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Interactions between Brettanomyces bruxellensis and other yeast species in the initial stages of winemaking
Abstract
Aims:
Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking.
Methods and results:
Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation.
Conclusions:
Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine.
Significance and impact of the study:
Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.
... Based on the surveyed literature it can be proposed that these yeasts have the ability to proliferate, survive and contaminate the wine during various processing phases of winemaking. These yeasts are also able to affect both alcoholic and malolactic fermentation (Renouf et al., 2006a), and they are mostly reported to occur mainly in aging processes, especially in oak barrels (Rubio et al., 2015). Therefore, the off-odours can appear during any of different wine production stages. ...
... Regarding that high ethanol concentration, D. bruxellensis is less sensitive in comparison to Saccharomyces cerevisiae (Abbott et al., 2004) and represents one of the rare species that can survive and develop in media which is nutritionally exhausted. Wine with low sugar content and high alcohol concentration after alcoholic fermentation (AF) is a good example of such media (Renouf et al., 2006a). Chandra et al. (2015) evaluated three red grape varieties for susceptibility to D. bruxellensis during both alcoholic and malolactic fermentations and found that both examined strains survived well during fermentations. ...
... Besides, these yeasts are also able to affect both fermentations, i.e. AF and MLF (Rodrigues et al., 2001;Renouf et al., 2006a). Therefore, the off-odours can appear during all wine production stages, but it has been reported that this occurs mainly during aging processes, especially in oak barrels (Froudiѐre and Larue, 1988;Chatonnet et al., 1992Chatonnet et al., , 1993Silva et al., 2004Silva et al., , 2005Malfeito-Ferreira et al., 2004;Rubio et al., 2015). ...
The aim of this research has been to examine the microbiological and technological parameters influencing volatile phenols (VP) production in wine of the autochthonous Montenegrin grape varieties Kratošija and Vranac and the international grape variety Cabernet Sauvignon. Within two consecutive vintages (2012 and 2013) grape must and wines have been analysed on the presence of Dekkera bruxellensis yeast and on hydroxycinnamic acids (HCA) as precursors of VP. Among technological parameters, the influence of commercial yeast and lactic acid bacteria (LAB) and the addition of oak alternatives on wine chemical composition and sensory profile has been examined. Caffeic acid represented the prevalent HCA compound for all varietal wines, with the exception of Kratošija in the 2012 vintage in which ferulic acid (93.2%) was the main HCA. Higher total content of all three examined HCA was observed in all varietal wines inoculated with commercial yeast in 2012, as compared to 2013, due to the high content of caffeic acid in the 2012 vintage. Statistically significant differences in VP content were found between commercial yeast, LAB and oak alternatives. The presence of Dekkera bruxellensis spp. has been noticed only in the 2013 vintage for Vranac control wine after alcoholic fermentation (AF), confirming that these yeasts are present in extremely low numbers at the beginning of AF only. Vranac wines, inoculated with commercial yeast, contained the highest content of p-coumaric acid in 2012 (1.54 mg/L), followed by Kratošija wines in the 2013 vintage (1.03 mg/L). The highest number of samples with detected VP from the vintage 2012 were found for Vranac wines (12 out of 28), while in 2013 vintage, Kratošija had the highest number of samples (18 out of 28). VP have appeared only in wines of autochthonous grape varieties, after long bottle aging and the number of infected samples were vintage dependent. Comparing autochthonous Montenegrin wines to Cabernet Sauvignon, it can be concluded that this international variety grown in agro-ecological conditions of Ćemovsko field does not show increased VP even after three or four years of aging in bottles. It has been concluded that VP are formed predominantly in wines with high alcohol content, low pH, low reducing sugars content, and even in wines with free SO2 above 30 mg/L
... Elles peuvent également se développer et être isolées en bouteille (Renouf, 2006). ...
... Pendant la FML, les faibles concentrations en SO2 libre, l'augmentation du pH et la libération de nutriments tels que le tréhalose par l'autolyse des levures seraient favorables à la croissance de B. bruxellensis (Renouf, 2006 ;Oelofse, 2008 (Fugelsang, 1997 ;Laureano et al., 2005). Cela leur fournit une protection contre le SO2 et leur permet de se développer grâce à l'hydrolyse des sources carbonées disponibles et notamment la libération de cellobiose (Fugelsang, 1997 ;Swaffield et al., 1997 ;Oelofse, 2008). ...
