O vinho é a bebida obtida por meio da fermentação alcoólica do mosto simples de uva sã, fresca e madura. Durante o processo fermentativo, realizado por leveduras Saccharomyces cerevisiae, ocorre uma sucessão de microrganismos. Espécies pertencentes aos gêneros Brettanomyces e Dekkera permanecem no produto final podendo influenciar de forma negativa ao produzirem compostos fenólicos voláteis. Estas leveduras apresentam como uma de suas características a lenta taxa de crescimento. Com isto, este trabalho teve como objetivo o desenvolvimento de meio de cultivo para isolamento e detecção destas leveduras e de metodologia molecular para detecção independente de cultivo. O meio de cultivo desenvolvido permitiu o isolamento e também a diferenciação de leveduras dos gêneros Brettanomyces e Dekkera das demais leveduras comumente encontradas em vinho. Uma metodologia molecular, baseada em PCR convencional, foi desenvolvida para a detecção dos gêneros em vinhos. Inicialmente foram construídos dois pares de oligonucleotídeos. Um par universal para leveduras e outro específico para D. bruxellensis. O primeiro par apresentou ampla aplicabilidade na avaliação da efetividade de diferentes processos de extração de DNA e na detecção de compostos inibidores presentes em amostras de vinho. A utilização de ambos os pares permitiu o desenvolvimento de um protocolo de extração e amplificação de DNA diretamente de amostras de vinho, facilitando a identificação de leveduras D. bruxellensis e permitindo a tomada de decisão em tempo hábil de evitar perdas econômicas. / The wine is a beverage obtained by alcoholic fermentation of simple must of healthy, fresh and mature grape. During the fermentation process, carried out by Saccharomyces cerevisiae yeasts, these is a succession of microorganisms. Species belonging to the genera Brettanomyces and Dekkera remain in the final product can influence negatively by producing volatile phenolic compounds. These yeasts present as one the features of the slow rate of growth. This work aimed at the development of culture media for isolation and detection of these strains and molecular methods for detection of independent culture. The medium developed also allowed the isolation and differentiation of yeasts of the genera Brettanomyces and Dekkera yeasts from other commonly found in wine. A molecular approach, based on conventional PCR, was developed for the detection of genera in wines. Initially we constructed two sets of primers. A universal pair for yeast and other specific for D. bruxellensis. The first pair showed a broad applicability in evaluating the effectiveness of various procedures for DNA extraction and detection of inhibitory compounds present in wine samples. The use of both pairs allowed development of a protocol for extracting and amplifying DNA directly from wine samples, facilitating the identification of yeasts D. bruxellensis and allowing the decision-making in a timely manner to avoid economic losses.

A levedura Brettanomyces/Dekkera pode causar alterações importantes em vinhos tintos, com a formação de etil-fenóis, compostos de aromas desagradáveis. Este estudo teve como objetivo determinar a presença dessa levedura e de etil-fenóis em vinhos tintos comerciais e durante a vinificação em escala industrial, além de observar sua possível inibição pelo ácido sórbico. Brettanomyces/Dekkera foi quantificada em meio seletivo, e etil-fenóis, por cromatografia gasosa. SO2 livre e total, álcool, extrato seco total, açúcares residuais, acidez total e volátil e pH também foram determinados. O crescimento durante a vinificação foi acompanhado usando diferentes meios seletivos. Dekkera bruxellensis (NRRL Y – 12961) e leveduras isoladas de vinhos brasileiros foram cultivadas em meio sintético e vinho, contendo ácido sórbico entre 0 e 250 mg/L. Das 126 amostras de vinhos comerciais, 26,98% apresentaram Brettanomyces/Dekkera, e 46,03%, etil-fenóis acima do limiar de 426 μg/L, com SO2 e álcool mostrando-se como fatores limitantes. A passagem dos vinhos por barricas e as variedades de uva não influenciaram os níveis de contaminação e de etil-fenóis. Durante a vinificação, Brettanomyces/Dekkera foi detectada a partir do