Table of Contents
TABLE OF CONTENTS
List of Figures
viii
List of Tables
xviii
Glossary of Abbreviations
xix
Acknowledgements
xx
1. Introduction
1.1
Yeast – Overview
1
1.2
General cellular characteristics of yeast
2
1.3
Yeast cell organelles and compartments
3
1.4
Protein trafficking in Saccharomyces cerevisiae
5
1.5
Endocytosis
5
1.6
Yeast metabolism
5
1.7
Yeast division and cell cycle
6
1.8
Important physiological marker substances in yeast
8
1.8.1 Glycogen
8
1.8.2 Neutral Lipids
9
1.8.3 Trehalose
10
1.8.4 Bud Scars
11
1.8.5 Yeast DNA
11
1.8.6 Intracellular proteinase activity
12
1.8.7 Measurement of intracellular proteinase activity- Overview
13
Proteinase A activation and secretion
14
1.9.1 The activation cascade of proteinase A
16
1.9.2 The mechanism of proteinase A secretion
19
Beer proteins and beer foam stability
21
1.10.1 Beer proteins – Overview
23
1.10.2 Hydrophobic polypeptides
23
1.10.3 Protein Z
25
1.10.4 Lipid Transfer Protein (LTP1) and the 17 kDa protein
26
1.11
High gravity brewing and beer foam stability
28
1.12
Beer flavour stability – Overview
31
1.13
Reactive Oxygen Species (ROS)
33
1.9
1.10
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1.14
Detection of ROS with Electron Spin Resonance (ESR)
35
1.15
Carbonyl compounds and flavour stability
37
1.16
Beer protein thiols and flavour stability
41
1.17
Metal catalysed oxidation (MCO) of beer proteins
41
1.18
Beer flavour stability and yeast
43
1.19
Fluorescence techniques for monitoring physiological changes
in Saccharomyces cerevisae
43
1.19.1 Flow cytometry
43
1.19.2 Fluorescence microscopy – Overview
45
1.19.3 The use of Green Fluorescent Protein (GFP) for studying
1.20
protein dynamics and function
49
Research objectives
50
1.20.1 Foam Stability
50
1.20.2 Flavour Stability
51
2. Materials and Methods
2.1
Chemicals, reagents and kits
52
2.2
Agar slopes, media, buffers and solutions
54
2.3
Yeast strains
55
2.4
Determination of yeast viability
58
2.5
Beer production in the ICBD pilot plant
58
2.6
Beer production at the Foster’s Brewery in Yatala, Brisbane,
Australia
59
2.7
Specific gravity and pH
60
2.8
Total protein concentration
61
2.9
Free alpha-amino nitrogen (FAN) in wort by colorimetry
61
2.10
Amino acid spectra of wort and beer by HPLC
62
2.11
Total polyphenols in wort and beer
62
2.12
Determination of pentose sugars with phloroglucinol
62
2.13
Determination of pentose sugars in freeze dried proteinaceous
material
63
2.14
Determination of pentose sugars by phloroglucinol in wort and beer
63
2.15
Total carbohydrates in wort and beer by spectrophotometry
63
2.16
Total carbohydrate determination in freeze dried proteinaceous
63
Material
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2.17
Determination of glucose, D-fructose, sucrose, maltose and
maltotriose by HPLC
64
2.18
Determination of ethanol concentration by GC
65
2.19
Determination of ethanol concentration by distillation
65
2.20
Determination of foam stability
65
2.21
Hydrophobic polypeptide analysis
66
2.22
Measurement of extra-cellular proteinase A activity
67
2.23
Fluorescence microscopy and confocal imaging
68
2.24
Staining of physiological parameters in yeast cells for confocal
2.25
imaging and flow cytometry
69
2.24.1 Staining of glycogen with acriflavine
69
2.24.2 Staining of neutral lipids with the fluorescent dye nile red
70
2.24.3 Staining of trehalose with the concancavalin A-fluorescein
71
2.24.4 Staining of bud scars with WGA-fluorscein
71
2.24.5 Fluorescent staining of yeast DNA with propidium iodide
72
2.24.6 Intracellular proteinase staining with BODIPY-FL-casein
73
2.24.7 Staining of the vacuolar membrane with FM4-64
74
Measurement of Reactive Oxygen Species (ROS) using Electron
Spin Resonance (ESR)
2.26
76
The Peroxide Challenge Test (PCT): A novel assay for predicting
beer flavour stability
2.27
77
3,3’,5,5’-tetramethylbenzidine (TMB) detection for rapid analysis of
beer protein thiol concentration
2.28
79
Labelling of protein thiols using MPB (N'-(3-maleimidylpropionyl)
biocytin)
80
2.29
Total thiol determination in beer using DTNB
80
2.30
Thiol determination in beer proteins using an enzyme-linked
immunosorbent assay (ELISA)
80
2.30.1 Preparation of reduced/oxidised BSA as standard protein
81
2.30.2 Oxidised and reduced BSA as standard protein: Estimation of
protein thiol content using DTNB
2.31
81
Detection of protein carbonyl groups with an enzyme linked
immunosorbent assay (ELISA)
83
2.31.1 Preparation of oxidised and reduced BSA standards
83
2.31.2 Blanking carbonyl reactivity with sodium borohydride
84
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2.31.3 Determination of protein oxidation with the standard
colorimetric technique for detection of carbonyl groups
84
2.31.4 DNPH derivatisation after absorbing the beer proteins on
the PVDF membrane: “On the plate derivatisation”
2.32
Protein extraction and purification from beer wort, hot water
extracts and other liquid process samples
2.33
