Brettanomyces bruxellensis est une levure particulièrement redoutée des vinificateurs pour ses capacités d’altération organoleptique des vins. Elle est également associée à de nombreux produits fermentés et présente une importante diversité génétique en lien avec son origine écologique. L’analyse des profils microsatellites d’une collection importante d’individus (1318) d’origines géographiques variées montre une diversité génétique importante parmi les isolats de vin. Elle met notamment en évidence la coexistence d’individus diploïdes et triploïdes dans différentes régions du monde ainsi qu’à l’échelle d’un chai et d’un vin. La présence de certains génotypes dans plusieurs régions à travers le monde suggère la dispersion de cette espèce et une adaptation importante au milieu difficile qu’est le vin.La relation entre diversité génétique, matrice d’origine et traits physiologiques a été explorée. La nature des sucres utilisables pour supporter la croissance ainsi que les capacités de production de phénols volatils sont peu variables entre les souches étudiées, indépendamment de leur niveau de ploïdie ou de leur origine écologique. Néanmoins, les profils de croissance et de production de phénols volatils (vitesses et rendements) varient et traduisent des différences dans l’adaptation des souches au milieu et aux conditions d’oxygénation. Nos données suggèrent notamment une adaptation plus importante des souches triploïdes aux conditions physico-chimiques du vin. D’un point de vue pratique, l’influence de certains facteurs physico-chimiques, tels que les sucres et la température, sur le développement de B. bruxellensis dans les vins a été étudiée. Dans les vins rouges, la composition en sucres résiduels ne peut pas être considérée comme un outil de diagnostic du risque « Brett ». Néanmoins, les variations importantes de température observées dans les chais, jusqu’alors sous-estimées, pourraient expliquer en partie les phénomènes d’altération de vins rouges fréquemment observés au cours du premier été d’élevage en barrique. / The yeast species Brettanomyces bruxellensis is the most dreaded wine spoilage microorganism because of its repercussions on wine organoleptic wine alteration. It is also present in numerous fermented beverages and its high genetic diversity is partly associated with its ecological origin. Microsatellite analysis of a large collection of isolates (1318) from various geographical origins shows the species’ high genetic diversity, namely among wine strains. Notably, it highlights the coexistence of diploid and triploid individuals worldwide as well as at the region, winery and wine level. Isolation of some of the genotypes in several wine regions in the world suggests this species’ dispersion as well as the putative adaptation of these individuals to the harsh wine environment.The relationship between genetic diversity, matrix type, and physiological traits was further explored. The type of consumable sugars in relation to growth and phenol volatile production capacities of the studied strains, are independent from the ploidy level or ecological origin of the latter. Nevertheless, growth and phenol volatile production profiles (rates and yields) vary, highlighting differences in strains’ growth capacity in different media and aeration conditions. In particular, our data suggests an important adaptation of triploid strains to wine-type environment. From a practical point of view, influence of physicochemical parameters (such as sugars and temperature) on B. bruxellensis’ development in wine has been investigated. In red wine, residual sugar profiles don’t seem to be a relevant tool to estimate the risk associated with “Brett” spoilage. However, the important temperature variations occurring in wine cellars could be a possible explanation for contamination frequency during the first summer of barrel-ageing.

Thesis (MScAgric (Viticulture and Oenology))--Stellenbosch University, 2008. / The production of good quality wine is essential to ensure competitiveness on an international level. Wine quality is usually evaluated for the visual, olfactory and taste characteristics of that specific wine. The winemaking process starts with the grapes in the vineyard followed by oenological practises in the winery until the final wine is bottled. Factors that could influence wine quality include the grape quality from which the wine is made and different techniques used during wine production. Other factors include the presence as well as the interaction between microorganisms found in the grape juice and wine, and the biochemical effect these microorganisms have on certain chemical compounds in the wine. The different microorganisms found in grape juice and wine can either have a negative or positive contribution to the final quality of the wine. During certain stages of the winemaking process the growth and metabolic activity of certain microorganisms is a necessity to produce good wine. During other stages the presence of certain microorganisms can lead to the development of compounds that is regarded as off-flavours and therefore lead to unpalatable wines of low quality. Yeast strains that naturally present on the grapes and in the winery can also contribute to the final quality of the wine. Brettanomyces yeasts are part of the natural flora of winemaking and can drastically influence the aroma characters of a wine through the production of volatile phenols. The general aroma descriptions of volatile phenols include "smoky", "spicy", "barnyard", "animal" and "medicinal". Although some wine drinkers believe that these characters can add to the complexity of a wine, high levels of volatile phenols is mostly regarded as off-flavours and mask the natural fruity flavours of a wine. With this study we wanted to generate a better understanding of the effect of different winemaking practises on the production of volatile phenols by B. bruxellensis. We evaluated the difference in volatile phenol production when B. bruxellensis was introduced before or after alcoholic fermentation. We have shown that B. bruxellensis could grow and produce volatile phenols during alcoholic fermentation. Results obtained also showed that commercial wine yeast strains could produce the vinyl derivatives that serve as precursors for Brettanomyces yeast to produce the ethyl derivatives. The commercial yeast strains differed in their ability to produce vinyl derivatives. Different malolactic fermentation scenarios were evaluated, namely spontaneous versus