mosto. A baixa população não foi suficiente para formar etil-fenóis durante cinco meses após o esmagamento. Os meios não foram completamente seletivos, especialmente para uvas e mostos. O ácido sórbico inibiu a cepa de Dekkera bruxellensis (NRRL Y – 12961), especialmente para concentrações acima de 150 mg/L, sendo variável o grau de inibição para os isolados. / The yeast Brettanomyces/Dekkera can cause significant spoilage in red wines, with the production of ethylphenols, compounds of unpleasant odors. This study aimed at determining the presence of this yeast and ethylphenols in commercial red wines during vinification in industrial scale and to observe its possible inhibition by sorbic acid. Brettanomyces/Dekkera was quantified on selective medium, while ethyphenols were quantified by gas chromatography. Free and total SO2, alcohol, total dry extract, residual sugar, total and volatile acidity, and pH were also determined. The growth during winemaking was followed using different selective media. Dekkera bruxellensis (NRRL Y – 12961) and Brazilian wines yeasts were grown in synthetic medium and in wine, containing sorbic acid between 0 and 250 mg/L. Brettanomyces/Dekkera was present in 26.98% of the 126 samples of commercial wines. The ethylphenols were above of the 426 μg/L threshold in 46.03% of the samples. SO2 and alcohol were limiting factors. The stage in barrels and the varieties did not affect the levels of contamination and ethylphenols. During winemaking, Brettanomyces/Dekkera was detected from the must. The low population was not enough to produce ethylphenols in five months after crushing. The media were not completely selective, especially for grapes and musts. Sorbic acid inhibited the strains of Dekkera bruxellensis (NRRL Y – 12961), especially at concentrations above 150 mg/L, with variable degree of inhibition for the isolates.

Leveduras do gênero Dekkera/Brettanomyces causam sérios problemas ao vinho, afetando as propriedades sensoriais do produto final. O objetivo deste trabalho foi investigar a capacidade de duas linhagens de Saccharomyces cerevisiae em inibir a atividade de Brettanomyces custersianus na vinificação em tinto e durante a fase inicial de envelhecimento, assim como investigar a capacidade deste microrganismo (Br. custersianus) em inibir a atividade metabólica de Sacch. cerevisiae. Foram realizadas vinificações com 4 inoculações diferentes. O tratamento 1 (T1) foi inoculado com a linhagem neutra Sacch. cerevisiae EMBRAPA 1vvt/97, T2-Sacch. cerevisiae EMBRAPA 91B/84 killer, T3-EMBRAPA 1vvt/97 e Br. custersianus, T4-EMBRAPA 91B/84 killer e Br. custersianus e T5-Br. custersianus. Também foram realizados, testes de velocidade fermentação, inibição ou estímulo do metabolismo e tolerância ao SO2. Durante a fase tumultuosa realizaram-se análises de açúcares redutores totais e etanol. Observou-se que durante todo o período da fermentação, o consumo de substrato de T1 e T2 foi mais rápido, do que em T3 e T4. A velocidade de fermentação da Br. custersianus (T5) foi muito inferior às demais linhagens. Mesmo com uma velocidade de crescimento baixa, a linhagem contaminante quando inoculada juntamente com Sacch. cerevisiae retarda a fermentação tumultuosa, podendo comprometer o processo de vinificação. Br. custersianus (T5) não teve sua atividade metabólica afetada na presença de 125 mg/L de SO2, logo conclui-se que as concentrações normalmente utilizadas no processo de vinificação, 30 a 70 mg/L, não seriam suficientes para impedir sua atividade. / Dekkera/Brettanomyces yeasts cause serious problems to the wine, affecting the sensorial properties of the final product. The objective of this work was to investigate the capacity of two strains of Saccharomyces cerevisiae in inhibiting the activity of Brettanomyces custersianus in the vinification in red wine and during the early stage of aging of the wine, as well as to investigate the ability of this microorganism (Br. custersianus) to inhibit metabolic activity of Saccharomyces cerevisiae. Vinifications were performed with 4 different inoculations. Treatment 1 (T1) was inoculated with the neutral strain Sacch. cerevisiae EMBRAPA 1vvt/97, T2-killer Sacch. cerevisiae