In der ersten Hälfte der Weingärung dominiert die Weinhefe Kloeckera apiculata (perfekte Form: Hanseniaspora uvarum), auch wenn eine Starterkultur der Reinzuchthefe Saccharomyces cerevisiae zugesetzt wurde. Sogenannte Spontangärungen ohne einen solchen Zusatz ergeben gelegentlich qualitativ bessere Weine. Allerdings überwiegt das Risiko von Gärstockungen (z.B. durch Essigsäure-Produktion) und Fehltönen bei der Spontangärung ihre potentiellen Vorteile in der großtechnischen Weinherstellung. Dort könnten wesentliche Qualitätssteigerungen durch die parallele Zugabe von Starterkulturen eines modifizierten K. apiculata Stammes erzielt werden, der die Aromakomposition verbessern könnte, ohne sich negativ auf die Produktqualität auszuwirken. Zu diesem Zweck ist ein vertieftes Verständnis der Genetik und Physiologie von K. apiculata nötig. Aufgrund dessen wurde daher mit dem Aufbau eines molekulargenetischen Systems dieser Hefe begonnen. Dafür wurde zunächst genomische DNA präpariert und zur Herstellung einer Genbank verwendet, mit deren Hilfe einige Marken (HIS3, URA3, TRP1 und LEU2) über heterologe Komplementation in entsprechenden S. cerevisiae-Stämmen erhalten wurden. Die so isolierten Gene sollen in Zukunft dazu dienen, um E.coli/K. apiculata „-Shuttle-Vektoren“ für Klonierungen zu konstruieren. Weiterhin soll versucht werden, Deletionen im Kloeckera Genom zu erhalten um gezielt Stoffwechselwege auszuschalten. So zum Beispiel den Stoffwechselweg, der zur Essigsäureproduktion führt. Erste Untersuchungen zur Phosphofructokinase, dem ersten für die Glykolyse spezifischen Enzym auf dem Weg zur alkoholischen Gärung, legen ähnlich wie bei S. cerevisiae einen heterooktameren Aufbau des Enzyms nahe. Erste Ergebnisse aus der enzymatischen Analyse nach heterologer Expression der kodierenden Gene in S. cerevisiae werden vorgestellt. Zudem wurden mit der Sequenzierung des vollständigen Genoms von K. apiculata begonnen und es konnten etwa 90% der Sequenz entschlüsselt werden. Damit konnten sehr viele Homologe zu Genen identifiziert werden, die in anderen Hefearten für Proteine mit bekannter Funktion kodieren. Darüber hinaus lassen die vorläufigen Ergebnisse vermuten, dass eine Genomduplikation, wie sie für Hefen aus der Saccharomyces-Gruppe postuliert wird, in K. apiculata noch nicht stattgefunden hat. Die ersten Ergebnisse aus vergleichenden FACS-Analysen deuten an, dass es sich bei K. apiculata um einen diploiden Organismus handelt. Es ist ebenfalls gelungen, die Chromosomen von K. apiculata mit Hilfe einer speziellen Gelelektrophorese- Technik (PFGE) zu trennen. Nach einem Southern-Blot konnten zudem durch Hybridisierung mit Sonden einzelne Gene den entsprechenden Chromosomen zugeordnet werden.