85
86
Gel electrophoresis and visualisation of protein bands using
silver staining
86
2.33.1 Silver staining of SDS-PAGE gels
89
3. Fluorescence techniques for monitoring physiological changes in S. cerevisiae
3.1
The potential of confocal imaging of physiological changes
in S. cerevisiae
92
3.1.1
Glycogen
92
3.1.2
Neutral lipids
93
3.1.3
Trehalose
93
3.1.4
Bud scars
93
3.1.5
DNA
94
3.1.6
Intracellular proteinase activity
94
3.1.7
Vacuolar membrane
94
3.1.8
Multicoloured confocal images
95
3.1.9
Visualisation of physiological parameters in yeast
95
3.1.10 Set up of the POC-chamber –system for live cell imaging
100
3.1.11 Confocal imaging of proteinase A (Pr A) expression in a
yeast GFP-clone
101
3.1.12 Live cell imaging of a yeast GFP clone: Proteinase A
expression as a response to ethanol, heat and during
fermentation
104
3.1.13 Discussion and conclusions
106
3.1.13.1 Proteinase A expression studies using a S.cerevisiae
3.2
GFP clone
107
3.1.13.2 Conclusions
107
Detection of proteinases in S. cerevisiae by flow cytometry
3.2.1
109
Visualisation of intracellular proteinases using
fluorescence microscopy
109
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3.2.2
3.2.3
3.3
Flow cytometric measurement of intracellular
proteinase activity
110
Discussion and conclusions
113
A novel procedure for determining yeast pitching rates employing
flow cytometry
3.3.1
117
Flow cytometric assessment of the physiological state
of the yeast population during wort fermentations with
different pitching rates
121
3.3.1.1 Determination of yeast DNA content and cell cycle
stages employing flow cytometry
121
3.3.1.2 Glycogen determination using flow cytometry
123
3.3.1.3 Flow cytometric determination of intracellular
3.3.2
proteinase content
125
Discussion and conclusions
127
4. Proteinase A and beer foam stability
4.1
4.2
The influence of pasteurisation on beer foam stability
131
4.1.1
135
Discussion and conclusions
The impact of bottle-conditioning of beer employing revitalised
yeast on extracellular Pr A levels and the consequences
for foam stability
138
4.2.1 Viability of yeast in bottle conditioned beer
139
4.2.2 Pr A activity in bottle conditioned beer
139
4.2.3 Hydrophobic polypeptides
139
4.2.4 Foam stability of bottle conditioned beer
140
4.2.5 Discussion and conclusions
143
5. Characterisation of polypeptides throughout the brewing process of lower (12
˚Plato) and high gravity wort (20 ˚Plato) and beer
5.1
Polypeptide characterisation of a selection of cereals and hop
varieties used for beer production
145
5.1.1 Cereals
145
5.1.2 Hop pellets and hop extract
148
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5.2
5.3
5.4
Changes in polypeptide composition during production of
sales and high gravity wort
149
5.2.1 55 kDa polypeptide
150
5.2.2 45 kDa polypeptide
150
5.2.3 40 kDa polypeptide
150
5.2.4 26 kDa polypeptide
151
5.2.5 17 kDa polypeptide
151
5.2.6 10 kDa polypeptide
151
5.2.7 Glycosylation of polypeptides during wort production
151
Changes in polypeptide composition during fermentation of
high and low gravity wort
152
5.3.1 Lower gravity fermentation
152
5.3.2 High gravity fermentation
153
5.3.3 Glycosylation of polypeptides during fermentation
153
Determination of polypeptide distribution in mash solids,
hot trub and fermenter residue retrieved during 12˚Plato and
20˚Plato wort production
154
5.4.1 Mash solids
154
5.4.2 Hot trub
154
5.4.3 Fermenter residue
154
5.4.4 Degree of glycosylation in hot trub, cold trub and fermenter
5.5
Residue
155
Discussion and conclusions
162
5.5.1 Protein Z
162
5.5.2 Lipid Transfer Protein (LTP1)
163
5.5.3 17 kDa polypeptide
164
5.5.4 Conclusions
166
6. The importance of proteins in beer staling
6.1
Novel procedures for detection of thiol groups in beer proteins
169
6.1.1
Detection of protein thiol groups using Western Blotting
171
6.1.2
Detection of protein thiol groups using an Enzyme-Linked
Immunosorbent Assay (ELISA)
171
6.1.2.1 Assay development for protein thiol determination
using ELISA: Variation of TMB development time
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6.1.2.2 Assay development for protein thiol determination
Using ELISA: Variation of protein loading
174
6.1.2.3 Assay development for protein thiol determination
using ELISA: Comparison of proteins as potentially suitable
6.1.3
6.2
6.3
standard proteins
175
Discussion and conclusions
177
A novel assay for the detection of carbonyl groups in beer proteins
184
6.2.1
Detection of carbonyl groups using Western Blotting
184
6.2.2
Detection of protein carbonyl groups with an enzyme
linked immunosorbent assay (ELISA)
185
6.2.3
187
Discussion and conclusions
The Peroxide Challenge Test (PCT): A novel method for holistic,
near real time measurement of beer flavour stability
190
6.3.1
202
Discussion and conclusions
7. Conclusions
205
8. Literature Cited
211
9. Appendices
239
10. Publications and Presentations
24
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