... Contrairement à S. cerevisiae (Goddard et al., 2010), B. bruxellensis n'a pas été isolée dans des environnements dits naturels (sols, écorces…) autour du vignoble. En revanche, elle a, dans de rares occasions, été isolée de raisins (Guerzoni et Marchetti, 1987 ;Renouf et Lonvaud-Funel, 2006 ;Barbin, 2006). Néanmoins, après le vin, l'habitat privilégié de cette levure est le chai (Fugelsang, 1998 ;Connel et al., 2002). ...
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.
... Especially when damaged berries are taken into consideration, they can carry a high number of undesirable yeast cell populations (Barata, Malfeito-Ferreira, & Loureiro, 2011;Guerzoni & Marchetti, 1987;Pretorius, 2000). Among these, Brettanomyces bruxellensis was isolated from several vineyards and in different stages of grape berry development, using mainly enrichment media (Renouf et al., 2006;Renouf & Lonvaud-Funel, 2007). The yeasts belonging to the genus Dekkera/Brettanomyces are mainly responsible for wine spoilage during its storage in cellars, particularly in red wines. ...
... Particularly, the reduction of B. bruxellensis is twice that of the apiculate yeast in treatments with EW and WO and even four times higher in GO treatments. In addition to this, Renouf et al. (2006Renouf et al. ( ), 2007, demonstrated that the concentration of B. bruxellensis found in fresh grape must after crushing is 2.0 Log CFU/mL. As a consequence, the reduction of 2.1 Log observed in grapes treated with GO for 24 h, could help to limit the spreading of this undesirable yeast and prevent the contamination of the winery from the vineyard. ...
... However, towards the end of fermentation, more B. bruxellensis cells were found as S. cerevisiae viable population started to decline. This is correlated with the higher ethanol tolerance of B. bruxellensis than S. cerevisiae in conditions of low sugar concentrations (Renouf et al., 2006). It is important to take into account that, at the end of fermentation, the population of B. bruxellensis was lower after GO treatments when compared with that after EW and WO treatments, this fact is reflected in the data of the acetic acid present in the wines. ...
In this study, we investigated the possible effect of electrolyzed water (EW), aqueous ozone (WO) and gaseous ozone (GO) on Brettanomyces bruxellensis DSM 7001 strain artificially inoculated on the grape surface and on its evolution during the subsequent, inoculated must fermentation. Culture-dependent and -independent techniques were used to evaluate the effectiveness of treatments against B. bruxellensis, as well as its presence during fermentation. Particularly, GO treatment of 24 h decreased its presence by about 2.1 Log, making it possible to reduce significantly the concentration of ethylphenols in the wine in relation to the control wine. EW and WO treatments caused less relevant reductions. The results showed that all the treatments reduced the presence of this yeast on grapes. However, in these experimental conditions it was not possible to achieve a complete removal of this undesirable yeast.
... When these reactions occur, 4-EP and 4-EG accumulate during the winemaking process (Berna et al. 2008;Suárez et al. 2007), particularly during barrel aging (Nikfardjam et al. 2009), and in bottles (though this occurs to a lesser extent) (Loscos et al. 2010). Several reports have indicated that 4-EG and 4-EP can be generated during alcoholic fermentation in a laboratory (Heresztyn 1986;Renouf et al. 2006), but similar reports do not exist for 4-EG and 4-EP generation in a winery. ...
... During alcoholic fermentation, Brettanomyces/Dekkera and Saccharomyces cerevisiae use ferulic acid and p-coumaric acid to generate 4-EG and 4-EP, respectively (Heresztyn 1986;Renouf et al. 2006;Ribéreau-Gayon et al. 2006). The first step was using cinnamic acid decarboxylase to change ferulic acid and p-coumaric acid into 4-vinylphenol (4-VP) and 4-vinylguaiacol (4-VG). ...
... The first step was using cinnamic acid decarboxylase to change ferulic acid and p-coumaric acid into 4-vinylphenol (4-VP) and 4-vinylguaiacol (4-VG). After that, under the effect of vinyl phenol reductase, 4-VP and 4-EG generated 4-EG and 4-EP (Renouf et al. 2006;Ribéreau-Gayon et al. 2006). Therefore, the dynamic changes of 4-EP, 4-EG, ferulic acid and p-coumaric acid during wine alcoholic fermentation in the winery were assessed, and the results are shown in Fig. 5. ...