inoculated, and with or without yeast lees. Results showed that spontaneous malolactic fermentation had higher volatile phenol levels in the wine than inoculated malolactic fermentation. The treatment with lees reduced the level of volatile phenols, probably due to absorption by yeast cells. The presence of the phenyl acrylic decarboxylase (PAD1) gene and the production of volatile phenols by S. cerevisiae commercial yeast strains were evaluated in Shiraz grape juice and in synthetic grape juice. The results indicated that the yeast strains differ in their ability to produce 4-vinylphenol and 4-vinylguaiacol. All the yeast strains tested had the PAD1 gene. We also evaluated the presence of the phenolic acid decarboxylase (padA) gene and the ability of different lactic acid bacteria strains to produce volatile phenols in synthetic wine media. Although some of these strains tested positive for the phenolic acid decarboxylase gene most of them only produced very low levels of volatile phenols. This study made a valuable contribution on the knowledge about the effect of Brettanomyces yeast on the volatile phenol content of red wines during different stages of the winemaking process and when applying different winemaking practices. It also showed the effect between Brettanomyces yeast and other wine microorganisms and the possible influence it could have on the final quality of wine. Research such as this can therefore aid the winemaker in making certain decisions when trying to manage Brettanomyces yeast spoilage of wines.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Brettanomyces bruxellensis has become of increasing interest over the past few decades yet this complex red wine spoilage yeast is still poorly understood and strain variance also leads to the contradictory results reported in literature. This yeast is responsible for the production of phenolic compounds, associated with off-flavours that render wine unpalatable. Sulphur dioxide (SO2) is the most commonly used antioxidant and antimicrobial preservative instrumental in the control of spoilage yeasts such as B. bruxellensis. However, its diploid/triploid genome is enriched for genes that provide the yeast a fortuitous advantage, under conditions permissive for growth, with genotype-dependent SO2 tolerance phenotypes observed among numerous strains. This study investigates the metabolic, physiological and genetic responses associated with SO2 exposure. It also explores the environmental cues responsible for the onset of non-SO2 induced morphological characteristics. These morphological characteristics were investigated using fluorescent probes and microscopy in the presence of SO2 and in the absence thereof, in YPD media. Pseudohyphae formation was observed to be a highly strain dependent feature and less pronounced in the presence of 0.6 mg/L molecular SO2. This study also reports on the metabolic response observed over a 3-week period, following exposure to SO2, in a synthetic wine medium. The following metabolites were consistently monitored during the course of the experiment: acetic acid, acetaldehyde, D-glucose and D-fructose. Utilization of sugars was retarded in the presence of SO2 for up to 10 days in the presence of 1.2 mg/L molecular SO2 and overproduction of acetaldehyde was prominent, with a peak at day 10. The study further highlights the expression profiles observed for the SSU1 gene (referring to SO2 tolerance) and the PAD gene (referring to production of volatile compounds) under SO2 induced conditions in SWM, using qRT-PCR. The co-involvement of increased acetaldehyde production and elevated gene expression were indicative of B. bruxellensis yeast adapting to the presence of molecular SO2, allowing survival of this fascinating yeast. Sequencing of the SSU1 and PAD genes suggests the probable existence of different alleles of these genes that could explicate SO2 tolerance and phenolic compound production associated differences among strains of this species. / AFRIKAANSE OPSOMMING: Hoewel Brettanomyces bruxellensis oor die afgelope paar dekades toenemende belangstelling gewek het, word hierdie komplekse rooiwynbederfgis steeds swak verstaan en lei rasvariasie ook tot teenstrydige resultate in die literatuur. Hierdie gis is verantwoordelik vir die produksie van fenoliese verbindings, wat geassosieer word met afgeure, wat die wyn onsmaaklik laat. Swaweldioksied (SO2) is die algemeenste preserveermiddel wat, weens antioksidant- en antimikrobiese eienskappe, instrumenteel in die beheer van bederforganismes, soos B. bruxellensis, gebruik word. Nogtans is die diploïede/triploïede genoom vir gene verryk, wat die gis ‘n toevallige voordeel bied tydens ongunstige toestande, met genotipe-afhanklike SO2 weerstandbiedende fenotipes wat onder verskeie rasse waargeneem word. Hierdie studie ondersoek die metaboliese, fisologiese en genetiese reaksies tydens SO2-blootstelling. Dit bestudeer verder die omgewingsleidrade wat vir die aanvang van die nie-SO2 geassosiseerde morfologiese eienskappe verantwoordelik is. Hierdie morfologiese eienskappe is ondersoek met behulp van fluoresserende bakens en mikroskopie in die teenwoordigheid van molekulêre SO2 en, in die afwesigheid daarvan, in YPD-medium. Pseudohyphae-vorming is as ʼn baie rasspesifieke eienskap waargeneem en is minder prominent in die teenwoordigheid van molekulêre SO2. Hierdie studie rappoteer ook oor die metaboliese reaksies waargeneem oor ‘n 3-weke tydperk, na blootstelling aan SO2, in ‘n sintetiese wynmedium. Die volgende metaboliete was voordurend gemonitor tydens die verloop van die eksperiment: asynsuur, asetaldehied, D-glukose en D-fruktose. Benutting van die suikers is in die teenwoordigheid van SO2 vertraag en oorproduksie van asetaldehied is prominent waargeneem. Hierdie studie beklemtoon verder die uitdrukkingsprofiele vir die SSU1-geen (verwys na SO2-weerstandbiedendheid) en die PAD-geen (verwys na die produksie van vlugtige verbindings) in SO2-geïnduseerde toestande in SWM, met behulp van qRT-PCR. Die gesamentlike invloed van beide verhoogde asetaldehied produksie en verhoogde uitdrukking van gene, was beduidend van B. bruxellensis-gis wat aanpas in die teenwoordigheid van molekulêre SO2, wat die oorlewing van hierdie fassinerende gis verseker. Volgordebepaling van die SSU1- en PAD-geen dui daarop dat daar waarskynlik meer as een verskillende alleel vir dié gene bestaan, wat die SO2-verdraagsaamheid en produksie van fenoliese verbindings, wat tans tussen verskeie spesies teenwoordig is, kan verduidelik.

