Tengvall
Oy Sinebrychoff Ab, PO Box 87, FI-04201 Kerava, Finland
R.A. Stafford
The South African Breweries Ltd, Engineering Development, Corporate Technical
Centre, PO Box 782178, Sandton, 2146, South Africa
G. Stanley
School of Food Science and Technology, Victoria University Werribee Campus,
P.O. Box 14428 Melbourne City, Victoria 8001, Australia
B. Taidi
Scottish Courage Brewing Ltd, Technical Centre, 160 Canongate, Edinburgh EH8
8DD, UK
K. Tanaka
Kirin Brewery Co., Ltd., Central Laboratories for Key Technology, 1-13-5, Fukuura,
Kanazawa-ku, Yokohama-shi, Kanagawa 236-0004, Japan
K. Tapani
Oy Sinebrychoff Ab, PO Box 87, FI-04201 Kerava, Finland
A. Tauschmann
BIOTECON Diagnostics GmbH, Hermannswerder Haus 17, 14473 Potsdam,
Germany
J.M. Thevelein
Laboratory of Molecular Cell Biology, Department of Biology, KU Leuven,
Kasteelpark Arenberg 31, B-3001 Leuven (Heverlee), Belgium
P. Thurston
Scottish Courage Brewing Ltd, Berkshire Brewery, Imperial Way, Reading
RG2 0PN, UK
L. Van Nedervelde
Department of Brewing Sciences and Fermentation Technologies, Institut Meurice,
1 Avenue E. Gryson, B-1070 Brussels, Belgium

S.M. Van Zandycke
SMART Brewing Services, Oxford Brookes Enterprises, School of Biological and
Molecular Sciences, Gipsy Lane, Oxford OX3 0BP, UK
D. Veal
Centre for Fluorimetric Applications in Biotechnology, Department of Biological
Sciences, Macquarie University, North Ryde, Sydney, NSW 2109, Australia
CONTRIBUTORS ix
H. Verachtert
Centre for Malting and Brewing Science, Faculty of Agricultural and Applied
Biological Sciences, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22,
3001 Heverlee, Belgium
K.J. Verstrepen
Centre for Malting and Brewing Science, Faculty of Agricultural and Applied
Biological Sciences, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22,
3001 Heverlee, Belgium
S. Vincent
Kent Brewery, Carlton and United Breweries, Broadway, Sydney, NSW 2001,
Australia
C.E. White
White Labs, Inc., 7564 Trade Street, San Diego, CA 92121, USA
L.R. White
White Labs, Inc., 7564 Trade Street, San Diego, CA 92121, USA
P.A. White
School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy
Lane Campus, Headington, Oxford OX3 0BP, UK
J. Winderickx
Laboratory of Molecular Cell Biology, Department of Biology, KU Leuven,
Kasteelpark Arenberg 31, B-3001 Leuven (Heverlee), Belgium
x CONTRIBUTORS
Contents

Contributors v
Preface to the second edition xxv
Katherine A. Smart
Part 1 Molecular Innovations 1
1 Analysis of karyotypic polymorphisms in a bottom-fermenting yeast
strain by polymerase chain reaction 3
K. Tanaka and O. Kobayashi
1.1 Introduction 3
1.2 Materials and methods 4
1.2.1 Strains and media 4
1.2.2 Pulsed field gel electrophoresis and Southern
hybridisation of chromosomal DNA 4
1.2.3 DNA manipulations and sequencing 4
1.2.4 Polymerase chain reaction procedures 4
1.3 Results and discussion 5
1.3.1 Chromosome length polymorphisms in a
bottom-fermenting yeast strain 5
1.3.2 Structure of the 840 kb chromosome 6
1.3.3 Structure of the 820 kb chromosome 6
1.3.4 Translocation point in the 960kb chromosome 8
1.3.5 Development of the method for detection of the 960 kb
chromosome by polymerase chain reaction 10
1.4 Conclusions 11
References 11
2 Fast detection of beer spoilage microorganisms by consensus
polymerase chain reaction with foodproof
®
beer screening 13
K. Berghof, M. Fandke, A. Pardigol, A. Tauschmann and M. Kiehne
2.1 Introduction 13

