Investigating the role of Brettanomyces and Dekkera during winemaking

[摘要] Wine quality is greatly influenced by the number of microorganisms, which occurthroughout the winemaking process. These microorganisms are naturally present onthe grapes and in the cellar from where they can be introduced to the winemakingprocess at any given time and consequently impart specific contributions to the winequality. However, these microorganisms can be seen either as beneficial or as winespoilage microorganisms, depending on the conditions under which they canproliferate during the winemaking process. Wine yeasts (Saccharomyces spp.) aretypically responsible for the alcoholic fermentation; lactic acid bacteria (LAB) areresponsible for malolactic fermentation (MLF), while acetic acid bacteria (AAB) andother wild yeasts (non-Saccharomyces spp.) are typically associated with theformation of off-flavours under poorly controlled winemaking conditions.In recent years, evidence from the wine industry has highlighted a specific groupof non-Saccharomyces yeast species as a serious cause for wine spoilage thatrequired more research investigations. Yeast of the genus Brettanomyces or itsteleomorph Dekkera has been identified as one of the most controversial spoilagemicroorganisms during winemaking as they can produce several compounds that aredetrimental to the organoleptic quality of wine. This has triggered the researchinitiative behind this doctoral study on the significance of Brettanomyces and Dekkerayeasts during winemaking.In this dissertation, various aspects of the detection, isolation and identificationmethods of Brettanomyces yeast from the winemaking environment wereinvestigated. As a first objective, a culture collection of Brettanomyces bruxellensiswine isolates had to be established. This followed after the isolation ofBrettanomyces yeasts from various red wine cultivars from South African wineriesfrom different stages of the winemaking process. Different conventionalmicrobiological methods such as plating on selective agar media and microscopywere investigated along with molecular identification techniques such as thepolymerase chain reaction (PCR) in this regard.Other focus areas of this study aimed at performing genetic characterisation anddifferentiation studies of B. bruxellensis wine isolates. For this purpose, differentintraspecific identification methods were investigated on several strains, includingstrains of European origin. The application of molecular techniques allowing strainidentification aided in the selection of specific strains that were evaluated for volatilephenol production in synthetic media and wine. The results obtained from this workindicated that a large degree of genetic diversity exists among B. bruxellensis strainsand that the volatile phenol production differed between the strains after evaluation insynthetic media and wine.In addition to the molecular intraspecific strain identification techniques that wereinvestigated, a feasibility study was also performed that focused on evaluating Fourier transform infrared (FTIR) spectroscopy combined with chemometrics as analternative approach for differentiating between B. bruxellensis strains.The two approaches of FTIR spectroscopy that were investigated involved theuse of firstly, Fourier transform mid-infrared (FTMIR) spectroscopy to obtain spectralfingerprints of spoiled wines by different B. bruxellensis strains; and secondly,Attenuated total reflectance (FTIR-ATR) to obtain spectral fingerprints from wholecells of B. bruxellensis on microbiological agar media. The results of this studyillustrated the potential of FTIR spectroscopy to become a reliable alternative tomolecular based methods for differentiating between B. bruxellensis strains and forcharacterisation studies.The formation of volatile phenols in wine by species of the genera Brettanomycesand Dekkera is one of the primary reasons for their classification as wine spoilageyeasts. The enzymatic activities of this reaction have been identified and involve aphenyl acrylic (phenolic) acid decarboxylase (PAD) and a vinyl phenol reductase(VPR). However, only a limited amount of information is available about theseenzymes from Brettanomyces/Dekkera yeasts and no genetic data have beendescribed. It was therefore imperative that this dissertation should include a geneticinvestigation into the phenylacrylic (hydroxycinnamic) acid decarboxylase from thespecies B. bruxellensis involved in the formation of volatile phenols. Strategies thatwere investigated included various molecular DNA techniques and protein purificationprocedures to obtain either genetic or protein sequence data. The decarboxylaseactivity of this yeast species towards p-coumaric acid was demonstrated andsubstantial genetic sequence data was obtained.The results from this dissertation made a substantial contribution to the currentavailable knowledge about Brettanomyces/Dekkera spp. and led to a betterunderstanding of this wine spoilage yeast. This research developed a platform fromwhich further investigations could follow and the knowledge gained will be invaluablefor future Brettanomyces research projects at the Institute for Wine Biotechnology atStellenbosch University.

[发布机构] Stellenbosch University