L’état Viable Non Cultivable (VNC) a été observé et décrit chez de nombreuses espèces bactériennes. Mais cet état métabolique a également été suggéré chez certaines cellules eucaryotes, et notamment chez les levures du vin comme Brettanomyces. L’état VNC chez cette levure a donc été étudié afin d’en déterminer les conditions d’entrée et de sortie, ainsi que les modifications morphologiques et métaboliques associées à cet état VNC. Une addition de sulfite (0,8 mg/L de SO2 moléculaire) induit un état VNC chez Brettanomyces, et une inactivation de ce sulfite par modification du pH du milieu permet une sortie de l’état VNC de la levure par un regain de cultivabilité. Dans les conditions VNC, la taille moyenne des cellules de Brettanomyces a été déterminée comme diminuée de 22% comparée à leur taille en condition contrôle. Ensuite, la capacité des cellules à produire des phénols volatils, éléments de contamination des vins, est conservée même lorsque les cellules sont en état Viable Non Cultivable. De plus, l’étude comparative des protéomes entre cellules de Brettanomyces témoin et cellules en état VNC montre une modification du métabolisme avec une diminution de la synthèse d’ATP compensée par une augmentation des protéines impliquées dans d’autres voies métaboliques de production d’énergie. Cette étude met donc en évidence pour la première fois l’existence de l’état VNC chez une espèce eucaryote et montre des points communs avec l’état VNC chez les cellules procaryotes. L’existence de cet état VNC chez Brettanomyces peut également engendrer des erreurs de détection. Un nouvel outil de détection par hybridation in situ et lecture par cytométrie en flux a donc été mis en place. Cette méthode permet ainsi la mise en évidence des cellules de Brettanomyces présentes en vin de façon efficace et rapide. / The viable but not culturable (VBNC) state has been studied in detail in bacteria. It has been suggested that the VBNC state also exists in eukaryote cells, such as wine yeasts, including Brettanomyces in particular. We investigated the VBNC state in this yeast, focusing on the conditions for entry and exit, and the morphological and metabolic modifications associated with this state. We added sulfite (0.8 mg.L-1 molecular SO2) to induce the VBNC state. Increasing the pH of the medium inactivated the sulfite, allowing the cells to exit from the VBNC state and to become culturable again. In these conditions, we found that Brettanomyces VBNC cells were smaller than culturable cells, and that spoilage by volatile phenols could persist during VBNC state. Furthermore, according to our proteome comparison, it seems that the blockade of ATP synthesis was compensated by an increase in energy-producing metabolism pathway. This study provides the first insight into the VBNC state in eukaryote cells, showing common trend to the VBNC state of prokaryotic cells. The existence of VBNC state in Brettanomyces cells can also provoke errors of detection. A new tool of detection by fluorescence in situ hybridization and reading by flow cyometry was thus set up. This method allows the revealing of Brettanomyces cells presence in wine in an efficient and fast way.