A simple and convenient HS-SPME-GC-based detection method with high detection limit, good recoveries, highly reproducibility, minimal matrix effects and a 30-min total run time for 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) in red wines has been developed. The 4-EG concentration that was generated during alcoholic fermentation exhibited a biphasic trend, with an initial increase that was followed by a decrease to approximately 40–50 μg/L after alcoholic fermentation in the winery. The content of one precursor substance (ferulic acid) strongly correlated with the 4-EG content, and the 4-EG decrease observed late in fermentation may be due to its adsorption by the yeast cell wall. 4-EP was undetectable during alcoholic fermentation in the winery. Furthermore, 4-EG and 4-EP were detected in most of the commercial wines sampled from the Chinese wine market. The regions of origin, the grape varieties, and the oak barrel aging times had strong impacts on 4-EP and 4-EG.
... bruxellensis (Figure 8), se révélant ainsi critiques pour le vinificateur (Wedral et al., 2010). (Nardi et al., 2010;Renouf et al., 2006). L'élevage, qui fait suite à la FML, est une étape caractérisée par une activité microbienne très faible et représente également un moment propice au développement de B. bruxellensis. ...
... This yeast is responsible for the production of volatile phenols and most importantly 4-ethylphenol, which contributes to undesirable aromas described as "Brett character" (Chatonnet et al., 1992;Oelofse et al., 2008;Wedral et al., 2010), leading to rejection by consumers and to heavy economic losses (Fugelsang, 1997;Lattey et al., 2010). This yeast can be found at several steps in the winemaking process (Chatonnet et al., 1992;Renouf et al., 2006Renouf et al., , 2009Rubio et al., 2015;Suárez et al., 2007) due to its resistance to multiple stress conditions (Avramova et al., 2018b;Conterno et al., 2006;Longin et al., 2016;Schifferdecker et al., 2014;Serpaggi et al., 2012;Smith and Divol, 2016). The ability to form biofilm is another potential resistance strategy (Tek et al., 2018;Verstrepen and Klis, 2006), although in the case of B. bruxellensis it has been given only little attention so far. ...
La gestion des contaminations par la levure d’altération Brettanomyces bruxellensis est un véritable défi pour la filière viti-vinicole. Le mode de vie biofilm, connu pour accroitre la résistance des micro-organismes et permettre leur persistance dans l’environnement, est une stratégie pouvant être adoptée par B. bruxellensis.Dans ce projet de thèse, des observations microscopiques ont permis de mettre en évidence la présence de matrice autour des cellules, un élément essentiel de la définition d’un biofilm. L’étude a également révélé que différents morphotypes sont impliqués dans la structure du biofilm, en particulier des filaments formant un véritable réseau. Des chlamydospore-like jusque-là jamais décrites chez l’espèce B. bruxellensis, ont été observées au sein du biofilm mais également dans des cultures planctoniques. La production de tels éléments pourrait être une stratégie de la levure pour mieux persister dans les environnements stressants. Des différences notables dans la quantité de cellules adhérées ont été observées en fonction de la nature des supports et des milieux utilisés, démontrant l’impact de l’environnement sur la formation de biofilm chez B. bruxellensis. En particulier, l’absence de glucose semble diminuer la capacité d’adhésion de plusieurs souches de B. bruxellensis.De plus, l’invasion de gélose chez B. bruxellensis nouvellement décrite au cours de ce travail se caractérise par le développement de structures multicellulaires diverses à l’intérieur du milieu gélosé, composées notamment de filaments. L’analyse optimisée à travers un pipeline d’acquisition et de traitement d’images révèle que la présence de glucose et d’oxygène favorise l’invasion de gélose. B. bruxellensis semble également capable de former des structures biofilm-like telles que des biofilms air-liquide et des colonies complexes.Enfin, les capacités d’adhésion, de formation de biofilm et d’invasion semblent souche dépendantes, étayant les connaissances à propos de l’importante diversité intraspécifique chez B. bruxellensis. Deux méthodologies rapides et fiables ont été adaptées afin de discriminer des souches au sein de groupes génétiques précédemment définis : un protocole de RAPD-PCR et un outil de deep learning. Ce dernier se base sur la diversité de morphologie cellulaire pour prédire le groupe génétique d’un isolat avec une précision de 96,6%. Cette approche nouvelle ouvre la voie pour la mise en place de méthodes de routine simples et accessibles aux acteurs de la filière viti-vinicole pour la prévention des risques de contamination par B. bruxellensis.