2.2 Materials and methods 14
2.2.1 LightCycler™ Technology 14
2.2.2 Design of the polymerase chain reaction 15
2.2.3 Analytical procedure 16
2.2.3.1 Microbiological enrichment 16
2.2.3.2 Sample preparation 16
2.2.3.3 Standard protocol for polymerase chain
reaction preparation 17
2.3 Results and discussion 18
2.3.1 Detection of bacteria 18
2.3.2 Identification of bacteria 19
2.4 Conclusions 20
References 21
Part 2 Brewing Yeast Stress Responses During Handling 23
3 The impact of ethanol stress on yeast physiology 25
A. Lentini, P. Rogers, V. Higgins, I. Dawes,
M. Chandler, G. Stanley and P. Chambers
3.1 Introduction 25
3.2 Materials and methods 26
3.2.1 Yeast storage trials 26
3.2.1.1 Membrane lipid composition 26
3.2.1.2 Trehalose content 26
3.2.1.3 Yeast slurry pH 26
3.2.1.4 Yeast protease 26
3.2.1.5 Yeast viability 26
3.2.1.6 Yeast vitality 26
3.2.2 Gene array technology 27
3.3 Results and discussion 27
3.3.1 Impact of ethanol and temperature on the
structure of the yeast cell membrane 27

3.3.2 Cell-wall trehalose 28
3.3.3 Yeast slurry pH 29
3.3.4 Protease release from yeast 30
3.3.5 Yeast vitality 31
3.3.5.1 Acidification power test 31
3.3.5.2 Oxygen uptake rate 32
3.3.6 Changes in gene expression 32
3.3.6.1 Observations on using gene array technology 36
3.4 Conclusions 36
Acknowledgements 37
References 37
4 Yeast physical (shear) stress: the engineering perspective 39
R.A. Stafford
4.1 Introduction 39
4.1.1 Yeast cell response to shear stress 40
4.1.2 Cell stimuli 40
4.1.3 Newton’s law of viscosity: a gross deforming force 41
4.1.4 Yeast rheology 41
4.1.5 Methods of estimating shear rate of agitated systems 42
4.1.6 Energy dissipation rate 43
4.1.7 Kolmogorov turbulence scale 43
4.1.8 Residence/exposure time 43
4.2 Conclusions 44
xii CONTENTS
Acknowledgements 44
References 44
5 The osmotic stress response of ale and lager brewing yeast strains 46
P.A. White, A.I. Kennedy and K.A. Smart
5.1 Introduction 46
5.2 Materials and methods 48

5.2.1 Yeast strains 48
5.2.2 Media and growth conditions 48
5.2.3 Osmotic challenge 48
5.2.4 Viability determinations 48
5.2.5 Glycerol determination 48
5.2.6 Preparation of cells for confocal microscopic analysis 49
5.2.6.1 Staining of vacuole lumen 49
5.2.6.2 Staining of tonoplast 49
5.2.6.3 Staining of plasma membrane 49
5.2.6.4 Visualisation of samples 49
5.3 Results and discussion 49
5.3.1 Osmotic stress tolerance of YPD-grown cells 49
5.3.1.1 Physiological state 49
5.3.1.2 Strain dependence 51
5.3.1.3 Solute considerations 51
5.3.2 Compatible solute accumulation 53
5.3.2.1 Physiological state 53
5.3.2.2 Strain dependence and glycerol accumulation 53
5.3.2.3 Solute considerations of glycerol accumulation 53
5.3.3 Vacuolar changes 56
5.3.3.1 Vacuolar morphology of YPD-grown cells 57
5.3.3.2 Vacuolar morphology of exponential-phase cells 57
5.3.3.3 Vacuolar fragmentation and osmotic stress 57
5.4 Conclusions 58
Acknowledgements 59
References 59
6 Brewing yeast oxidative stress responses: impact of brewery handling 61
V. Martin, D.E. Quain and K.A. Smart
6.1 Introduction 61
6.2 Materials and methods 62