EMBRAPA 91B/84, T3-EMBRAPA 1vvt/97 and Br. custersianus, T4-EMBRAPA 91B/84 and Br. custersianus and T5-Br. custersianus. There were also carried out tests of speed fermentation, inhibition or stimulation of metabolism and tolerance to SO2. During the tumultuous phase analysis was performed of total reducing sugars and ethanol. It was observed that throughout the period of fermentation, the substrate consumption in T1 and T2 was faster than in T3 and T4. The speed of fermentation of Br custersianus (T5) was much lower than the other strains. Even with a low rate of growth, the strain contaminant when inoculated with Saccharomyces cerevisiae. delays the tumultuous fermentation, being able to compromise the vinification process. Br. custersianus (T5) did not have its metabolic activity affected in the presence of 125 mg/L of SO2, then it was concluded that the concentrations normally used in the vinification process, 30 to 70 mg/L, would not be enough to prevent their activity.

O crescimento do mercado de cervejas artesanais tem demandado inovações. Uma abordagem que se destaca neste contexto é o uso de leveduras não convencionais em processos controlados de fermentação. Para ter melhores resultados neste cenário é fundamental que o produtor conheça as capacidades e limitações das leveduras que serão utilizadas na produção de cervejas. Assim, este estudo teve como objetivo avaliar características fisiológicas, essenciais e complementares, de uma cepa de Brettanomyces anomalus e uma Torulaspora delbrueckii para a produção de cervejas, comparando-as com duas cepas de Saccharomyces cerevisiae já utilizadas na indústria cervejeira. Avaliou-se o perfil cromossômico das leveduras (cariotipagem), a capacidade de crescimento em diferentes substratos e fontes de carbono, bem como em diferentes concentrações de etanol e compostos de lúpulo, a capacidade de esporulação, floculação, produção de sulfeto de hidrogênio (H2S), formação de espuma e a evolução da fermentação em função do tempo. Foram observadas diferenças entre os padrões cromossômicos das quatro leveduras. A intensidade de produção de H2S foi maior para a B. anomalus (WLP640) quando comparada com T. delbrueckii (WLP603), que foi classificada com a mesma intensidade de produção da S-33 (S. cerevisiae). As duas leveduras não convencionais atenderam às características fisiológicas essenciais para fermentação de cervejas. A B. anomalus foi capaz de metabolizar diversas fontes de carbono, como glicose, frutose, sacarose, maltose, matotriose e celobiose, ao passo que a T. delbrueckii cresceu apenas em glicose, frutose e sacarose, apontando sua potencial aplicação para produção de cervejas com teor alcoólico reduzido ou seu uso em inoculações sequenciais ou co-inoculações com outras leveduras. Ambas apresentaram crescimento em teores alcoólicos de 4% e 8%, ao passo que T. delbrueckii tolerou maior concentração de compostos do lúpulo em relação à B. anomalus, que não foi capaz de crescer em meio com as maiores concentrações combinadas de álcool (8% v/v) e α-ácidos (80 mg/L). Os resultados permitem concluir que as leveduras B. anomalus e T. delbrueckii possuem potencial para a produção de cervejas, desde que seja observada a compatibilidade de suas características fisiológicas com a expectativa acerca das características da cerveja. / The booming in the craft beer market worldwide has demanded innovations to bring up distinctive products. An approach that stands out in this context is the use of non-conventional yeasts in controlled beer fermentation processes. To have better outcomes in this scenario, it is essential that the producer knows the capabilities and limitations of those yeasts. Thus, this study aimed to assess essential and complementary physiological traits of one strain of Brettanomyces anomalus and one of Torulaspora delbrueckii for beer brewing, comparing them with two Saccharomyces cerevisiae strains already used in commercial breweries. The characteristics assessed were chromosome profile (karyotyping technique), growth capacity in different substrates and carbon sources, as well as under different concentrations of ethanol and hop compounds, the capability of