This thesis is concerned with the comparing of sensory and analytical characteristic of red wines infected by Brettanomyces/Dekkera. For the purposes of the research twenty four samples of red wines were selected, that had been evidently affected by Brettanomyces/Dekkera yeasts. This yeast genus produces the volatile phenols linked to the distinctive aromas/sensory compounds in red wines characterised as being reminiscent of farmyards, animals or phenolic notes. The theoretical part covers the genesis and development of the infection in red wine, and how the sensory qualities of wine are affected by Brettanomyces/Dekkera yeasts. Possible causes of the infection and ways of preventing wines from becoming infected are discussed. The experimental part focuses on the analysis and statistical assessment comparation of the sensory evaluation resulst and the chemical analysis of volatile compounds results. The level of correlation between sensory perception of volatile phenols, which are linked to these undesirable aromas, and their real content was compared.

This thesis deals with possibilities of isolation DNA yeasts genus Brettanomyces from wine with following identification using PCR. Fourteen wine samples with assumption of occurrence Brettanomyces were analysed. The literary section describes characteristic of yeasts Brettanomyces and their impact on wine. Further is described principle of PCR and use of this method in detection of Brettanomyces. Four different methods of isolation DNA directly from wine were tested. Wine samples were analysed using of five different primers by PCR. In this work were tested designed probe and primers for identification of Brettanomyces by real-time PCR.

Dissertation (PhD)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

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.

Dekkera and the closely related Brettanomyces are important yeasts in food and beverage production in part due to the metabolism of hydroxycinnamic acids (HCAs). There is a dearth of information concerning the role Brettanomyces spp. play in the food or beverage from which they are isolated and although Dekkera spp. have been investigated further there are discrepancies and questions yet to be answered. Representatives of both genera were examined to define growth and metabolism of individual HCAs in synthetic media. In addition, growth with combinations of HCAs was investigated for the first time. The results provide a comprehensive overview of HCA metabolism and volatile product formation for these genera. Furthermore, results have been confirmed in a semidefined wine medium that more closely resembled the physio-chemical parameters found in the typical wine environment. The enzymes responsible for the metabolism of HCAs were examined in Dekkera and Brettanomyces. Dekkera yeasts are known to enzymatically convert HCAs into vinylphenols (VPs) and ethylphenols (EPs). These products are indicative of Dekkera contamination. The first enzyme in the two-step HCA ─ VP ─ EP biochemical pathway is a hydroxycinnamic acid decarboxylase (HCD). This enzyme has been previously characterised from a single Dekkera strain. The second enzyme, vinylphenol reductase (VPR) has never been isolated or characterised from any microorganism. In order to further elucidate the HCA ─ VP ─ EP pathway, cell extracts were prepared from all five Dekkera and Brettanomyces spp. to evaluate activity against HCAs and VPs. Brettanomyces spp. were unable to metabolise HCAs indicating that these yeast do not have a functional HCD enzyme. Both Dekkera spp. have substrate inducible HCD activity. Temperature and pH optima were 40ºC and 5.75-6.00, respectively. The active protein was purified from cell extracts of D. anomala CBS 77 and a partial sequence was obtained. 3’RACE PCR was performed and a near complete gene sequence determined. This sequence does not have homology to HCA decarboxylase enzymes previously characterised from yeasts and bacteria and thus may represent a novel enzyme not previously described. Biochemical characterisation of the vinylphenol reductase (VPR) enzyme was also undertaken. VPR activity was found for all 5 Dekkera and Brettanomyces spp. Activity was greatest at pH 6 and between 40-50ºC and was induced by both VPs and HCAs. Data obtained during growth experiments indicated that HCAs, and in particular ferulic acid, inhibited the growth of Dekkera and Brettanomyces spp. On this basis a more detailed study was carried out to determine the concentrations required to prevent growth in various media. In a modified red wine a concentration 0.1 mM ferulic acid inhibited growth and 2 mM prevented cultures of both D. anomala and D. bruxellensis from becoming established even when re-inoculated into to a fresh HCA-free medium. Scanning electron micrographs revealed that ferulic acid caused physical damage to Dekkera cells upon exposure. This work could lead to the development of an alternative method for the control of Dekkera in wine or other food products. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1454852 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2010

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 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.

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.