6.2.1 Yeast strains and growth conditions 62
6.2.2 Yeast sample collection 62
6.2.3 Determination of response to oxidative stress 62
6.2.4 Glutathione concentration 62
6.2.5 Protein extraction for enzymic assays by glass bead 62
cell lysis method
6.2.6 Catalase activity 63
6.2.7 Glycogen and trehalose concentration 63
CONTENTS xiii
6.3 Results and discussion 63
6.3.1 Oxidative stress resistance is dependent on growth
phase, strain and medium 63
6.3.2 Defence mechanisms against hydrogen peroxide are
dependent on strain and medium 63
6.3.3 Cellular damage 66
6.3.4 Oxidative stress during the brewing process 67
6.3.5 Propagation 67
6.3.6 Pitching 68
6.3.7 Storage and acid washing 69
6.3.8 Serial repitching 70
6.4 Conclusions 71
Acknowledgements 71
References 72
Part 3 Wort Composition: Impact on Yeast Metabolism and
Performance 75
7 Wort composition and beer quality 77
C.W. Bamforth
7.1 Introduction 77
7.2 The relationship of wort composition to beer quality 78
7.3 The key components of wort 78

7.4 The impact of wort on the production of flavour
compounds by yeast 79
7.5 Models 81
7.6 Sources of variability in wort composition 83
7.7 Conclusions 84
Acknowledgements 84
References 84
8 Wort substitutes and yeast nutrition 86
B. Taidi, A.I. Kennedy and J.A. Hodgson
8.1 Introduction 86
8.2 Materials and methods 87
8.2.1 Materials 87
8.2.2 Fully defined medium 87
8.2.3 Semi-defined medium 89
8.2.4 Analytical methods 89
8.3 Results and discussion 90
8.3.1 Fully defined medium 90
8.3.2 Semi-defined medium 92
8.4 Conclusions 95
Acknowledgements 95
References 95
xiv CONTENTS
9 Wort supplements: from yeast and for yeast 96
M. Dillemans, L. Van Nedervelde and A. Debourg
9.1 Introduction 96
9.2 Materials and methods 97
9.2.1 Yeast strains 97
9.2.2 Fermentations 97
9.2.3 Measurement of glucose uptake 97
9.2.4 Measurement of fructose-2,6-biphosphate 98

9.2.5 Acidification power test 98
9.2.6 Determination of enzyme activities 98
9.2.7 Measurement of glycerol 98
9.2.8 Protein determination 99
9.2.9 Lipid extraction 99
9.2.10 Glycogen determination 99
9.2.11 Farnesol-induced growth inhibition 100
9.2.12 Effect of ethanol and osmotic pressure on
growth on glucose and maltose 100
9.2.13 Effect of ethanol and osmotic pressure on
fermentation power 100
9.3 Results and discussion 100
9.3.1 Influence of yeast peptide complex on fermentation rate 100
9.3.2 Influence of yeast peptide complex on
glucose metabolism 101
9.3.3 Influence of yeast peptide complex on anabolic
enzyme activities 103
9.3.4 Influence of yeast peptide complex on yeast synthesis 105
9.3.5 Mode of action of yeast peptide complex 106
9.3.6 Influence of yeast peptide complex on ethanol and
osmotic stresses of growing cells 107
References 108
10 Unsaturated fatty acid supplementation of stationary-phase
brewing yeast and its effects on growth and fermentation ability 110
N. Moonjai, K.J. Verstrepen, F.R. Delvaux, G. Derdelinckx and
H. Verachtert
10.1 Introduction 110
10.2 Materials and methods 111
10.2.1 Yeast strain and maintenance 111
10.2.2 Growth medium 111

