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 3-3 Experimental plan 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 yeasts Table 4-6 Concentrations of sulphur-containing compounds in end products 57 obtained from grape juice fermentation with non-Sccharomyces yeasts 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 4-13 Composition of the wines produced by fermentation of unclarified 70 Riesling grape juice fermented with different inoculation protocols Table 4-14 Concentrations of metabolites binding sulphur dioxide in wines from 71 clarified Riesling grape juice fermented with different inoculation protocols Table 4-15 Concentrations of metabolites binding 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 4-17 Concentration of amino acids in unclarified 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 4-19 Concentrations of odouriferous 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 4-21 Concentrations of sulphur-containing compounds in wines from 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 OD600 values of synthetic media containing different sources of 156 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 yeasts 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 Table A-8 Assimilable nitrogen expressed as free α-amino nitrogen (FAN) in 161 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-13 Concentration of amino acids in unclarified 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-15 Concentrations of odouriferous compounds in wines from unclarified 175 Riesling grape juice fermented with different inoculation protocols Table A-16 Assimilable nitrogen expressed as free α-amino nitrogen (FAN) in 177 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-18 Concentrations of sulphur-containing compounds in wines from 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 Figure 2-3 Reaction of ammonia with α-keto-gutarate to incorporate inorganic 31 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 Figure 4-9 Assimilable nitrogen expressed as free α-amino