Impact of protease activity of yeasts on wine fermentation and formation of volatile and non-volatile metabolites [Elektronische Ressource] / submitted by Ni-orn Chomsri
198 Pages
English
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Impact of protease activity of yeasts on wine fermentation and formation of volatile and non-volatile metabolites [Elektronische Ressource] / submitted by Ni-orn Chomsri

Downloading requires you to have access to the YouScribe library
Learn all about the services we offer
198 Pages
English

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Institute of Nutritional Science Justus Liebig-University Giessen, Germany and Section of Microbiology and Biochemistry Geisenheim Research Center, Germany Impact of protease activity of yeasts on wine fermentation and formation of volatile and non-volatile metabolites Thesis submitted in partial fulfilment of the requirements for the degree of Doctor oeconomiae trophologiaeque (Dr. oec. troph.) Submitted by Ni-orn Chomsri Lampang/Thailand 2008 This Ph.D. work was approved by the committee (Faculty 09: Agricultural and Nutritional Sciences, Home Economics and Environmental Management) of Justus Liebig-University Giessen, as a thesis to award the Doctor degree of oeconomiae trophologiaeque (Dr. oec. troph.) 1. Supervisor: Professor Dr. Hans Brueckner 2. Supervisor: Professor Dr. Doris Rauhut 3. Supervisor: Professor Dr. Sylvia Schnell Date of disputation: 10.07.2008 Contents 1 INTRODUCTION 1 1.1 Statement and significance of the study 1 1.2 Objectives 3 2 LITERATURE REVIEW 4 2.1 Yeasts 4 2.2 Proteolytic enzymes of yeasts 12 2.3 Nitrogen-containing compounds in winemaking 18 3 MATERIALS AND METHODS 33 4 RESULTS 44 4.1 Screening of wine yeasts that secrete extracellular proteases for the use in 44 grape juice fermentation 4.1.

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Published 01 January 2008
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Institute of Nutritional Science
Justus Liebig-University Giessen, Germany
and
Section of Microbiology and Biochemistry
Geisenheim Research Center, Germany







Impact of protease activity of yeasts on
wine fermentation and formation of volatile
and non-volatile metabolites



















Thesis submitted in partial fulfilment of
the requirements for the degree of
Doctor oeconomiae trophologiaeque
(Dr. oec. troph.)





Submitted by
Ni-orn Chomsri
Lampang/Thailand
2008







































This Ph.D. work was approved by the committee
(Faculty 09: Agricultural and Nutritional Sciences,
Home Economics and Environmental Management) of
Justus Liebig-University Giessen, as a thesis to award the
Doctor degree of oeconomiae trophologiaeque (Dr. oec. troph.)


1. Supervisor: Professor Dr. Hans Brueckner
2. Supervisor: Professor Dr. Doris Rauhut
3. Supervisor: Professor Dr. Sylvia Schnell

Date of disputation: 10.07.2008








Contents



1 INTRODUCTION 1
1.1 Statement and significance of the study 1
1.2 Objectives 3
2 LITERATURE REVIEW 4
2.1 Yeasts 4
2.2 Proteolytic enzymes of yeasts 12
2.3 Nitrogen-containing compounds in winemaking 18
3 MATERIALS AND METHODS 33
4 RESULTS 44
4.1 Screening of wine yeasts that secrete extracellular proteases for the use in 44
grape juice fermentation
4.1.1 Protease production in synthetic medium 44
4.1.2 Protease production in grape juice 47
4.1.3 Effect of proteins isolated from must on yeast growth 48
4.2 Fermentative characteristics of non-Saccharomyces yeasts exhibiting 53
extracellular proteases in enological fermentation
4.3 Effect of yeast producing protease in mixed cultures for winemaking 62
4.3.1 Influence of yeasts expressing protease activity on fermentation activity 62
4.3.2 Influence of inoculation treatment on fermentation of Riesling grape juice 67
4.4 Influence of yeasts on polypeptides and proteins in winemaking 81
4.4.1 Production of yeast proteins in synthetic medium 81
4.4.2 Investigation of polypeptides and proteins in fermented grape juice and 82
wine
4.4.3 Application of electrophoresis to study polypeptide and protein profiles 85