10.2.3 Yeast propagation 111
10.2.4 Preparation of stationary-phase cells and unsaturated
fatty acid supplementation 111
10.2.5 Analysis of pitching yeast 112
10.2.6 Test fermentations 112
10.2.7 Monitoring of fermentation 113
10.2.8 Analysis of volatile esters and higher alcohols 113
CONTENTS xv
10.3 Results and discussion 113
10.3.1 Unsaturated fatty acid supplementation of
pitching yeast 113
10.3.2 Fermentation with unsaturated fatty
acid-supplemented yeast 115
10.4 Conclusions 118
References 118
11 Impact of wort composition on flocculation 120
B. Axcell
11.1 Introduction 120
11.2 Molecular mechanism of yeast flocculation 121
11.3 Premature flocculation and beer quality 123
11.4 The antimicrobial peptide hypothesis 124
11.5 Possible mechanism for premature flocculation 125
11.6 Conclusions 126
References 127
Part 4 Yeast Quality Maintenance and Assessment 129
12 Management of multi-strain, multi-site yeast storage and supply 131
A.I. Kennedy, B. Taidi, A. Aitchison and X. Green
12.1 Introduction 131
12.1.1 Historical perspective 131
12.2 Yeast culture management 132

12.2.1 Aims 132
12.2.2 Strategies for strain maintenance 132
12.2.3 Selection of master cultures 133
12.2.4 Testing procedures 133
12.2.4.1 Flocculation (Tullo) and adhesion 133
12.2.4.2 Sedimentation (Helm’s test) 133
12.2.4.3 Sugar utilisation 133
12.2.4.4 Head formation 133
12.2.4.5 Petite stability 134
12.2.4.6 Fermentation performance 134
12.2.5 Deposition in liquid nitrogen 134
12.2.6 Cascade storage system 134
12.2.7 Retrieval from liquid nitrogen and slope preparation 134
12.2.8 Quality assurance 135
12.2.8.1 Freedom from contamination 135
12.2.8.2 Petite mutants 135
12.2.8.3 Viability 135
12.2.8.4 Genetic confirmation of identity 135
12.2.9 Integrity of supply 136
12.2.10 Statistics 136
xvi CONTENTS
12.3 Conclusions 136
Acknowledgements 136
References 136
13 Comparison of yeast viability/vitality methods and their
relationship to fermentation performance 138
L.R. White, K.E. Richardson, A.J. Schiewe and C.E. White
13.1 Introduction 138
13.2 Materials and methods 139
13.2.1 Yeast 139

13.2.2 Citrate methylene blue 139
13.2.3 Alkaline methylene blue 139
13.2.4 Alkaline methylene violet 139
13.2.5 Acidification power 140
13.2.6 Standard plate count 140
13.2.7 Fermentation 140
13.3 Results and discussion 140
13.3.1 Citrate methylene blue 140
13.3.2 Alkaline stains 142
13.3.2.1 Alkaline methylene blue 142
13.3.2.2 Alkaline methylene violet 142
13.3.2.3 Acidification power test 145
13.3.2.4 Standard plate count 145
13.3.2.5 Yeast performance 145
13.4 Conclusions 145
References 147
14 Yeast quality and fluorophore technologies 149
S.M. Van Zandycke, O. Simal, S. Gualdoni and
K.A. Smart
14.1 Introduction 149
14.2 Materials and methods 153
14.2.1 Yeast strains and growth conditions 153
14.2.2 Yeast starvation and heat treatment 153
14.2.3 Citrate methylene violet 153
14.2.4 MgANS 154
14.2.5 Oxonol 154
14.2.6 Propidium iodide 154
14.2.7 Sytox orange 154
14.2.8 Berberine 154
14.2.9 FUN1 155