sporulation, flocculation, hydrogen sulfide (H2S) production and foam formation and, finally, the fermentation evolution pattern. Differences in the chromosomal profile were observed among the four strains. The potential for H2S production was higher for B. anomalus (WLP640) when compared to T. delbrueckii (WLP603), which had the same potential than S-33 (S. cerevisiae). Both non-conventional yeasts have met the essential physiological traits demanded to carry beer wort fermentation. B. anomalus was able to metabolize many of the assessed carbon sources, as glucose, fructose, sucrose, maltose, maltotriose and cellobiose, while the T. delbrueckii strain was able to grow only in glucose, fructose and sucrose, pointing its potential application for low alcohol beer production, as well as its use in sequential inoculations or co-inoculations with other yeasts. Both showed satisfying growth under alcohol contents of 4% and 8%. T. delbrueckii tolerated higher hop compounds concentration when compared to B. anomalus, that was unable to grow at the highest combined concentrations of ethanol (8% v/v) and α-acids (80 mg/L). The results lead to the conclusion that the yeasts B. anomalus and T. delbrueckii can be explored in beer brewing, provided that an alignment between their physiological traits and the expectations around the beer characteristics are observed.

Brettanomyces bruxellensis est un microorganisme qui est considéré comme la cause majeure des défauts microbiologiques du vin. L’importance de cette levure à l’échelle industrielle est liée au fait qu’elle est isolée à partir de substrats différents tels que la bière, le kombucha, les molasses utilisées pour la production de bioéthanol et autres. Ce projet a pour objectif d’étudier la diversité génétique de l’espèce en se basant sur une large population d’isolats provenant de niches écologiques et géographiques variées. Pour ce faire, une méthode de génotypage robuste (analyse microsatellite) a été optimisée et appliquée sur la population, mettant en évidence la coexistence de populations diploïdes et triploïdes à l’échelle globale. Puis, la relation entre regroupement génétique et traits physiologiques a été explorée. Notamment, l'étude de la tolérance aux sulfites a été effectuée sur un sous-ensemble de souches représentatif de la population. Les résultats obtenus mettent en évidence un lien entre groupes génétiques et comportement vis-à-vis des sulfites. Des expériences de compétition en présence de dioxyde de soufre montrent un avantage sélectif des souches tolérantes aux sulfites par rapport aux souches sensibles, suggérant ainsi une adaptation spécifique au principal antiseptique utilisé en œnologie. Ce travail contribue à une meilleure connaissance de cette levure d’altération du vin en termes de diversité génétique et phénotypique et permet d’émettre des hypothèses sur les stratégies évolutives d'adaptation au milieu anthropique de cette espèce modèle non conventionnelle. / Brettanomyces bruxellensis is a microorganism described as the first cause of microbial spoilage of wine. Its industrial relevance is highlighted by the fact that this yeast is isolated from different substrates such as beer, kombucha, bioethanol fermentation molasses and others. This project aims to explore the genetic diversity of the species by studying a large population of isolates from various geographical and ecological niches. For this purpose, a robust genotyping method (microsatellite analysis) was optimized and applied on the population, thus highlighting the coexistence of diploid and triploid populations worldwide. Further, the relation between genotypic clustering and physiological traits was studied. Namely, sulphite tolerance assay was performed on a subset of strains representative of the total population. The results reveal a link between genetic group and growth profile in the presence of sulphur dioxide. Competition experiments in presence of sulphites highlight a selective advantage of sulphite tolerant strains compared to sulphite sensitive ones, thus suggesting a specific adaptation to the main antimicrobial used in winemaking. This work contributes to a deeper understanding of this wine spoilage microorganism in means of genetic and phenotypic diversity and sheds light on putative evolutionary strategies for adaptation to human related environment of this non-conventional model yeast species.