In this diploma thesis we investigate how wine characteristics is influenced by the apiculate wine yeast Metschnikowia pulcherrima. For this purpose, two wines of a grape variety Welschriesling were manufactured using an identical technological approach with the only distinction: two separated musts were supplied with broth containing different yeasts. The literary part of the thesis discusses yeasts used in winery in general. We describe both apiculate yeasts and Saccharomyces. In this part, we also further discuss the polymer chain reaction and similar methods. The experimental part deals with possibilities of Metschnikowia pulcherrima DNA isolation from fermenting must and the subsequent quantification of yeasts with help of the real-time PCR method. After evaluation and comparison of the wines, where both general and expert public participated, it was concluded that the yeasts substantially influence the wine cha-racteristics.

The oleaginous yeasts have an ability to accumulate an increased number of lipids, under certain circumstances. These microbial lipids differentiate in the number of fat acids present, which enables their wide application in biotechnological industry. This master’s thesis is aimed on lipid production, number of the fat acid groups present, and squalene production by Metschnikowia yeasts, based on the cultivating conditions. Biomass and lipid production was observed in separate cultivation media, with the addition of the different waste substrates. Production properties were observed by method of the gas chromatography. For the squalene production observation, a HLPC method was chosen. All production groups were able to accumulate lipids on the waste substrate, although in different values. These values were very individual, especially in the areas of the specific groups and growth on the given substrate. The lipid composition was different, which was caused by differences in the waste substrates. With regards to the squalene production – the yeasts from the Metschnikowia family were not able to produce squalene in the presence of the terbinafine and its increasing concentration. Therefore, the same procedure was chosen, as it was for the Yarrowia lipolytica yeast, with the difference in the sterol synthesis, however squalene was still not produced this way.

2-phenylethanol (2PE) is a valuable fragrance compound which gives a rose-like aroma. As such 2PE is one the highest used fragrances globally. While 2PE is predominantly produced from petrochemical resources, there is a growing market for a naturally derived alternative for food products. 2PE from natural sources is priced so highly due to limited supply from rose petals. Recently, a few reports have demonstrated the production in yeasts through both the de novo production from glucose and ex novo biosynthesis with L-phenylalanine as a precursor. While these are promising most of the yeasts used can only produce low titres under optimal conditions, and the fermentation still appears to be too expensive. In this investigation the wine yeast M. pulcherrima was selected to be assessed for 2PE production. M. pulcherrima is known to produce 2PE in small titres in wine production though has yet to be explored as a platform for this product. M. pulcherrima has several advantages as a yeast platform, in that it produces a range of antimicrobials which can ward off invasive species, allowing for less sterile control in any large scale fermentation. M. pulcherrima was demonstrated to be able to produce 2PE in high titres in the batch mode through de novo synthesis of glucose, producing up to 1 g/L in shake flasks on the lab scale. Arabitol was also observed in the fermentation broth and was produced up to 20 g/L. The fermentation was then scaled up to 2L in batch mode. From these experiments, up to 700 mg/L of 2PE was produced. This is substantially more than any other yeast in the literature to date. Though when xylose or glycerol was present then 2PE production was severely limited. M. pulcherrima was also demonstrated to be able to produce 2PE by bioconversion from phenylalanine up to 1.5 g/L. This 2PE concentration is suggested to be threshold of toxicity to M. pulcherrima by the toxicity study. The production of 2PE could be increased substantially by introducing an absorbent into the process. Liquid solvents and solid adsorbents were assessed to increase 2PE production, used as in-situ 2PE adsorbents. Oleyl alcohol was found to be a good solvent for in-situ extractive solvent in M. pulcherrima culture and increase the production to 3.3 g/L which is higher than 2PE tolerance threshold of the yeast. Activated carbon was also found to be an excellent 2PE adsorbents, with maximum Langmuir adsorption capacity up to 0.807 g/g. 2PE synthesis with activated carbon as an in-situ adsorbent can increase 2PE production to 14 g 2PE/L. Finally, the process was scaled to 2L and run in batch, continuous and semi-continuous modes. This study demonstrates that not only is M. pulcherrima a viable organism to produce 2PE but it has the potential to be scaled up and run in a more cost effective semi-continuous mode when coupled to a continuous extraction technique.

L'élimination des protéines est une étape clé lors de la production du vin blanc afin d'éviter l'apparition éventuelle d'un voile inoffensif mais inesthétique. Des solutions de rechange à l'utilisation de la bentonite sont activement recherchées en raison des problèmes technologiques, organoleptiques et de durabilité associés à son utilisation. Dans cette étude, MpAPr1, une protéase aspartique extracellulaire préalablement isolée et partiellement caractérisée à partir de la levure Metschnikowia pulcherrima, a été clonée et exprimée de manière hétérologue dans la levure Komagataella pastoris. Les propriétés enzymatiques de MpAPr1 ont été initialement caractérisées dans un extrait brut. Après plusieurs essais faisant appel à différentes techniques, MpAPr1 a été purifié avec succès par chromatographie échangeuse de cations. Son activité contre les protéines de raisin a été initialement testée dans une solution modèle dans des conditions environnementales optimales pour l'activité de MpAPr1 puis dans celles qui règnent lors de la vinification. Ensuite, l'activité de MpAPr1 a été évaluée dans du moût de raisin et au cours de la fermentation alcoolique. La présence de MpAPr1, supplémenté au moût de raisin, a entraîné une dégradation partielle des protéines de raisin tout au long de la fermentation et une légère différence dans la composition en composés volatils du vin. L'étude a confirmé que les protéases aspartiques pourraient représenter une alternative à la bentonite pour l'industrie du vin et que les levures non-Saccharomyces telles que M. pulcherrima pourraient avoir un impact bénéfique sur les propriétés du vin. / Protein removal is a key step during the production of white wine in order to avoid the possible appearance of a harmless but unsightly haze. Alternatives to the use of bentonite are actively sought because of technological, organoleptic and sustainability issues associated with its use. In this study, MpAPr1, an extracellular aspartic protease previously isolated and partially characterised from the yeast Metschnikowia pulcherrima, was cloned and expressed heterologously in Komagataella pastoris. Enzymatic properties of MpAPr1 were initially characterised in a crude extract. After several attempts using different techniques, MpAPr1 was successfully purified via cation exchange chromatography. Its activity against haze-forming grape proteins was initially tested in a model solution under optimal environmental conditions for MpAPr1 activity and under those occurring during winemaking. Thereafter, MpAPr1 activity was evaluated in grape must and throughout alcoholic fermentation. The presence of MpAPr1, supplemented to grape must, resulted in the partial degradation of grape proteins throughout fermentation and ultimately in a slight difference in the wine’s composition in volatile compounds. The study provides further evidence that aspartic proteases could represent a potential alternative to bentonite for the wine industry and that non-Saccharomyces yeasts such as M. pulcherrima could have a beneficial impact on wine properties.