14.2.10 Plate count 155
14.2.11 Photographs 155
14.3 Results and discussion 155
CONTENTS xvii
14.3.1 Can fluorophores differentiate between viable and
non-viable populations? 155
14.3.1.1 Lager strain L138 156
14.3.1.2 Ale strain 2593 157
14.3.2 Determination of yeast cell viability of starved
populations 158
14.4 Conclusions 160
Acknowledgements 160
References 160
15 Vitality assessment using the fluorescent stain FUN1 162
S.M. Van Zandycke, O. Simal and K.A. Smart
15.1 Introduction 162
15.2 Materials and methods 164
15.2.1 Yeast strains and growth conditions 164
15.2.2 Starvation and oxidative stress 164
15.2.3 Acidification power test 164
15.2.4 Glycogen and trehalose 164
15.2.5 FUN1 stain for vitality assessment 165
15.3 Results and discussion 165
15.3.1 Determination of yeast cell vitality of
starved stressed populations 165
15.3.2 Determination of yeast cell vitality of oxidatively
stressed populations 166
15.4 Conclusions 167
Acknowledgements 167
References 168

16 Flow cytometry: a new tool in brewing technology 169
K.J. Hutter and C. Lange
16.1 Introduction 169
16.2 Materials and methods 170
16.2.1 Glycogen content 170
16.2.2 DNA content 170
16.2.3 Detection of beer spoilage contaminants 170
16.2.4 Flow cytometry 170
16.3 Results and discussion 171
Acknowledgement 173
References 173
17 Comparison of the methylene blue assay with a new
flow-cytometric method for determining yeast viability in a
brewery 174
A. Boyd, T. Gunasekera, P. Attfield, K. Simic, S. Vincent and
D. Veal
17.1 Introduction 174
xviii CONTENTS
17.2 Materials and methods 175
17.2.1 Trial location and yeast analysed 175
17.2.2 Methylene blue staining and microscopic analysis 175
17.2.3 Oxonol staining and flow-cytometric analysis 175
17.2.4 Statistical analyses 176
17.3 Results and discussion 176
17.3.1 Comparison of viability assays 176
17.3.2 Operator error and reproducibility of viability data 177
17.4 Conclusions 178
Acknowledgements 179
References 179
Part 5 The Role of Brewing Yeast in Beer Flavour Development 181

18 Formation and disappearance of diacetyl during lager
fermentation 183
C. Boulton and W. Box
18.1 Introduction 183
18.2 Materials and methods 184
18.3 Results and discussion 184
18.4 Conclusions 193
Acknowledgements 194
References 194
19 The formation of higher alcohols 196
J.R. Dickinson
19.1 Introduction 196
19.2 Conclusions 204
References 205
20 Methionine: a key amino acid for flavour biosynthesis in beer 206
P. Perpète, L. Gijs and S. Collin
20.1 Introduction 206
20.2 Materials and methods 207
20.2.1 Reagents 207
20.2.2 Strains 207
20.2.3 Culture media and sampling 208
20.2.4 Methanethiol quantification 208
20.3 Results and discussion 208
References 211
21 Control of ester synthesis during brewery fermentation 213
J P. Dufour, Ph. Malcorps and P. Silcock
21.1 Introduction 213
21.2 Ester formation and excretion during fermentation 215
CONTENTS xix
21.3 The rate-limiting factors of ester synthesis and the relationship