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: In the current study, two isolates showing killer activity against several wine yeast species in a previous study were identified to strain level and found to belong to the yeast species Candida pyralidae. The identified yeast strains and a Kluyveromyces wickerhamii yeast strain used as a control exhibited killer activity against B. bruxellensis known for its spoilage characteristics in red wine, and against several strains of the genus Brettanomyces on white and red grape juice medium. The killer yeasts inhibited neither the growth of S. cerevisiae nor that of the lactic acid bacteria Oenococcus oeni and Lactobacillus plantarum strains. Yeasts are reported to secrete killer toxins, which can play a role in yeast microbial interactions under winemaking conditions. The C. pyralidae strains were found to secrete two novel killer toxins, designated CpKT1 and CpKT2. These killer toxins were stable and active under winemaking conditions, pH 3.5 - 4.5 and temperature ranges between 15 and 25°C. Ethanol and sugar concentrations found during winemaking did not affect the activity and stability of these killer toxins. Although, the killer toxins differed with regards to their biochemical and environmental stability and activity, they were found to have a similar mode of action. The killer toxins induced a fungistatic effect on B. bruxellensis sensitive cells in addition to binding to the cell wall of the sensitive cells, inducing cell surface and plasma membrane damage as did the Kwkt killer toxin secreted by K. wickerhamii. According to the author’s knowledge this is the first report on the identification of novel killer toxins secreted by C. pyralidae strains isolated from a wine environment as well as the identification of the mode of action of killer toxins on B. bruxellensis cells. This indeed provides great research scope in this field. The exoproteomes consisting of the killer toxins Kwkt, CpKT1 and CpKT2 revealed the presence of exo-glucanases and glucosidases, respectively. The enzymes KwExg1 (exoglucanase) and KwSun4 (glucosidase) retrieved from K. wickerhamii’s exoproteome were identified as the potential toxins, but their killer activity could not be confirmed. These findings suggest that hydrolytic enzymes possess killer activity, as previously reported in literature. However, further investigation is needed to identify the killer toxins characterized in this study. / AFRIKAANSE OPSOMMING: In die huidige studie is twee isolate wat in ’n vorige studie “killer” aktiwiteit teenoor verskeie wyngisspesies vertoon het, tot op rasvlak geïdentifiseer en daar is gevind dat hulle aan die gisspesie Candida pyralidae behoort. Die geïdentifiseerde gisrasse en ’n Kluyveromyces wickerhamii gisras wat as kontrole gebruik is, het “killer” aktiwiteit getoon teenoor B. bruxellensis, wat bekend is vir sy bederfkarakter in rooi wyn, en ook teenoor verskeie rasse van die genus Brettanomyces in wit en rooi druiwesapmedium. Die “killer” giste het nie die groei van óf S. cerevisiae óf van die melksuurbakteria Oenococcus oeni en Lactobacillus plantarum-rasse geïnhibeer nie. Giste word berig om “killer” gifstowwe uit te skei, wat ’n rol kan speel in gis mikrobiese interaksies onder wynbereidingstoestande. Die C. pyralidae-rasse is gevind om twee nuwe “killer” gifstowwe af te skei, wat CpKT1 en CpKT2 genoem is. Hierdie “killer” gifstowwe was stabiel en aktief onder wynbereidingstoestande, pH 3.5 - 4.5 en temperatuur tussen 15 en 25°C. Die etanol- en suikerkonsentrasies wat onder wynbereiding voorkom, het nie die aktiwiteit en stabiliteit van hierdie “killer” gifstowwe beïnvloed nie. Hoewel die “killer” gifstowwe met betrekking tot hulle biochemiese en omgewingstabiliteit en aktiwiteit verskil het, is daar gevind dat hulle ’n eenderse modus van aksie het. Die “killer” gifstowwe het ’n fungistatiese effek op B. bruxellensis sensitiewe selle geïnduseer, buiten dat dit aan die selwand van die sensitiewe selle