Made available in DSpace on 2017-07-21T18:53:19Z (GMT). No. of bitstreams: 1 Caroline M E Santos.pdf: 1575163 bytes, checksum: 7a4f0fd823c8bbe2f2632a343ded69ca (MD5) Previous issue date: 2011-03-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Brazilian cider, sparkling apple wine with low-alcohol content, has little color and low aromatic quality, requiring correction of the final sugar content. French cider is considered the best in the world due to its physical-chemical and aromatic sensations, through the fermentation process that enhances oxidative step that contributes intensively to the formation of aromatic compounds. Thus, this study aimed to evaluate the profile of volatile compounds of fermented apple prepared under action of different oxidative and fermentation yeasts. We used apple juice from Gala and monitored the growth of the strains, the physicochemical and kinetic aspects over 10 days of fermentation. The technique of headspace was used for collect of volatiles that were identified and quantified by gas chromatography.Oxidative yeasts used, Hanseniaspora uvarum and Hanseniaspora guilliermondii, when kept with no other strains in the wort, showed fermentation activity and remained residual sugar in the finished product, so thus can be used in the preparation of fermented products with low alcohol content. The volatile compounds were obtained in small amounts, but all have floral and fruity aromas that contribute to the aromatic quality of the product. Fermentative strains used were Saccharomyces cerevisiae Bouquet, Saccharomyces cerevisiae Arome Plus, Saccharomyces cerevisiae Fermol Aromatic and Saccharomyces cerevisiae UFLA CA-11. These showed expected fermentation activity with maintenance of residual sugar but the volatile compounds were not pleasant aromatic quality. The mixed fermentations were carried out under action of Saccharomyces cerevisiae Bouquet whit Hanseniaspora uvarum and Saccharomyces cerevisiae Bouquet with Hanseniaspora guilliermondii, using different sequential inoculations. The results showed that there was good interaction between fermentative and oxidative yeasts, because in addition to presenting appropriate alcoholic, maintained the presence of residual sugar, the volatile compounds contributed to the aromatic quality. In this study it was observed that the headspace technique is not efficient for volatile compounds with high points of volatilization, thereby detecting a small amount of compounds in each product. The beverage prepared most appreciated in relation to the aromatic profile in the sensory analysis was prepared under the exclusive action of Hanseniaspora guilliermondii, which is considered a good choice of strain to be used in the fermented apple production because it was highly producing esters, compounds that contribute to fruity and sweet aromas in fermentation processes. / A sidra brasileira, bebida frisante de maçã de baixo grau alcoólico, apresenta cor pouco intensa e baixa qualidade aromática, necessitando de correção do teor de açúcar final. A bebida francesa é considerada a melhor do mundo devido às suas características físicoquímicas e qualidade aromática, através de processo que valoriza a fermentação oxidativa, etapa que contribui intensivamente para a formação de compostos aromáticos da bebida. Desta forma, este trabalho teve como objetivo avaliar o perfil de compostos voláteis de fermentados de maçã elaborados sob ação de diferentes leveduras oxidativas e fermentativas. Foi utilizado mosto de maçã da cultivar Gala e monitorado o crescimento das cepas, parâmetros físico-químicos e cinéticos ao longo de 10 dias de processo fermentativo. A técnica de headspace foi utilizada para a coleta dos voláteis que foram identificados e quantificados por cromatografia gasosa. As leveduras oxidativas, Hanseniaspora uvarum e Hanseniaspora guilliermondii, quando mantidas no mosto sem adição de outras cepas, apresentaram atividade fermentativa e mantiveram açúcares residuais no produto final, podendo assim serem usadas na elaboração de produtos fermentados com baixa graduação alcoólica. Os compostos voláteis foram captados em baixa quantidade, no entanto todos com aromas frutais e florais que contribuem para a qualidade aromática da bebida. As cepas fermentativas utilizadas foram: Saccharomyces cerevisiae Bouquet, Saccharomyces cerevisiae Arome Plus, Saccharomyces cerevisiae Fermol Aromatic e Saccharomyces cerevisiae UFLA CA-11. As cepas fermentativas apresentaram atividade de fermentação desejada com manutenção de açúcares residuais mas os compostos voláteis não apresentaram qualidade aromática agradável. As fermentações mistas foram realizadas sob ação de accharomyces cerevisae Bouquet com Hanseniaspora uvarum, e de Saccharomyces cerevisae Bouquet com Hanseniaspora guilliermondii, utilizando-se de inoculações sequenciais. Os resultados evidenciaram que houve uma boa interação entre as leveduras fermentativas e oxidativas, pois os fermentados obtidos além de apresentarem grau alcoólico adequado, mantiveram a presença de açúcares residuais e os compostos voláteis que contribuíram para a qualidade aromática da bebida. Neste trabalho foi possível perceber que a técnica de headspace utilizada não foi eficiente para compostos voláteis com altos pontos de volatilização, detectando assim uma pequena quantidade de compostos em cada produto. A bebida elaborada que mais agradou em relação ao perfil aromático na análise sensorial foi a elaborada sob ação de processo puro com a cepa Hanseniaspora guilliermondii, sendo esta considerada uma boa opção a ser utilizada na produção de fermentados de maçã, pois foi altamente produtora de ésteres, compostos que contribuem para aromas frutados e doces em processos fermentativos.