Thermal processing has been used as a means of modifying the sensory aspects of wine. Cabernet Sauvignon wines were heated prior to dejuicing (3C per day from 25C to 42C) or after bottling (42C for 21 days) to determine the effects on total glycosides and glycosidic fractions. Total and phenol-free glycosidic concentrations in the wine and skins were quantified by analysis of glycosyl-glucose. Pre-dejuicing thermal vinification resulted in higher total glycosides (12%), phenol-free glycosides (18%), total hydroxycinnamates (16%), large polymeric pigments (LPP) (208%) small polymeric pigments (SPP) (41%), and lower monomeric pigments (42%) in wines. Skins had lower total glycosides (-16%), and no significant difference in phenol-free glycosides. Post-bottling heat treatment resulted in lower total (-15%) and phenol-free (-16%) glycosides, increased hue (25%), a 62% increase in LPP and a 29% decrease in monmeric pigments. A second study investigated the potential of enological spoilage microorganisms to affect wine aroma, flavor, and color. The activities of b-glucosidase were determined in model systems for fourteen strains of Brettanomyces bruxellensis yeast and nine strains of lactic acid bacteria (Oenococcus oeni). All Brettanomyces strains and seven Oenococcus strains exhibited enzymatic activity. B. bruxellensis b-glucosidase activity was primarily intracellular; O. oeni showed some extracellular activity. Yeasts and bacteria showing activity greater than 1000 nmole mL-1 g -1 for Brettanomyces, or 100 nmole mL-1 g -1 for Oenococcus, were evaluated for their effect on Viognier grape glycosides. Neither was active on native grape glycosides. / Master of Science

Les altérations sensorielles des vins dues à la présence des levures du genre Brettanomyces se caractérisent par une augmentation de la teneur en phénols volatils tel que le 4-éthylphénol. Le premier objectif de ce travail était de faire un état des lieux sur le risque « éthylphénol » au Liban en s'intéressant à la présence d'un précurseur (acide p-coumarique), du microorganisme responsable (Brettanomyces) et du produit final (4-éthylphénol) dans les vins élaborés dans ce pays. Une forte hétérogénéité de concentrations en acide p-coumarique a été observée avec des valeurs variant de 0 à 31,4 mg.L-1. Des niveaux importants de 4-éthylphénols de l'ordre de 1,367 mg.L-1 ont été détectés sur certains vins. Un dépistage du contaminant microbien a permis de confirmer pour la première fois la présence de Brettanomyces au Liban, les proportions restant toutefois assez faibles (3 % des échantillons testés). Une étude génétique a caractérisé les souches retenues qui se sont montrées diverses au sein de l'espèce. Le travail a porté ensuite sur l'analyse cinétique des étapes réactionnelles constituant le processus enzymatique de la bioconversion des substrats acide p-coumarique et 4-vinlphénol en 4-éthylphénol pour 5 souches de Brettanomyces bruxellensis d'origines libanaises et françaises. La variabilité entre les souches s'est exprimée aux niveaux génétique et cinétique. Des profils hétérogènes de bioréaction ont été mis en évidence en fonction de la nature des souches. L'analyse du bilan-matière a révélé l'existence probable de phénomènes d'adsorption sur les parois des Brettanomyces qui sont souche-dépendants. La dernière partie a été consacrée à l'évaluation du lien entre quantité de biomasse et production de 4-éthylphénol ainsi qu'à l'influence de quelques paramètres environnementaux (pH, source d'ammonium et milieu de culture) sur la cinétique réactionnelle. / Wine sensory alterations due to the presence of Brettanomyces yeasts are characterized by an increased content of volatile phenols such as 4-ethylphenol. The first aim of this work was to make an inventory of the "ethylphenol" risks in Lebanon by focusing on the presence of one precursor (p-coumaric acid), the microorganism provoking these risks (Brettanomyces) and the final product (4-ethylphenol) in wines produced in this country. High heterogeneity of p-coumaric acid concentration was observed with values ranging from 0 to 31,4 mg.L-1. Significant levels of 4-ethylphenols of about 1,367 mg L-1 have been detected in some wines. Screening of microbial contaminants confirmed the presence of Brettanomyces for the first time in Lebanon, with proportions remaining relatively low (3 % of samples tested). A genetic study has characterized the selected strains which are shown to be various within the species. The second objective of this study was the kinetic analysis of the reaction steps constituting the bioconversion enzymatic process of both substrates p-coumaric acid and 4-vinlphenol into 4-ethylphenol for 5 strains of Brettanomyces bruxellensis of different origins (Lebanon and France). Variability between strains was expressed at both levels, genetic and kinetic. Heterogeneous bioreaction profiles were identified according to strain's nature. The mass balance analysis revealed the possible existence of adsorption phenomena on the cell walls of Brettanomyces which are strain-dependent. The last part was devoted to the evaluation of the relationship between biomass concentration and production of 4-ethylphenol as well as the influence of some environmental parameters (pH, ammonium source and culture medium) on the reaction's kinetic.