gebind het, en het seloppervlak- en plasma-membraanskade geïnduseer, net soos die Kwkt “killer” gifstof wat deur K. wickerhamii afgeskei is. So ver die skrywer weet, is hierdie die eerste verslag van die identifisering van nuwe “killer” gifstowwe wat deur C. pyralidae rasse afgeskei word wat uit ’n wynomgewing geïsoleer is, asook van die identifikasie van die modus van aksie van “killer” gifstof op B. bruxellensis selle. Dit verbreed dus beslis die navorsingsomvang van hierdie gebied. Die eksoproteome, bestaande uit die “killer” gifstowwe Kwkt, CpKT1 en CpKT2, het die teenwoordigheid van ekso-glukanases en glukosidases onderskeidelik onthul. Die ensieme KwExg1 (eksoglukanase) en KwSun4 (glukosidase) wat vanuit K. wickerhamii se eksoproteoom herwin is, is as die potensiële gifstowwe geïdentifiseer, maar hulle “killer” aktiwiteit kon nie bevestig word nie. Hierdie bevindings suggereer dat hidrolitiese ensieme “killer” aktiwiteit besit, soos voorheen in die literatuur berig is. Verdere ondersoeke word egter benodig om die “killer” gifstowwe wat in hierdie studie gekarakteriseer is, te identifiseer.

No description available.

Memoria para optar al título profesional de Ingeniero Agrónomo Mención Enología y Vitivinicultura / El propósito del estudio fue evaluar la eliminación de Brettanomyces bruxellensis por medio de aplicaciones de ozono en mezcla gaseosa a presiones de trabajo y tiempos de exposición diferentes. La investigación se realizo con bloques de madera de roble americano en formato viniblock, los cuales fueron inoculados con las dos cepas de Brettanomyces bruxellensis utilizadas a lo largo del ensayo (cepa 1009 y 1451), donde se evaluó la mortalidad a través de un conteo directo en placas Petri post un periodo de incubación. Los mayores promedios de mortalidad obtenidos para la cepa 1009 (100%) fueron aquellos donde los bloques contaminados fueron expuestos a tiempos de 20 minutos de exposición y a una presión de trabajo de 1,8 bares, para ambas mediciones realizadas (efecto superficial y subsuperficial). En el caso de la cepa 1451 los mejores resultados fueron obtenidos al aplicarse las mismas condiciones anteriormente señaladas. La cepa 1009 tuvo una cinética de crecimiento mayor a la presentada en la cepa 1451, donde el número de colonias fue más numerosa, pero con lo que respecta al comportamiento que tuvieron frente al agente sanitizante sus promedios de mortalidad tendieron a igualarse, lo cual nos indica que ambas cepas son igualmente sensibles a las aplicaciones de ozono en mezcla gaseosa. Por lo que se concluye que el ozono es un buen sanitizante para las maderas de guarda ya que bajo ciertas condiciones de trabajo elimina la totalidad de las levaduras presentes, tanto a nivel superficial como bajo la madera, lo cual otorga la completa esterilidad de las maderas de guarda. / The purpose of this study was to evaluate the elimination of Brettanomyces bruxellensis through applications of ozone gas mixture at different pressures of work and exposure times. The research was carried out with blocks of American oak in viniblock format, which were inoculated with two strains of Brettanomyces bruxellensis used along the assay (strain 1009 and 1451), where mortality was evaluated through a direct count in Petri dishes post an incubation period. The highest average mortality obtained for strain 1009 (100%) were those contaminated blocks were exposed to times of 20 minutes of exposure and a working pressure 1.8 bar for both measurements (surface and subsurface effect) in the case of strain 1451 the best results were obtained when applying the same conditions stated above. The 1009 strain had higher growth kinetics to that presented in strain 1451, where the number of colonies was larger, but with regard to behavior which sanitizing agent were compared to averages of mortality tended to level out, which indicates that both strains are equally sensitive to ozone applications in a gaseous mixture. It that concludes the ozone is a good sanitizer for woods barrels because under certain working conditions eliminated all of yeast presents at both the surface and under the wood, which gives the complete sterility of the woods barrels.