This research investigated yeast populations and diversity during pre-fermentation cold maceration and alcoholic fermentation of Vitis vinifera L. cv. Pinot noir grapes from a commercial vineyard (Dayton, OR). Fermentations were conducted at the Oregon State University research winery in 100 L tanks while grapes from the same vineyard lot were fermented at a commercial winery. Samples were taken daily during pre-fermentation maceration (9°C) and alcoholic fermentation (27°C) and plated on WL and lysine media to determine Saccharomyces and non-Saccharomyces populations and diversity. Total non-Saccharomyces populations increased from 1 x 10³ cfu/mL to 1 x 10⁵ cfu/mL during pre-fermentation cold maceration and reached a maximum of 1 x 10⁷ cfu/mL during alcoholic fermentation. Thirteen distinct yeast species were tentatively identified based on appearance on WL media and were initially screened for β-glucosidase activity using 4-methyllumbelliferyl-β-D-gluconopyranoside (4-MUG) plates. The identity of the isolates screening positive for β-glucosidase activity was determined by sequence analysis of the D1/D2 domain of the 26S rDNA gene. The five isolates identified were Metschnikowia pulcherrima, Hanseniaspora uvarum, Kluveromyces thermotolerans, and two Saccharomyces cerevisiae isolates. β-glucosidase activity was further characterized and quantified using a liquid media representing grape must conditions (pH 3.5, 20° Brix) at two temperatures (25°C and 8°C). While increasing sugar concentration suppressed the β-glucosidase activity of H. uvarum (-99%), β-glucosidase activity still remained relatively high for M. pulcherrima, S. cerevisiae isolate 1, and S. cerevisiae isolate 2. At 8°C, β-glucosidase activity was reduced for M. pulcherrima compared to activity at 25°C, but activity increased for K. thermotolerans, S. cerevisiae isolate 1, and S. cerevisiae isolate 2. The yeast isolates possessing β-glucosidase activity were used in fermentations of Vitis vinifera L. cv. Pinot Noir grapes. The grapes were treated with high hydrostatic pressure (HHP) to inactivate naturally occurring yeast and bacteria. All yeast isolates grew during pre-fermentation cold maceration (7 days at 9°C) and populations increased 3 to 4 logs. Following pre-fermentation cold maceration, all ferments were warmed to 27°C and inoculated with S. cerevisiae RC212. Alcoholic fermentations were all complete within eight days and after pressing wines were analyzed for volatile aroma compounds by SPME-GC-MS. The presence of different yeast isolates during pre-fermentation cold maceration resulted in wines with unique aroma profiles. Ethyl ester concentrations were highest in the wine that did not undergo a pre-fermentation cold maceration, while concentrations of branch-chained esters were higher in the treatments with yeast present during pre-fermentation cold maceration. Pre-fermentation cold maceration with yeast isolates demonstrating β-glucosidase did not affect the concentration of β-damascenone or β-ionone. Wines that had undergone pre-fermentation cold maceration with S. cerevisiae isolate 1, S. cerevisiae isolate 2, and a combination of all isolates resulted in over twice the concentration of β-citronellol over wines that did not undergo a pre-fermentation cold maceration. / Graduation date: 2013