Les nouvelles pratiques utilisées pour l’élaboration du vin amènent à une recrudescence des altérations microbiennes. C’est pourquoi, de nouvelles méthodes doivent être développées afin de quantifier ces microorganismes de façon précise, rapide et avec de faibles coûts. Les principales altérations du vin sont dues aux bactéries acétiques (BA) (A. aceti, A. pasteurianus, G. oxydans et Ga. liquefaciens) et à Brettanomyces bruxellensis. Par l’action d’enzymes, les 1ères transforment l’éthanol en acide acétique alors que B. bruxellensis transforme les acides hydroxycinnamiques en éthyles phénols (EP) (molécules odorantes désagréables). La cytométrie en flux couplée à la technique d’hybridation in situ en fluorescence a tout d’abord été étudiée. Aucun résultat reproductible n’a été développé pour les BA en vin rouge alors que pour B. bruxellensis, le protocole existant a été amélioré avec une quantification possible en 18 h. La PCR en temps réel a également été utilisées afin de quantifier ces microorganismes. Un protocole a été développé pour la quantification des BA en vin rouge (103 cellules/mL) avec l’utilisation d’un témoin interne microbiologique permettant de valider le rendement de l’extraction de l’ADN. Pour B. bruxellensis, trois kits commerciaux ont été analysés lors d’une étude interlaboratoires. Les quantifications se sont révélées significativement différentes des énumérations sur boite de Pétri avec une quantification des cellules mortes. De plus, il a été étudié et validé l’effet population de B. bruxellensis sur l’efficacité du SO2. Il ressort également de ces expérimentations que les cellules en état viable mais non cultivable ne produisent pas d’EP. / New practices used to elaborate wine lead to an increase of wine spoilage due to microorganisms. That is why, new technics have to be developed to quantify these microorganisms accurately, quickly and with low costs. The main wine spoilages are due to acetic acid bacteria (AAB) (A. aceti, A. pasteurianus, G. oxydans and Ga. liquefaciens) and Brettanomyces bruxellensis development. AAB transforms ethanol to acetic acid while B. bruxellensis transforms hydroxycinnamic acids to ethyl phenols (EP) (unpleasant odor molecules). In order to detect these wine spoilage microrganisms, flow cytometry coupled to fluorescent in situ hybridization has been assessed. No reproducible results have been developed for AAB in red wine while for B. bruxellensis, the existing protocol has been improved with a possible quantification after 18 h compared to 48-72 h in the previous protocol. The real-time PCR was also used to quantify these microorganisms. A protocol has been developed for the AAB quantification in red wine (103 cells/mL) with the use of a microbiological internal control to validate the DNA yield after extraction. For B. bruxellensis, three commercial kits were analyzed in an interlaboratory study. Quantifications were significantly different to the enumerations by Petri dish, with dead cell quantifications. Moreover, we demonstrated that the effectiveness of sulfite is dependent of the B. bruxellensis population. It also appears from these experiments that cells in viable but not culturable state do not produce EP.

Thesis (PhD (Genetics. Plant Biotechnology))--Stellenbosch University, 2008. / Wine quality is greatly influenced by the number of microorganisms, which occur throughout the winemaking process. These microorganisms are naturally present on the grapes and in the cellar from where they can be introduced to the winemaking process at any given time and consequently impart specific contributions to the wine quality. However, these microorganisms can be seen either as beneficial or as wine spoilage microorganisms, depending on the conditions under which they can proliferate during the winemaking process. Wine yeasts (Saccharomyces spp.) are typically responsible for the alcoholic fermentation; lactic acid bacteria (LAB) are responsible for malolactic fermentation (MLF), while acetic acid bacteria (AAB) and other wild yeasts (non-Saccharomyces spp.) are typically associated with the formation of off-flavours under poorly controlled winemaking conditions. In recent years, evidence from the wine industry has highlighted a specific group of non-Saccharomyces yeast species as a serious cause for wine spoilage that required more research investigations. Yeast of the genus Brettanomyces or its teleomorph Dekkera has been identified as one of the most controversial spoilage microorganisms during winemaking as they can produce several compounds that are detrimental to the organoleptic quality of wine. This has triggered the research initiative behind this doctoral study on the significance of Brettanomyces and Dekkera yeasts during winemaking. In this dissertation, various aspects of the detection, isolation and identification methods of Brettanomyces yeast from the winemaking environment were investigated. As a first objective, a culture collection of Brettanomyces bruxellensis wine isolates had to be established. This followed after the isolation of Brettanomyces yeasts from various red wine cultivars from South African wineries from different stages of the winemaking process. Different conventional microbiological methods such as plating on selective agar media and microscopy were investigated along with molecular identification techniques such as the polymerase chain reaction (PCR) in this regard. Other focus areas of this study aimed at performing genetic characterisation and differentiation studies of B. bruxellensis wine