between ester synthesis, lipid metabolism and growth 215
21.3.1 Synthesis of the acetate esters 216
21.3.2 Synthesis of the medium-chain fatty
acid esters (C6–C10) 217
21.4 Parameters influencing the synthesis of beer esters 218
21.5 Influence of the yeast characteristics on the synthesis of esters 219
21.5.1 Yeast strain 219
21.5.2 Pitching rate 219
21.5.3 Genetic and physiological instability of brewing yeast 219
21.6 Physicochemical and technological parameters affecting
the production of esters during brewing fermentation 221
21.6.1 Influence of lipids on ester synthesis 221
21.7 Influence of oxygen/air on ester synthesis 222
21.7.1 Influence of the trace element: zinc 223
21.8 Influence of fermentation conditions 224
21.8.1 Stirring 224
21.8.2 Effect of carbon dioxide pressure 224
21.8.3 Fermentation in cylindroconical fermenters 224
21.8.4 Continuous fermentation and maturation 225
21.8.5 Temperature 226
21.9 Contribution of esterase activities to beer ester levels 226
21.10 Conclusions 227
References 228
22 Genetic regulation of ester synthesis in yeast: new facts,
insights and implications for the brewer 234
K.J. Verstrepen, N. Moonjai, F.F. Bauer, G. Derdelinckx,
J P. Dufour, J. Winderickx, J.M. Thevelein,
I.S. Pretorius and F.R. Delvaux
22.1 Introduction 234
22.2 Materials and methods 236

22.2.1 Microbial strains, media and culturing conditions 236
22.2.2 DNA manipulations 237
22.2.3 Fermentation experiments 237
22.2.4 Sensory analysis 238
22.2.5 Headspace analysis for the measurement of
acetaldehyde, ethyl acetate, n-propanol, isobutanol,
isoamyl alcohol, isoamyl acetate and ethyl caproate 238
22.2.6 Liquid chromatography for the measurement of
wort sugars 238
22.2.7 Carbon starvation 238
22.2.8 RNA extraction and Northern analysis 239
xx CONTENTS
22.3 Results and discussion 239
22.3.1 Activity of ATF1, ATF2 and EHT1 during
brewery fermentations 239
22.3.2 Overexpression of ATF1 and ATF2 in brewing yeast: genetic
modification allows management of ester production 240
22.3.3 ATF1 is regulated by glucose through the
cyclic AMP/protein kinase A signalling pathway 242
22.4 Conclusions 245
Acknowledgements 246
References 246
Part 6 Yeast Handling: Objectives, Obstacles and Opportunities 249
23 Yeast Propagation 251
G.A. Hulse
23.1 Introduction 251
23.2 Historical perspective 252
23.3 Current perspective 252
23.4 Future perspectives 255
23.5 Conclusions 255

References 256
24 Serial repitching fermentation performance and functional biomarkers 257
C.L. Jenkins, A.I. Kennedy, P. Thurston,
J.A. Hodgson and K.A. Smart
24.1 Introduction 257
24.2 Materials and methods 259
24.2.1 Yeast strains and growth conditions 259
24.2.2 Citrate methylene violet 259
24.2.3 MgANS 260
24.2.4 Viability plate counts 260
24.2.5 Intracellular glycogen and trehalose determination 260
24.2.6 Determination of frequency of petite mutation 260
24.2.7 Propensity to form petites 260
24.2.8 Budding index 261
24.2.9 Percentage of yeast solids 261
24.2.10 Flocculation 261
24.2.11 Cell-surface charge 262
24.2.12 Hydrophobicity 262
24.2.13 Vicinal diketone uptake 262
24.3 Results and discussion 262
24.3.1 Impact of serial repitching on yeast quality 262
24.3.2 Impact of serial repitching on petite mutation 265
CONTENTS xxi
24.3.3 Impact of serial repitching on the fermentation
performance of lager brewing yeast 266
24.3.4 Impact of fermentation on the replicative capacity of
lager brewing yeast 266
24.3.5 Impact of serial repitching on the attenuation of
lager brewing yeast 267
24.3.6 Impact of serial repitching on the flavour

