Thesis (PhDAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Stuck alcoholic fermentations are a major enological problem for the international winemaking industry. Incomplete wine fermentations are frequently characterized by high residual fructose concentrations and the near-absence of residual glucose, a fact that is due to the glucophilic character of the wine yeast Saccharomyces cerevisiae. Wines with high contents of post fermentation sugar are very susceptible for microbial spoilage since residual fructose and/or glucose can be metabolized by bacteria and yeast to undesired by-products such as volatile acid and off-flavours, resulting in wine spoilage and considerable economic losses. It has been reported that stuck fermentations are usually caused by several synergistically acting inhibition factors, and the glucose to fructose ratio (GFR) is thought to play an important role in this context. This study is aimed at contributing towards a better understanding of this industrial problem, and at finding industrially applicable solutions. In a first part, this study describes the isolation of two appropriate strains of the fructophilic yeast Zygosaccharomyces bailii from the natural microflora of grapevine, followed by trials in small scale test fermentations using stuck industrial fermentations as model media. These experiments were expanded to also investigate large scale industrial fermentations. As a result, a strategy for the treatment of stuck fermentations was developed and successfully applied in several wineries with fermentation problems. This methodology represents an entirely novel and industrially applicable solution to high residual fructose levels. In a second part, the data contributes to elucidating the molecular nature of the fructophilic phenotype of Z. bailii by characterizing some of the genes and proteins that may be responsible for the fructophilic character. In particular, the investigation focused on the first two steps of hexose metabolism, the transport of sugar into the cell by permeases and sugar phosphorylation by hexokinases, which combined are thought to be primarily responsible for sugar preference. One result of this study was Fructoferm W3©, a dry yeast product which is commercially available. Fructoferm W3 was awarded with the innovation medal for enological products at Intervitis/Interfructa, Stuttgart, Germany in 2007. / AFRIKAANSE OPSOMMING: Die voorkoms van steek alkoholiese fermentasies is ‘n ernstige problem in die internasionale wyn industrie. Onvolledige fermentasies word dikwels gekenmerk deur hoë residuele fruktose konsentrasies en die veitlike afwesigheid van residuele glukose. Die kenmerke kan meestal toegeskryf word aan die glukofilliese kakakter van die wyngis Saccharomyces cerevisiae. Wyne met ‘n hoë suiker inhoud na die afloop van fermentasie is vatbaar vir mikrobiese bederf aangesien residuele fruktose en/of glukose gemetaboliseer kan word deur bakterië en gis om ongewenste byprodukte soos vlugtige sure en bygeure te vorm wat kan lei tot wyn bederf en aansienlike ekonomies verlies. Dit is vasgestel dat steek fermentasies gewoonlik veroorsaak word deur verskeie sinergisties werkende inhibisie faktore, waartoe die glukose/fruktose verhouding ‘n noemenswaardiege bydrae lewer. Die mikpunt van hierdie studie was om ‘n bydrae te lewer tot die begrip van steek fermentasies en die daarstelling van moontlike industriële oplossings. Die eerste deel van die werk beskryf die isolasie van twee rasse van die gis Zygosaccharomyces baillie uit die natuurlike wingerd mikroflora, gevolg deur steekproewe in die vorm van kelinskaalse fermentasies met steek industriële fermentasies gebruik as model media. Hierdie ekserimente is vervolgens uitgebrei om grootskaalse industriële steek fermentasies te bestudeer. Die uitkoms van hierdie werk het gelei tot die ontwikkeling van ‘n strategie vir die behandeling van steek fermentasies wat susksesvol toegepas is in verskeie wynmakerye. Die metodiek bring ‘n nuwe en industrieel toepasbare oplossing vir hoë residuele fruktose vlakke. Die data aangebied in die tweede afdeling dra by tot die verheldering van die molekulêre natuur van die fruktofilliese fenotipe van Z. baillie deur die tipering van gene en protiëne wat moontlik verantwoordelik is vir die fruktofilliese karakter van die gis. Die ondersoek het spesifiek op die eerste twee stappe van heksose metabolisme, naamlik die invoer van suiker in die sel deur permeases en suiker fosforilering deur heksokinases, gekonsentreer. Die kombinasie van die twee prosesse is vermoedelik verantwoordelik vir die regulering van suiker voorkeur. ‘n Gevolg van die studie was die ontwikkeling van ‘n droë gisproduk, Fructferm W3©, wat kommersieel beskikbaar gestel is. Fructoferm W3 is in 2007 toegeken met die innovasie medalje vir wynkundige produkte by Intervittis/Interfructa in Stuttgart, Duitsland.

Au Mexique l’état de Tamaulipas est susceptible de devenir l'un des plus grands producteurs de mezcal. Contrairement à d’autres boissons alcoolisées, la mycoflore responsable de la fermentation alcoolique du mezcal n'a pas été pleinement identifiée ou caractérisée métaboliquement. Il est d'un grand intérêt industriel de savoir si les levures indigènes impliquées dans la fermentation de moûts d’agave ont une forte nature fructophilique qui serait induite par la composition naturelle riche en fructose du moût. De plus, la caractérisation cinétique des levures et au niveau moléculaire des transporteurs impliqués dans la consommation de fructose par cette flore est pertinente étant donné que des études sur l'utilisation des hexoses par Saccharomyces cerevisiae n’ont pas montré que les transporteurs de glucose et de fructose étaient différents. Dans ce travail, la population levurienne étudiée était de 17 isolats, à partir de 103 initialement isolés de la mezcaleria "El Palmar" Emilio Lozoya, situé à San Carlos (Tamaulipas, Mexique) au niveau de différentes étapes. Seules les levures de l’espèce Saccharomyces cerevisiae ont été retenues et étudiées. En particulier de polymorphismes des 2 gènes des transporteurs principaux de sucres associes au fructose consume HXT1 et HXT3, a été évalué en comparaison à une souche de référence. Les résultats obtenus dans cette étude suggèrent la nécessité d'une analyse approfondie afin de préciser les liens entre la fructophilie et l'expression et / ou la structure des gènes des transporteurs de sucres lors de la fermentation alcoolique. / The Mexican state of Tamaulipas has the potential to become one of the main producers of mezcal. But contrary to what is known about other alcoholic beverages, the mycoflora involved in this alcoholic fermentation has not been completely identified nor characterised from the metabolic point of view. There is an increasing industrial interest on knowing if the native yeasts associated to agave must fermentations possess a fructophilic behaviour, favoured by the natural high fructose composition of the agave musts. Moreover, the kinetic characterisation of these yeasts and the molecular analysis carried out on the hexose transporters genes reported to be involved in the glucose and fructose consumption is pertinent, and several studies on Saccharomyces cerevisiae have demonstrated a differential preference for each hexose. In this study, a set of 17 yeast isolates were chosen from an original collection of 103 yeast isolated at the mezcalería El Palmar Emilio Lozoya, which is located at San Carlos (Tamaulipas, Mexico) from different stages of the fermentation. Only those belonging to S. cerevisiae specie were studied, mainly concerning the polymorphisms found on their two main hexose transporter genes associated to a preferential consumption of fructose, HXT1 and HXT3, and results compared to a control strain. The results obtained showed that there is no a direct link between the fructophilic phenotype and/or the structure of these genes and that more studies are needed to establish such relationships