isolates. For this purpose, different intraspecific identification methods were investigated on several strains, including strains of European origin. The application of molecular techniques allowing strain identification aided in the selection of specific strains that were evaluated for volatile phenol production in synthetic media and wine. The results obtained from this work indicated that a large degree of genetic diversity exists among B. bruxellensis strains and that the volatile phenol production differed between the strains after evaluation in synthetic media and wine. In addition to the molecular intraspecific strain identification techniques that were investigated, a feasibility study was also performed that focused on evaluating Fourier transform infrared (FTIR) spectroscopy combined with chemometrics as an alternative approach for differentiating between B. bruxellensis strains. The two approaches of FTIR spectroscopy that were investigated involved the use of firstly, Fourier transform mid-infrared (FTMIR) spectroscopy to obtain spectral fingerprints of spoiled wines by different B. bruxellensis strains; and secondly, Attenuated total reflectance (FTIR-ATR) to obtain spectral fingerprints from whole cells of B. bruxellensis on microbiological agar media. The results of this study illustrated the potential of FTIR spectroscopy to become a reliable alternative to molecular based methods for differentiating between B. bruxellensis strains and for characterisation studies. The formation of volatile phenols in wine by species of the genera Brettanomyces and Dekkera is one of the primary reasons for their classification as wine spoilage yeasts. The enzymatic activities of this reaction have been identified and involve a phenyl acrylic (phenolic) acid decarboxylase (PAD) and a vinyl phenol reductase (VPR). However, only a limited amount of information is available about these enzymes from Brettanomyces/Dekkera yeasts and no genetic data have been described. It was therefore imperative that this dissertation should include a genetic investigation into the phenylacrylic (hydroxycinnamic) acid decarboxylase from the species B. bruxellensis involved in the formation of volatile phenols. Strategies that were investigated included various molecular DNA techniques and protein purification procedures to obtain either genetic or protein sequence data. The decarboxylase activity of this yeast species towards p-coumaric acid was demonstrated and substantial genetic sequence data was obtained. The results from this dissertation made a substantial contribution to the current available knowledge about Brettanomyces/Dekkera spp. and led to a better understanding of this wine spoilage yeast. This research developed a platform from which further investigations could follow and the knowledge gained will be invaluable for future Brettanomyces research projects at the Institute for Wine Biotechnology at Stellenbosch University.

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2009. / Grape juice is transformed into wine through the complex processes of alcoholic and malolactic fermentation that is performed by yeasts, lactic acid bacteria and acetic acid bacteria. However, the microbes involved in these processes do not only take part in ensuring the successful production of wine, but also cause spoilage of the wine if their growth is not controlled. Conventional, culture-dependent methods of microbiology have been used as the main technique in detecting and identifying these spoilage microbes. Cultureindependent techniques of molecular biology have recently become more popular in detecting possible spoilage microbes present in must and wine, since it allows the detection and identification of viable, but non-culturable microbes and are not as timeconsuming as conventional microbiological methods. The aim of this study was to investigate the sustainability of polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis in detecting wine spoilage microbes inoculated into sterile saline solution (SSS) (0.85% (m/v) NaCl) and sterile white wine and red wine as single microbial species and as part of mixed microbial populations. Three methods of DNA isolation from SSS, sterile white wine and sterile red wine inoculated with reference microbial strains were compared in terms of DNA concentration and purity, as well as simplicity of the technique. These three DNA isolation methods were the TZ-method, the proteinase K-method and the phenol extraction method. DNA could not successfully be isolated from red wine using any of the three DNA isolation methods. The TZ-method was the method of choice for the isolation of DNA from inoculated SSS and sterile white wine as this technique gave the best results in terms of simplicity, DNA concentration and purity. PCR and DGGE conditions were optimised for the universal primer pair, HDA1-GC and HDA2, the wine-bacteria specific primer pair, WBAC1-GC and WBAC2, and the yeast specific primer pair, NL1-GC and LS2. DNA from Acetobacter pasteurianus, Lactobacillus plantarum, Pediococcus pentosaceus, Oenococcus oeni, Brettanomyces bruxellensis and Saccharomyces cerevisiae were amplified with the appropriate primers and successfully resolved with DGGE analysis. PCR and DGGE detection limits were successfully determined when 106 cfu.ml-1 of the reference microbes, A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis were separately inoculated into SSS and sterile white wine. It was possible to detect low concentrations (101 cfu.ml-1) with PCR for