... Saccharomyces et Saccharomyces cerevisiae (Renouf et al., 2006) : elle est moins adaptée dans un milieu riche en sucre (Barata et al., 2008b;Nardi et al., 2010). Dans le cas de fermentations alcooliques languissantes, Brettanomyces bruxellensis peut profiter de la baisse d'activité de Saccharomyces cerevisiae pour se développer. ...
... Dans le cas de fermentations alcooliques languissantes, Brettanomyces bruxellensis peut profiter de la baisse d'activité de Saccharomyces cerevisiae pour se développer. Elle est également capable de subsister dans les vins et donc finir par être dominante notamment pendant l'élevage des vins ou même en bouteille (Renouf et al., 2006). ...
L’utilisation de conservateur chimique dans les produits agro-alimentaires est source de controverse, et l’œnologie n’en fait pas l’exception notamment avec le dioxyde de soufre (SO2). Cet adjudant est utilisé depuis de nombreuses années au cours du processus de vinification pour ses propriétés antimicrobiennes, antioxydantes et antioxydasiques. Face aux attentes sociétales et aux problèmes de normes, les viticulteurs s’engagent vers la production de vins à faibles teneurs en sulfites voire zéro sulfites. Pour les praticiens, s’en détacher demeure d’une décision hasardeuse et dont les conséquences sur la conduite des fermentations et les caractéristiques chimiques et sensorielles des vins restent peu connues. L’ajout de levure non-Saccharomyces en tant que bioprotection est considéré actuellement comme alternative au premier sulfitage. Ce travail a pour objectifs d’évaluer les conséquences de la suppression du sulfitage et de l’utilisation de la bioprotection sur la communauté microbienne des phase préfermentaires et fermentaires, mais également sur la composition chimique et sensorielle des vins.En absence de sulfitage, les populations de Metschnikowia pulcherrima et de Hanseniaspora spp augmentent pendant la phase préfermentaire et les indices de diversité sont significativement plus faibles du fait de la colonisation du milieu par certaines espèces, notamment Hanseniaspora dans les moûts rouges. De plus, le dioxyde de soufre impacte principalement la communauté fongique et levure du point de vue de l’abondance des OTUs mais également la diversité des souches indigènes de S. cerevisiae en particulier dans le cas des moûts blancs. Les deux espèces utilisées en bioprotection, Torulaspora delbrueckii et dans une moindre mesure Metschnikowia pulcherrima s’implantent dans le moût de raisin au cours des étapes préfermentaires, avec pour conséquences une forte diminution des abondances relatives des communautés fongiques en comparaison avec le témoin sans SO2 probablement du fait de phénomènes de compétition vis-à-vis de l’occupation du milieu. Cet effet occupation de la niche est moins important dans le cas de vendanges à maturité avancée. En revanche, aucun effet négatif n’est observé sur les niveaux de populations de Hanseniaspora spp. Dans nos conditions expérimentales en blanc, l’absence de SO2 entraine une oxydation des flavonoïdes dans le moût, une évolution de la couleur et une concentration en glutathion plus faible dans les vins blancs finis. Les deux espèces utilisées en tant que bioprotection consomment l’oxygène dans le moût de raisin permettant ainsi une protection partielle vis-à-vis de l’oxydation tout en limitant le développement des bactéries acétiques. Une étude préliminaire basée sur des analyses métabolomiques montrent que la composition des vins rouges sans sulfites est significativement différente de celle des vins élaborés avec un sulfitage classique à l’encuvage. D’un point de vue sensoriel, les vins sans sulfites sont caractérisés par des notes de cerises noires cuites et une perception de fraicheur au nez et en bouche alors que les vins sulfités sont corrélés à des notes de fumée. Enfin, l’utilisation précoce des levures non-Saccharomyces en bioprotection impacte significativement les caractéristiques chimiques des vins avec, au niveau sensoriel, une augmentation du caractère fruité des vins jeunes qui reste néanmoins limitée par rapport à l’utilisation des mêmes souches en fermentation mixe avec Saccharomyces cerevisiae.