Grape-derived aroma and flavor precursors exist partially as non-volatile, sugar-bound glycosides. Hydrolysis of these compounds may modify sensory attributes and potentially enhance wine quality. In the first study, four levels of shoot thinning (control, 20, 25, and 30 shoots per meter) with and without basal leaf removal (2-4 leaves per shoot) were established on mid-wire (90 cm), bilateral cordon-trained, mature Cabernet Sauvignon (Vitis vinifera L.) grapevines in eastern Virginia in 1996 to determine the effects on grape chemistry, glycoconjugates, and conjugate fractions. Reduced shoot density generally resulted in higher berry weight and lower soluble solids (°Brix) at each sampling date. Titratable acidity and pH were generally unaffected by shoot thinning. The 25 shoots per meter treatment displayed the greatest rate of increase in total, red-free, and phenolic-free glycoconjugates, expressed as glycosides (μmol).. Leaf removal resulted in increased pH, total phenolics, and total anthocyanins at each sampling date and a higher concentration of total, red-free, and phenolic-free glycosides. In a second study, three crop levels [high (6.4 and 5.3 kg/vine), medium (5.1 and 4.9 kg/vine), and low (3.2 and 2.6 kg/vine) ] were established on mature Cabernet Sauvignon grapevines during the 1995 and 1997 seasons, respectively. Cluster thinning of vines trained to a mid-wire (90cm), bilateral cordon-system was performed by hand three weeks post-bloom to determine the effects on grape glycoconjugates and conjugate fractions (expressed as glycosyl-glucose). In 1995, reduced crop level resulted in higher soluble solids concentration, pH, and total and red-free glycosides but did not affect berry weight or titratable acidity. In 1997, the reduced crop level treatment had higher berry weight and lower soluble solids, sugar per berry, and anthocyanins compared with the high treatment throughout the sampling period. The low treatment had the highest concentration of total, red-free, and phenolic-free glycosides per gram of fresh fruit weight on the last sampling date and the highest total, red-free, and phenolic-free glycosides per gram of fresh fruit weight when compared at similar soluble solids concentrations. Duo-trio significance testing resulted in no sensory differences among the treatments in 1997. In a third study, Pinot noir (Vitis vinifera L.) wines were inoculated with one of six genetically different strains of Brettanomyces intermedius (Ave, M, 216, Vin 1, Vin 4, and Vin5). Wines stored sur lie and those racked immediately following the completion of secondary fermentation were analyzed to determine the influence of B. intermedius strains on total, red-free, and phenolic-free glycoside concentrations (estimated by the analysis of glycosyl-glucose), and on selected free volatiles. Sur lie wines inoculated with strain Vin 4 and racked wines inoculated with Vin 4 and Vin 5 had the lowest total glycoside concentration. Hydrolysis of red-free glycosides appeared greatest in sur lie wines inoculated with Vin 4 and racked wines inoculated with Vin 4 and Vin 5. Wines stored sur lie that were inoculated with M and Vin 1 and racked wines inoculated with Vin 1, Vin 4, and Vin 5 had the lowest concentration of phenolic-free glycosides. Wines were analyzed for volatile compounds known to be produced by Brettanomyces spp. Inoculated wines were found to have detectable concentrations of ethyl-2-methylbutyrate, isoamyl alcohol, ethyldecanoate, isovaleric acid, guaiacol, 2-pheylethanol, 4-ethylguaiacol and 4-ethylphenol. There were significant differences in the concentrations of these compounds among strains. Duo-trio testing demonstrated sensory differences between the control and all inoculated wines. Differences were also found between wines inoculated with strains Ave and Vin 5, strains M and 216, and strains M and Vin 4. / Master of Science