A. pasteurianus, Lb. plantarum, Grape juice is transformed into wine through the complex processes of alcoholic and malolactic fermentation that is performed by yeasts, lactic acid bacteria and acetic acid bacteria. However, the microbes involved in these processes do not only take part in ensuring the successful production of wine, but also cause spoilage of the wine if their growth is not controlled. Conventional, culture-dependent methods of microbiology have been used as the main technique in detecting and identifying these spoilage microbes. Cultureindependent techniques of molecular biology have recently become more popular in detecting possible spoilage microbes present in must and wine, since it allows the detection and identification of viable, but non-culturable microbes and are not as timeconsuming as conventional microbiological methods. The aim of this study was to investigate the sustainability of polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis in detecting wine spoilage microbes inoculated into sterile saline solution (SSS) (0.85% (m/v) NaCl) and sterile white wine and red wine as single microbial species and as part of mixed microbial populations. Three methods of DNA isolation from SSS, sterile white wine and sterile red wine inoculated with reference microbial strains were compared in terms of DNA concentration and purity, as well as simplicity of the technique. These three DNA isolation methods were the TZ-method, the proteinase K-method and the phenol extraction method. DNA could not successfully be isolated from red wine using any of the three DNA isolation methods. The TZ-method was the method of choice for the isolation of DNA from inoculated SSS and sterile white wine as this technique gave the best results in terms of simplicity, DNA concentration and purity. PCR and DGGE conditions were optimised for the universal primer pair, HDA1-GC and HDA2, the wine-bacteria specific primer pair, WBAC1-GC and WBAC2, and the yeast specific primer pair, NL1-GC and LS2. DNA from Acetobacter pasteurianus, Lactobacillus plantarum, Pediococcus pentosaceus, Oenococcus oeni, Brettanomyces bruxellensis and Saccharomyces cerevisiae were amplified with the appropriate primers and successfully resolved with DGGE analysis. PCR and DGGE detection limits were successfully determined when 106 cfu.ml-1 of the reference microbes, A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis were separately inoculated into SSS and sterile white wine. It was possible to detect low concentrations (101 cfu.ml-1) with PCR for A. pasteurianus, Lb. plantarum, iv Pd. pentosaceus, and B. bruxellensis in SSS when amplified with the HDA1-GC and HDA2 primer pair. A PCR detection limit of 102 cfu.ml-1 was determined in sterile white wine for Pd. pentosaceus and 103 cfu.ml-1 for B. bruxellensis using this primer pair. The results obtained from the PCR amplification with the WBAC1-GC and WBAC2 primer pair compared well with the results of the HDA1-GC and HDA2 primer pair. The results from the DGGE detection limits indicated that it was possible to detect lower concentrations (101 – 102 cfu.ml-1) of A. pasteurianus, Lb. plantarum and Pd. pentosaceus with the HDA1-GC and HDA2 primer pair than the WBAC-GC and WBAC2 primer pair (102 – 104 cfu.ml-1). Lower detection limits were also determined for B. bruxellensis amplified with the HDA1-GC and HDA2 primer pair (103 – 104 cfu.ml-1) than with the NL1-GC and LS2 primer pair (105 cfu.ml-1). PCR and DGGE detection limits for the inoculation of A. pasteurianus, Lb. plantarum and B. bruxellensis at an inoculum of 108 cfu.ml-1 as part of mixed populations in SSS and sterile white wine compared well with the results obtained from the reference microbes inoculated as single microbial species. PCR detection limits of 101 cfu.ml-1 were determined for all three reference microbes inoculated as part of mixed populations when amplified with the HDA1-GC and HDA2 and the WBAC1-GC and WBAC2 primer pairs. It was observed that similar or higher DGGE detection limits were obtained for the reference microbes inoculated in sterile white wine (101 – 107 cfu.ml-1) than when inoculated into SSS (101 – 105 cfu.ml-1). PCR-based DGGE analysis proved to be a technique that could be used successfully with the universal, wine-bacteria and yeast specific primer pairs for the detection of A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis. The culture-independent technique makes the early detection of possible spoilage microbes at low concentrations in wine possible.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The yeast Brettanomyces was isolated from beer in 1904 and associated with wine thereafter. A sporulating form, Dekkera, was discovered later. Brettanomyces bruxellensis produces high levels of volatile phenol off-flavours in wine. Sulphur dioxide (SO2) is the most widely used chemical preservative in wine. Yeasts have several mechanisms to cope with the SO2, namely Ssu1p, a membrane bound SO2 transporter; sulphite reduction, sulphite oxidation and acetaldehyde production. In unfavourable environmental conditions, certain yeasts can enter a viable-but-non-culturable (VBNC) state which is characterised by reduced metabolic rate, inability to reproduce on solid media and a reduction of cell size. VBNC can be triggered by chemical stress such as high SO2 levels. The objectives of this study were to examine the SO2 tolerance of B. bruxellensis and Saccharomyces cerevisiae, to quantify their rates of SO2 accumulation and efflux, determine the effect of SO2 on their energy metabolism