... Molecular techniques are useful to obtain a representative image of the species diversity. The most frequent methods are PCR based techniques like restriction fragment length polymorphism (PCR-RFLP) (Mitrakul et al., 1999) and polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) (Renouf et al., 2006), which are used for species identification. Furthermore, fingerprinting techniques are liable for intraspecific species characterization like random amplified polymorphism (RAPD), amplified fragment length polymorphism (AFLP), pulsed field electrophoresis (REA-PFGE), amplification of internal transcribed spacer (ITS) and microsatellite markers analysis (Albertin et al., 2014;Avramova et al., 2018aAvramova et al., , 2018bMiot-Sertier and Lonvaud-Funel, 2007;Mitrakul et al., 1999;Oelofse et al., 2009). ...
... B. bruxellensis can resist during all the winemaking steps, but is mostly known for growing and expressing the ethyl phenol character during stuck alcoholic or malolactic fermentation and during aging in oak barrels. The genetic and physiological characterization of B. bruxellensis strains is a way for understanding and unrevealing the resistance mechanism to antimicrobial agents and persistence capacity to various niches (Borneman et al., 2014;Conterno et al., 2010;Curtin et al., 2012;Hellborg and Piškur, 2009;Renouf et al., 2006;Wedral et al., 2010). This is the first study that focuses on the strains of B. bruxellensis isolated from Greek wine. ...
Brettanomyces bruxellensis is the most common spoilage wine yeast which can provoke great economic damage to the wine industry due to the production of undesirable odors. The capacity of the species to adapt in various environmental conditions offers a selective advantage that is reflected by intraspecific variability at genotypic and phenotypic level. In this study, microsatellite analysis of 22 strains isolated from Greek wine revealed the existence of distinct genetic subgroups that are correlated with their geographical origin. The response of these strains to increasing levels of sulfur dioxide confirmed the presence of both sensitive and tolerant strains, which belong to distinguished genetic clusters. The genetic categorization of B. bruxellensis strains could be used by the winemakers as a diagnostic tool regarding sulfur dioxide sensitivity.
... All of these factors narrow the spectrum of culturable organisms present, although next-generation sequencing analysis suggest a persistence of diversity. Numerous studies have categorized the changes and persistence of non-Saccharomyces strains during inoculated and uninoculated fermentations (Vezinhet et al. 1992;Querol et al. 1994;Schutz and Gafner 1994;Constanti et al. 1997;Gutierrez et al. 1997Gutierrez et al. , 1999Van der Westhuizen et al. 2000b;Torija et al. 2001;Beltran et al. 2002;Van Keulen et al. 2003;Hierro et al. 2006;Renouf et al. 2006b;Xufre et al. 2006). Yeast population dynamics are impacted by winemaking conditions. ...
... As crush progresses juices may pick up 10 3 -10 4 cells/mL of S. cerevisiae which represents a striking alteration of the juice microbial community (Boulton et al. 1996). Analysis of the surfaces of barrels indicated high numbers of Saccharomyces, with Candida, Cryptococcus, and Brettanomyces also commonly present, although in lower concentrations (Renouf et al. 2006b(Renouf et al. , 2007. Sanitation practices can therefore have a dramatic effect on the organisms present during fermentation by either allowing or restricting the buildup of high populations on equipment and in tanks. ...
Wine is the end product of the fermentative activity of yeast and bacteria. The microbiota of grape juice fermentation can vary significantly as over 40 genera and 100 different species of yeast have been isolated from grapes or wine (Table 3.1). Although the genera listed are commonly identified in surveys of grape mycobiota, some yeast species are more universally found than others, and numerous factors impact the composition of the yeast microbial community of grapes and their persistence during fermentation. Saccharomyces cerevisiae is the primary agent responsible for the conversion of grape sugars into alcohol but other yeast, collectively known as non-Saccharomyces yeast, and bacteria may also contribute to the aroma and flavor profile of the wine. Thus interspecies as well as intraspecies diversity plays an important role in the evolution of wine composition.
... In the first phases of vinification, it is usually present at lower concentrations than other yeasts responsible for alcoholic fermentation. Successively and during malolactic fermentation (MLF), B. bruxellensis may increase (Renouf et al. 2006) and become the dominant yeast, therefore seriously affecting the sensorial traits of wine. The ability to tolerate environmental stresses such as high ethanol concentrations (up to 14.5-15%), low pH and oxygen, low sugar (smaller than 300 mg/l) and fermentable nitrogen concentrations suggests that B. bruxellensis adapted to this peculiar niche . ...