development of lager brewing yeast 267
24.3.7 Impact of serial repitching on the flocculation capacity and
cell-surface characteristics of lager brewing yeast 268
24.4 Conclusions 269
Acknowledgements 269
References 269
25 The impact of yeast cell age on fermentation, attenuation and
flocculation 272
C.D. Powell, D.E. Quain and K.A. Smart
25.1 Introduction 272
25.2 Materials and methods 273
25.2.1 Yeast strains 273
25.2.2 Preparation of aged cell fractions 273
25.2.3 Sucrose gradients 273
25.2.3.1 Preparation of virgin cells 273
25.2.4 Fermentations 273
25.2.5 Measurement of cell flocculation 274
25.2.5.1 Helm’s test 274
25.2.6 Cell-surface hydrophobicity 274
25.2.7 Cell-surface charge 274
25.3 Results and discussion 274
25.3.1 Age synchronisation of yeast 274
25.3.2 Influence of cell age on the rate of sugar
utilisation during fermentation 274
25.3.3 Impact of age on cell flocculation 276
25.3.4 Relationship between age and cell hydrophobicity
and cell surface charge 276
25.4 Conclusions 279
Acknowledgements 279
References 279

26 Chronological and replicative lifespan in lager brewing yeast 281
D.L. Maskell, A.I. Kennedy, J.A. Hodgson and
K.A. Smart
26.1 Introduction 281
26.2 Materials and methods 283
26.2.1 Yeast strains 283
26.2.2 Media and growth conditions 283
xxii CONTENTS
26.2.3 Micromanipulation 283
26.2.3.1 Data analysis 284
26.2.4 Extended stationary phase 284
26.2.5 Production of sucrose gradients 284
26.2.6 Production of virgin and non-virgin populations 284
26.2.7 Viability assessment 284
26.2.7.1 Citrate methylene violet 284
26.2.7.2 Oxonol 285
26.2.7.3 Plate counts 285
26.3 Results and discussion 285
26.3.1 Replicative lifespan of four strains of lager brewing yeast 285
26.3.2 Chronological lifespan of four strains of lager brewing yeast 286
26.3.3 Is there a correlation between replicative and
chronological lifespan? 287
26.3.4 Do chronologically aged brewing yeast cells
demonstrate a reduced replicative lifespan? 288
26.4 Conclusions 289
Acknowledgements 290
References 290
27 Continuous primary fermentation of beer with immobilised yeast 293
K. Tapani, P. Soininen-Tengvall, H. Berg, B. Ranta and E. Pajunen
27.1 Introduction 293

27.2 Materials and methods 294
27.2.1 Yeast and wort 294
27.2.2 Carrier 294
27.2.3 Pilot plant unit 294
27.2.4 Start-up procedures 294
27.2.5 Basis for continuous fermentation 295
27.2.6 Process conditions 295
27.2.7 Analytical methods 295
27.2.7.1 Fermentation analyses 295
27.2.7.2 Flavour compounds and vicinal diketones 296
27.2.7.3 Fermentable sugars 296
27.2.7.4 Microbiological analysis 296
27.3 Results and discussion 296
27.3.1 Fermentation 296
27.3.2 Flavour formation 296
27.3.3 Vicinal diketones 298
27.3.4 Free amino nitrogen 299
27.4 Conclusions 299
Acknowledgements 300
References 300
Index 303
CONTENTS xxiii
Preface to the second edition
Controlling the impact of stress on brewing biomass, predicting yeast activity and
ensuring consistent fermentation performance through successive fermentations
remain areas of active interest for the brewing industry.
To be able to control and perhaps even manipulate yeast activity, it is necessary to
identify factors that affect its functionality during fermentation. Genetic stability and
integrity are crucial to maintaining predictable performance. The brewing yeast
genome is inherently unstable, leading to the formation of nuclear and mitochondrial

