Thesis (MScAgric (Viticulture and Oenology))--Stellenbosch University, 2008. / The production of good quality wine is essential to ensure competitiveness on an international level. Wine quality is usually evaluated for the visual, olfactory and taste characteristics of that specific wine. The winemaking process starts with the grapes in the vineyard followed by oenological practises in the winery until the final wine is bottled. Factors that could influence wine quality include the grape quality from which the wine is made and different techniques used during wine production. Other factors include the presence as well as the interaction between microorganisms found in the grape juice and wine, and the biochemical effect these microorganisms have on certain chemical compounds in the wine. The different microorganisms found in grape juice and wine can either have a negative or positive contribution to the final quality of the wine. During certain stages of the winemaking process the growth and metabolic activity of certain microorganisms is a necessity to produce good wine. During other stages the presence of certain microorganisms can lead to the development of compounds that is regarded as off-flavours and therefore lead to unpalatable wines of low quality. Yeast strains that naturally present on the grapes and in the winery can also contribute to the final quality of the wine. Brettanomyces yeasts are part of the natural flora of winemaking and can drastically influence the aroma characters of a wine through the production of volatile phenols. The general aroma descriptions of volatile phenols include "smoky", "spicy", "barnyard", "animal" and "medicinal". Although some wine drinkers believe that these characters can add to the complexity of a wine, high levels of volatile phenols is mostly regarded as off-flavours and mask the natural fruity flavours of a wine. With this study we wanted to generate a better understanding of the effect of different winemaking practises on the production of volatile phenols by B. bruxellensis. We evaluated the difference in volatile phenol production when B. bruxellensis was introduced before or after alcoholic fermentation. We have shown that B. bruxellensis could grow and produce volatile phenols during alcoholic fermentation. Results obtained also showed that commercial wine yeast strains could produce the vinyl derivatives that serve as precursors for Brettanomyces yeast to produce the ethyl derivatives. The commercial yeast strains differed in their ability to produce vinyl derivatives. Different malolactic fermentation scenarios were evaluated, namely spontaneous versus inoculated, and with or without yeast lees. Results showed that spontaneous malolactic fermentation had higher volatile phenol levels in the wine than inoculated malolactic fermentation. The treatment with lees reduced the level of volatile phenols, probably due to absorption by yeast cells. The presence of the phenyl acrylic decarboxylase (PAD1) gene and the production of volatile phenols by S. cerevisiae commercial yeast strains were evaluated in Shiraz grape juice and in synthetic grape juice. The results indicated that the yeast strains differ in their ability to produce 4-vinylphenol and 4-vinylguaiacol. All the yeast strains tested had the PAD1 gene. We also evaluated the presence of the phenolic acid decarboxylase (padA) gene and the ability of different lactic acid bacteria strains to produce volatile phenols in synthetic wine media. Although some of these strains tested positive for the phenolic acid decarboxylase gene most of them only produced very low levels of volatile phenols. This study made a valuable contribution on the knowledge about the effect of Brettanomyces yeast on the volatile phenol content of red wines during different stages of the winemaking process and when applying different winemaking practices. It also showed the effect between Brettanomyces yeast and other wine microorganisms and the possible influence it could have on the final quality of wine. Research such as this can therefore aid the winemaker in making certain decisions when trying to manage Brettanomyces yeast spoilage of wines.

Thesis (PhD (Genetics. Plant Biotechnology))--Stellenbosch University, 2008. / Wine quality is greatly influenced by the number of microorganisms, which occur throughout the winemaking process. These microorganisms are naturally present on the grapes and in the cellar from where they can be introduced to the winemaking process at any given time and consequently impart specific contributions to the wine quality. However, these microorganisms can be seen either as beneficial or as wine spoilage microorganisms, depending on the conditions under which they can proliferate during the winemaking process. Wine yeasts (Saccharomyces spp.) are typically responsible for the alcoholic fermentation; lactic acid bacteria (LAB) are responsible for malolactic fermentation (MLF), while acetic acid bacteria (AAB) and other wild yeasts (non-Saccharomyces spp.) are typically associated with the formation of off-flavours under poorly controlled winemaking conditions. In recent years, evidence from the wine industry has highlighted a specific group of non-Saccharomyces yeast species as a serious cause for wine spoilage that required more research investigations. Yeast of the genus Brettanomyces or its teleomorph Dekkera has been identified as one of the most controversial spoilage microorganisms during winemaking as they can produce several compounds that are detrimental to the organoleptic quality of wine. This has triggered the research initiative behind this doctoral study on the significance of Brettanomyces and Dekkera yeasts during winemaking. In this dissertation, various aspects of the detection, isolation and identification methods of Brettanomyces yeast from the winemaking environment were investigated. As a first objective, a culture collection of Brettanomyces bruxellensis wine isolates had to be established. This followed after the isolation of Brettanomyces yeasts from various red wine cultivars from South African wineries from different stages of the winemaking process. Different conventional microbiological methods such as plating on selective agar media and microscopy were investigated along with molecular identification techniques such as the polymerase chain reaction (PCR) in this regard. Other focus areas of this study aimed at performing genetic characterisation and differentiation studies of B. bruxellensis wine isolates. For this purpose, different intraspecific identification methods were investigated on several strains, including strains of European origin. The application of molecular techniques allowing strain identification aided in the selection of specific strains that were evaluated for volatile phenol production in synthetic media and wine. The results obtained from this work indicated that a large degree of genetic diversity exists among B. bruxellensis strains and that the volatile phenol production differed between the strains after evaluation in synthetic media and wine. In addition to the molecular intraspecific strain identification techniques that were investigated, a feasibility study was also performed that focused on evaluating Fourier transform infrared (FTIR) spectroscopy combined with chemometrics as an alternative approach for differentiating between B. bruxellensis strains. The two approaches of FTIR spectroscopy that were investigated involved the use of firstly, Fourier transform mid-infrared (FTMIR) spectroscopy to obtain spectral fingerprints of spoiled wines by different B. bruxellensis strains; and secondly, Attenuated total reflectance (FTIR-ATR) to obtain spectral fingerprints from whole cells of B. bruxellensis on microbiological agar media. The results of this study illustrated the potential of FTIR spectroscopy to become a reliable alternative to molecular based methods for differentiating between B. bruxellensis strains and for characterisation studies. The formation of volatile phenols in wine by species of the genera Brettanomyces and Dekkera is one of the primary reasons for their classification as wine spoilage yeasts. The enzymatic activities of this reaction have been identified and involve a phenyl acrylic (phenolic) acid decarboxylase (PAD) and a vinyl phenol reductase (VPR). However, only a limited amount of information is available about these enzymes from Brettanomyces/Dekkera yeasts and no genetic data have been described. It was therefore imperative that this dissertation should include a genetic investigation into the phenylacrylic (hydroxycinnamic) acid decarboxylase from the species B. bruxellensis involved in the formation of volatile phenols. Strategies that were investigated included various molecular DNA techniques and protein purification procedures to obtain either genetic or protein sequence data. The decarboxylase activity of this yeast species towards p-coumaric acid was demonstrated and substantial genetic sequence data was obtained. The results from this dissertation made a substantial contribution to the current available knowledge about Brettanomyces/Dekkera spp. and led to a better understanding of this wine spoilage yeast. This research developed a platform from which further investigations could follow and the knowledge gained will be invaluable for future Brettanomyces research projects at the Institute for Wine Biotechnology at Stellenbosch University.