5 DISCUSSION 91
6 CONCLUSION AND PERSPECTIVES 112
7 SUMMARY 114
8 REFERENCES 120
9 APPENDIX 153






























List of Tables


Table 2-1 List of the Saccharomyces yeasts, with their teleomorphic and 6
anamorphic names
Table 2-2 List of the Non-Saccharomyces yeast species, with their teleomorphic 9
and anamorphic names
Table 2-3 Families of proteolytic enzymes 15
Table 2-4 The identity and concentration of amino acids found in the whole 21
grape and juice at harvest
Table 2-5 Concentration of nitrogen containing compounds found in grape juice 25
and wine
Table 3-1 List of the yeasts used in the study 33
Table 3-2 Experimental plan of yeast inoculation 39
Table 3-3plan of the inoculation of yeasts 40
Table 3-4 Description of samples for the investigation of polypeptides and 42
proteins and SDS-PAGE analysis
Table 4-1 Proteolytic activity of yeasts grown in synthetic medium 45
Table 4-2 Effect of protease production of yeasts on concentration of total 47
soluble solids (TSS) of fermented grape juice and on their viable
population after fermentation
Table 4-3 Nitrogen-containing components in the isolated proteins (IP) from 49
must and in the synthetic grape juice (SGJ) supplemented with the IP
Table 4-4 Composition of end products obtained from grape juice fermentation 55
with non-Saccharomyces yeasts
Table 4-5 Concentrations of metabolites binding sulphur dioxide in end products 56
obtained from grape juice fermentation with non-Sccharomyces
ye as ts
Table 4-6 Concentrations of sulphur-containing compounds in end products 57
ofermentation with non-Sccharomyces
y eas ts
Table 4-7 Concentration of amino acids in fermented grape juice at the end of 59
fermentation
Table 4-8 Concentration of odouriferous compounds found in fermented grape 60
juice at the end of fermentation

Table 4-9 Composition of wines from fermentation of single and sequential 64
inoculation
Table 4-10 Concentration of amino acids in wines from fermentation of single 66
and sequential inoculation
Table 4-11 Composition of clarified and unclarified musts 67
Table 4-12 Composition of the wines produced by fermentation of clarified 69
Riesling grape juice fermented with different inoculation protocols
Table 4-13by fermentation of unclarified 70
Riesling grape juice fermculation protocols
Table 4-14 Concentrations of metabolites binding sulphur dioxide in wines from 71
clarified Riesling grape juice fermented with different inoculation
protocols
Table 4-15ding sulphur dioxide in wines from 71
unclarified Riesling grape juice fermented with different inoculation protocols
Table 4-16 Concentration of amino acids in clarified must and wines fermented 74
with different inoculation protocols
Table 4-17unclarified must and wines 75
fermented with different inoculation protocols
Table 4-18 Concentrations of odouriferous compounds in wines from clarified 77
Riesling grape juice fermented with different inoculation protocols
Table 4-19iferous compounds in wines from unclarified 78
Riesling grape juice fermented with different inoculation protocols
Table 4-20 Concentrations of sulphur-containing compounds in wines from 79
clarified Riesling grape juice fermented with different inoculation protocols
Table 4-21ur-containinom 80
unclarified Riesling grape juice fermented with different inoculation protocols
Table A-1 Proteolytic activity of yeasts grown in synthetic medium 153
Table A-2 Protease activity, total soluble solid (TSS) and viable yeast cells in 155
grape juice after 3 days of growth
Table A-3 OD values of synthetic media containing different sources of 156 600
nitrogen
Table A-4 Composition of end products obtained from grape juice fermentation 158
with non-Saccharomyces yeasts