variants during yeast handling and fermentation. Although recent molecular innovations
may allow rapid detection of such occurrences, the causes and nature of the DNA
damage remain to be elucidated.
During handling and fermentation the yeast is subjected to a rapidly changing
environment. There are many stresses to be considered, including physical stresses
such as shear, cold shock and hydrostatic pressure, and those created by the yeast’s
own biochemical activity such as oxidative stress, nutrient limitation, anaerobiosis,
osmotic stress, low pH, excess carbon dioxide and the formation of toxic metabolites.
In addition, wort composition is a critical determinant of yeast performance and final
product quality. Batch-to-batch changes in component ratios inevitably contribute to
the variability in performance exhibited by a given slurry, yet few extensive studies
have been conducted in this area. This very variability in both wort composition and
yeast quality is reflected in final beer quality and in particular beer flavour. The role
of the yeast cell in flavour attributes is therefore dependent on both intrinsic and
extrinsic factors.
It is not unreasonable to suggest that the physiological condition of brewing yeast
influences fermentation performance, therefore brewers require consistent yeast
quality and quantity. Ensuring the correct quality can be achieved by adequate strain
selection and maintenance though master culture storage regimes and effective
propagation and yeast handling during serial repitching. Preventing slurry deteriora-
tion through the use of immobilisation may prove successful but there is still a
requirement to identify adequate biomarkers for slurry deterioration and potential
to perform.
Katherine A. Smart
Royal Society Industrial Fellow
Scottish Courage Reader in Brewing Science
Part 1 Molecular Innovations
1 Analysis of Karyotypic Polymorphisms in a
Bottom-fermenting Yeast Strain by Polymerase
Chain Reaction

K. TANAKA and O. KOBAYASHI
Abstract Chromosomal rearrangement causes karyotypic variation in bottom-fermenting
yeast. However, the molecular basis of this phenomenon has not yet been clearly defined.
The complete genome sequence of Saccharomyces cerevisiae, which has been published,
can be used for genome analysis of bottom-fermenting yeast. The chromosomal organisa-
tion of a bottom-fermenting yeast strain is being investigated by pulsed field gel electro-
phoresis and Southern hybridisation using more than 100 genes from all 16 chromosomes
of S. cerevisiae as probes. In this study, the same techniques were used to detect the karyotypic
polymorphisms of single colonies isolated from a bottom-fermenting yeast strain.
Although the karyotypes of the isolated clones were almost the same, chromosome
length polymorphisms were observed in three chromosomes. These chromosomes were
investigated in detail and found to be chimeras, constructed from two different chromo-
somes. In the junction of the chimeric chromosomes, either a retrotransposon Ty or the
subtelomeric gene COS was found to exist. This suggested that translocation resulting
from homologous recombination produced these chimeric chromosomes.
Making use of the sequences of the junction regions, a new method to detect karyotypic
changes by polymerase chain reaction was developed. This new method is highly sensitive,
and able to detect karyotypic changes within 2 days, from a single colony. This method led to
the observation that translocation occurred at a frequency of 10
Ϫ5
during yeast cultivation.
1.1 Introduction
Genetic changes of bottom-fermenting yeast have been reported.
1,2
Such changes
may give rise to instabilities and, therefore, affect the performance of the bottom-
fermenting yeast during fermentation. To control the quality of yeast for fermentation,
it is important to know the environmental factors that affect the occurrence of such
changes. However, little is known of the mechanism by which genetic changes in bottom-
fermenting yeasts occur. To investigate the mechanism of karyotypic changes, highly

sensitive methods to detect genetic changes are required. Two types of method for
the detection of chromosomal rearrangement have been developed. One type uses
selectable marker genes on artificial loci.
3,4
Although such methods give a rapid and a
highly sensitive analysis, naturally occurring chromosomal rearrangements cannot be
detected. The other type of method detects chromosome length polymorphisms using
pulsed field gel electrophoresis (PFGE).
2
However, this latter method requires as long
as 8 days to obtain results from the start of culture. Moreover, bottom-fermenting yeasts
have many more chromosomes than laboratory yeasts, preventing adequate separation
of each chromosome.
Bottom-fermenting yeasts are known to have an unusual genomic background.
5
Not only are they polyploid strains, but they have at least two different genomic sets.
Brewing Yeast Fermentation Performance:
Second edition
Edited
by:
KATHERINE SMART
Copyright
0
Blackwell Science 2003