and investigate if B. bruxellensis possesses an orthologue to S. cerevisiae SSU1. In this study, the identity of a number of Brettanomyces/Dekkera strains was confirmed using 5.8S rDNA-ITS RFLP analysis and DNA sequencing. Sporulation assays were used to confirm whether these strains belonged to the Dekkera or Brettanomyces genus. A method to accurately quantify SO2 in laboratory conditions was optimised. Molecular SO2 tolerance was tested by spotting fresh yeast cultures on media with SO2 and/or ethanol. Tolerance to SO2 and/or ethanol showed highly strain dependent results with S. cerevisiae showing the highest tolerance levels while B. bruxellensis tolerated SO2 and ethanol poorly but certain strains grew well with only SO2. The SO2 accumulation and efflux rates of 3 S. cerevisiae strains and 3 B. bruxellensis strains were determined. It was shown that the S. cerevisiae strains followed the same trends as previously found in literature whereas B. bruxellensis strains showed similar trends but displayed highly variable strain-dependent results. B. bruxellensis CB63 and S. cerevisiae VIN13 were investigated for their response to SO2 in two different media, TA and SWM, over a 48-hour and 32-day period respectively. Acetic acid, acetaldehyde, D-glucose, D-fructose (only in SWM) and ethanol (only in TA) were regularly monitored over the time course of each experiment. SO2 had the greatest impact on B. bruxellensis with decreased rates of glucose consumption and ethanol production as well as increased acetic acid. Acetaldehyde peaked shortly after SO2 addition with the subsequent restarting of sugar consumption for certain samples. This suggests that sufficient acetaldehyde was produced to bind free SO2 to reduce SO2 stress. Volatile phenols were quantified for day 32 of the SWM experiment. An increase of 4-ethyl guaiacol was correlated to higher molecular SO2 levels. SO2 negatively affected both yeasts energy metabolism, forcing the yeasts metabolism to adapt to ensure survival. In general, SO2 was shown to have a negative impact on all aspects of a yeasts growth and metabolism and that SO2 tolerance is highly strain dependent and a far more complicated characteristic than currently understood. / AFRIKAANSE OPSOMMING: Die gis Brettanomyces is in 1904 uit bier geïsoleer en daarna met wyn geassosieer. 'n sporulerende vorm, Dekkera, is later ontdek. Brettanomyces bruxellensis produseer hoë vlakke van vlugtige fenol afgeure in wyn. Swaweldioksied (SO2) is die mees gebruikte chemiese preserveermiddel in wyn. Giste het verskeie meganismes om SO2 te hanteer, naamlik Ssu1p, 'n membraan-gebonde SO2 transporter, sulfietvermindering, sulfiet-oksidasie en asetaldehiedproduksie. In ongunstige omgewingstoestande kan sekere giste 'n lewensvatbare, maar nie-kultiveerbare (LMNK)-toestand aanneem wat gekenmerk word deur verlaagde metaboliese tempo, onvermoë om voort te plant op soliede media en 'n vermindering van die selgrootte. LMNK kan veroorsaak word deur chemiese stres, soos hoë SO2-vlak. Die doelwitte van hierdie studie was om die SO2 -bestandheid van B. bruxellensis en Saccharomyces cerevisiae te ondersoek, hul spoed van SO2 -opneming/akkumulasie en -uitskeiding te kwantifiseer, die invloed van SO2 op energiemetabolisme te bepaal en te ondersoek of B. bruxellensis oor ‘n soortgelyke geen as die S. cerevisiae SSU1 beskik. In hierdie studie is die identiteit van 'n aantal Brettanomyces/Dekkera-stamme bevestig deur 5.8S rDNA-ITS RFLP-analise en DNA-opeenvolging te gebruik. Sporulasietoetse is gebruik om te bevestig of hierdie stamme aan die genus Dekkera of Brettanomyces behoort. 'n Metode om SO2 onder laboratoriumtoestande akkuraat te kwantifiseer, is geoptimiseer. Molekulêre SO2- bestandheid is getoets deur vars giskulture op media met SO2 en/of etanol te groei. Bestandheid teen SO2 en/of etanol het stam-afhanklike resultate getoon, S. cerevisiae wat die hoogste toleransievlakke getoon het, terwyl B. bruxellensis SO2 en etanol swak tolereer, maar sekere stamme het goed gegroei met slegs SO2. Die SO2-akkumulasie en -uitskeidingtempo van 3 S. cerevisiae-rasse en 3 B. bruxellensis-stamme is bepaal. Daar is gevind dat die S. cerevisiae-rasse dieselfde tendens soos voorheen in die literatuur beskryf, gevolg het, terwyl B. bruxellensis-stamme soortgelyke tendense getoon het,maar hoogs veranderlike stamafhanklike resultate vertoon. B. bruxellensis CB63 en S. cerevisiae VIN13 is ondersoek vir hul reaksie tot SO2 in twee verskillende media, TA en SWM, oor 'n tydperk van 48-uur en 32-dae onderskeidelik. Asynsuur, asetaldehied, D-glukose, D-fruktose (slegs in SWM) en etanol (slegs in TA) is gereeld gemoniteer oor die verloop van elke eksperiment. SO2 het die grootste impak op B. bruxellensis met ‘n verlaagde tempo van glukoseverbruik en etanolproduksie, sowel as verhoogde asynsuur. ‘n Asetaldehiedhoogtepunt is bereik kort na die SO2-byvoeging met die daaropvolgende hervatting van suiker wat vir sekere monsters gebruik is. Dit dui daarop dat voldoende asetaldehied geproduseer is om vry SO2 te bind om SO2-stres te verminder. Vlugtige fenole is op dag 32 van die SWM-eksperiment gekwantifiseer. 'n Toename van 4-etiel-guajakol korreleer met hoër molekulêre SO2-vlakke. SO2 het beide giste se energiemetabolisme negatief beïnvloed, wat die gis dwing om sy metabolisme aan te pas om oorlewing te verseker. Oor die algemeen het SO2 'n negatiewe impak op alle aspekte van giste se groei en metabolisme, en SO2-bestandheid is hoogs stam–afhanklik. Dit is ook 'n baie meer ingewikkelde kenmerk as wat tans verstaan word.