Table A-5 Concentrations of sulphur-containing compounds in end products 159
obtained from grape juice fermentation with non-Sccharomyces
yeas ts
Table A-6 Concentrations of assimilable nitrogen in fermented grape juice at 160
the end of fermentation expressed as free α-amino nitrogen (FAN)
Table A-7 Composition of wines from fermentation of single and sequential 161
inoculation
161 Table A-8 Assimilable nitrogen expressed as free α-amino nitrogen (FAN) in
wines from fermentation of single and sequential inoculation
Table A-9 Composition of the wines produced by fermentation of clarified 166
Riesling grape juice fermented with different inoculation protocols
Table A-10 Composition of the wines produced by fermentation of unclarified 167
Riesling grape juice fermented with different inoculation protocols
Table A-11 Concentrations of metabolites binding sulphur dioxide in wines from 168
clarified and unclarified Riesling grape juice fermented with different
inoculation protocols
Table A-12 Concentration of amino acids in clarified must and wines fermented 169
with different inoculation protocols
Table A-13unclarified must and wines 171
fermented with different inoculation protocols
Table A-14 Concentrations of odouriferous compounds in wines from clarified 173
Riesling grape juice fermented with different inoculation protocols
Table A-15iferous compounds in wines from unclarified 175
Riesling grape juice fermculation protocols
177 Table A-16 Assimilable nitrogen expressed as free α-amino nitrogen (FAN) in
wines from clarified and unclarified musts fermented with different inoculation protocols
Table A-17 Concentrations of sulphur-containing compounds in wines from 178
clarified Riesling grape juice fermented with different inoculation protocols
Table A-18ur-containinom 180
unclarified Riesling grape juice fermented with different inoculation protocols
Table A-19 Viable yeast cells in the medium 182
Table A-20 Concentrations of extracellular proteins released by species of 182
non-Saccharomyces into synthetic defined medium

Table A-21 Estimation of the concentrations of polypeptides and proteins in 183
grape juice fermented with different yeast strains
Table A-22 Concentrations of polypeptides and proteins in wines with different 183
fermentation conditions

































List of Figures


Figure 2-1 Generalized growth of yeasts during spontaneous fermentation of 6
wine
Figure 2-2 Factors affecting the concentration of peptides and proteins in 26
must and wine
31 Figure 2-3 Reaction of ammonia with α-keto-gutarate to incorporate inorganic
forms of nitrogen by Saccharomyces
Figure 4-1 Proteolytic activity in supernatants obtained from yeasts grown in 48
grape juice
Figure 4-2 Chromatogram of amino acids found in isolated proteins (IP) from 50
grape juice
Figure 4-3 Chromatogram of amino acids found in the synthetic grape juice 51
(SGJ) supplemented with the isolated proteins (IP)
Figure 4.4 Yeast growth in the synthetic grape juice (SGJ) containing isolated 52
proteins (IP)
Figure 4.5 Optical density at wavelength 600 nm of the synthetic grape juice 53
(SGJ) containing different sources of nitrogen
Figure 4-6 Fermentation kinetics of non-Saccharomyces yeasts during 54
fermentation of grape juice
Figure 4-7 Concentrations of assimilable nitrogen in fermented grape juice at 58
the end of fermentation
Figure 4-8 Growth kinetics of grape juice fermentation with single and 63
sequential inoculation
65 Figure 4-9 Assimilable nitrogen expressed as free α-amino nitrogen (FAN) in
wines from fermentation of single and sequential inoculation
Figure 4-10 Effect of inoculation protocols on fermentation kinetics during 68
fermentation of Riesling musts
72 Figure 4-11 Assimilable nitrogen expressed as free α-amino nitrogen (FAN) in
wines from clarified and unclarified musts fermented with different inoculation protocols
Figure 4-12 Total number of yeast cells at inoculation and after three days of 81
cultivation in synthetic medium
Figure 4-13 Concentrations of extracellular proteins released by species of 82
non-Saccharomyces into synthetic defined medium

Figure 4-14 Estimation of the concentrations of polypeptides (a) and proteins in 84
grape juice fermented with different yeast strains
Figure 4-15ntrations of oteins 86
(b) in wines from different conditions of fermentation
Figure 4-16 SDS-PAGE protein patterns of grape juice proteins 87
Figure 4-17 SDS-PAGE patterns of proteins isolated from fermented musts and 88
wines
Figure 4-18proteins isolated from Riesling grape juices 89
and wines
Figure 4-19 SDS-PAGE patterns of Weissburgunder grape juice fermentation 90
Figure A-1 Fermentation kinetics of non-Saccharomyces yeasts during 157
fermentation of grape juice
Figure A-2 Growth kinetics of grape juice fermentation with single and 160
sequential inoculation
Figure A-3 Effect of inoculation treatment on fermentation kinetics during 162
fermentation of clarified Riesling must (R1)
Figure A-4 Effect of inoculation treag 163
f Riesling must (R2)
Figure A-5 Effect of inoculation treag 164
fermentation of unclarified Riesling must (R1)
Figure A-6 Effect of inoculation treatment on fermentation kinetics during 165
fd Riesling must (R2)