Les levures du genre Brettanomyces sont considérées depuis longtemps comme des contaminants par les industries vinicoles et brassicoles dû aux composés phénoliques indésirables qu’elles produisent. Cependant, l’utilisation des espèces B. bruxellensis et B. anomalus en brasserie est maintenant plus répandue grâce à leur capacité à diversifier les composés aromatiques produits par la traditionnelle levure Saccharomyces cerevisiae. Malgré cette émergence dans le domaine brassicole, peu d’informations sont disponibles en ce qui concerne la diversité génétique des souches et leur potentiel en fermentation. À notre connaissance, aucune méthode permettant de caractériser, d’identifier rapidement les souches de Brettanomyces spp. et pouvant facilement être mise en place dans un contexte industriel est présentement disponible. L’objectif de l’étude était donc de développer une nouvelle méthode rapide et fiable pour le typage moléculaire des levures Brettanomyces spp. dans l’optique de différencier les espèces et les souches, ainsi que de prédire leur potentiel brassicole. À cet effet, la méthode de typage génétique Random Amplification of Microsatellites (RAM)-PCR utilisant l’amorce (CGA)₅ a été développée et validée avec la méthode Restriction Endonuclease Analysis-Pulsed-Field Gel Electrophoresis (REA-PFGE) sur vingt-deux (22) souches de Brettanomyces spp., une (1) souche de Pichia kluyveri, une (1) souche de Candida parapsilosis, une (1) souche de Schizosaccharomyces pombe et une (1) souche de Saccharomyces cerevisiae, toutes isolées de bières, ferments commerciaux, vin rouge, levain et kombucha. Des essais de fermentation primaire ont révélé que les souches ayant un bon potentiel brassicole se trouvent dans les mêmes groupes phylogénétiques générés avec la méthode RAM-PCR et l’amorce (CGA)₅. Le projet a donc permis de fournir un outil efficace pour les brasseurs et les fournisseurs de levures pour l’identification rapide des espèces, des souches et du potentiel brassicole de levures Brettanomyces spp. provenant de différents substrats, sans avoir à faire appel aux techniques de séquençage et d’essais de fermentation qui sont plus coûteux ou qui demandent plus de temps à réaliser. / The yeasts of the genus Brettanomyces are often considered as a major contaminant in the wine and beer industries because of their production of phenolic off-flavors. Recently, few species of this genus, especially B. bruxellensis and B. anomalus, have been used in beers to enlarge the pool of aromatic compounds produced by the traditional Saccharomyces cerevisiae starter used for decades in breweries. The characterization of Brettanomyces species is therefore crucial to identify the strains showing interesting technological traits. However, to our knowledge, no fast method is currently available to evaluate the genetic diversity of the genus Brettanomyces which can be easily adapted in an industrial context. The aim of this study was to develop and optimize a new, fast and reliable typing method used for the species and strains differentiation of Brettanomyces spp. isolates and for the prediction of their brewing potential. Twenty-two (22) strains of Brettanomyces, one (1) Pichia kluyveri, one (1) Candida parapsilosis, one (1) Schizosaccharomyces pombe and one (1) Saccharomyces cerevisiae were isolated from beer, red wine, kombucha, sourdough, chicha and commercial starters and used for the development of a method based on the Random Amplification of Microsatellites (RAM)-PCR technique with (CGA)₅. This method was validated with the already existent and reliable Restriction Endonuclease Analysis coupled with Pulsed-Field Gel Electrophoresis (REA-PFGE) method. Further fermentation assays revealed that potential brewing isolates were clustered in the same phylogenetic group generated with RAM-PCR electrophoretic profiles. The study allowed the development of a useful tool for brewers and yeast suppliers for the identification at the species and strains levels and the brewing potential of various Brettanomyces spp. isolates, without the need for sequencing and laborious fermentation assays.