... During the last 20 years the molecular and functional diversity of B. bruxellensis isolates collected worldwide has been extensively studied (see Oelofse et al. 2008;Curtin et al. 2015). Different strains of B. bruxellensis can produce variable yields of volatile phenols not always correlated with its growth (Silva et al. 2004;Conterno et al. 2006;Renouf et al. 2006;Curtin et al. 2007;Vigentini et al. 2008;Romano et al. 2008;Joseph et al. 2013). Barata et al. (2008), studying the effect of sugar concentration and temperature on cellular viability and 4-ethylphenol production, found that the levels of ethylphenols were intrinsically linked to B. bruxellensis growth. ...
Yeasts belonging to the Brettanomyces/Dekkera genus are non-conventional yeasts, which affect winemaking by causing wine spoilage all over the world. This mini-review focuses on recent results concerning the presence of Brettanomyces bruxellensis throughout the wine processing chain. Here, culture-dependent and independent methods to detect this yeast on grapes and at the very early stage of wine production are encompassed. Chemical, physical and biological tools, devised for the prevention and control of such a detrimental species during winemaking are also presented. Finally, the mini-review identifies future research areas relevant to the improvement of wine safety and sensory profiles.
... Spoilage by B. bruxellensis occurs mainly during wine aging in oak barrels [5]. However, this yeast may also affect alcoholic fermentation (AF) or malolactic fermentation (MLF) [6,7]. ...
... Contrarily, S. cerevisiae started to diminish after completion of AF and could not be detected during MLF and storage period. These observations corroborate the study of Renouf et al. [6] showing B. bruxellensis to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar depletion. ...
Three red grapevine varieties, Touriga Nacional, Syrah and Cabernet Sauvignon, were evaluated for their susceptibility to spoilage by two strains of Brettanomyces bruxellensis during grape juice fermentation and in finished wine. B. bruxellensis ISA 2211 survived well during both alcoholic and malolactic fermentations. The 4-ethylphenol concentration at the end of malolactic fermentation was 176 µg L−1 for T. Nacional, 190 µg L−1 for C. Sauvignon and 149 µg L−1 for Syrah. These levels were slightly increased during 3 months of incubation, even though no culturable cells of B. bruxellensis could be detected after malolactic fermentation. However, a viable but non-culturable (VBNC) population of 3–4 log cells mL−1 was observed in all grape varieties when analyzed by flow cytometry. On the other hand, commercial varietal wines, inoculated with of B. bruxellensis ISA 1791, could attain levels of 6–8 log CFU mL−1 and 4-ethylphenol reached 1750, 1512 and 2707 µg L−1 in T. Nacional, C. Sauvignon and Syrah, respectively. Overall, the production of 4-ethylphenol was mostly dependent on the existence of growing B. bruxellensis populations, regardless of the grape variety. In finished wines, 4-ethylphenol was always produced in concentrations far above its preference threshold, indicating that T. Nacional, Syrah and C. Sauvignon are equally susceptible to the “horse sweat” taint.
... The yeast Dekkera bruxellensis [1] is well-known for its participation in industrial processes, being fairly linked to the production of alcoholic beverages, such as wine and beer [2]. Previous studies have identified D. bruxellensis as a main contaminant in the bioethanol production process in northeast Brazil, with differential factors in the establishment and adaptability to this environment, such as the advantageous assimilation of nitrate [3][4][5]. ...
Dekkera bruxellensis has been studied for several aspects of its metabolism over the past years, which has expanded our comprehension on its importance to industrial fermentation processes and uncovered its industrial relevance. Acetate is a metabolite often found in D. bruxellensis aerobic cultivations, whereas its production is linked to decreased ethanol yields. In a previous work, we aimed to understand how acetate metabolism affected the fermentation capacity of D. bruxellensis. In the present work, we evaluated the role of acetate metabolism in respiring cells using ammonium or nitrate as nitrogen sources. Our results showed that galactose is a strictly respiratory sugar and that a relevant part of its carbon is lost, while the remaining is metabolised through the Pdh bypass pathway before being assimilated into biomass. When this pathway was blocked, yeast growth was reduced while more carbon was assimilated to the biomass. In nitrate, more acetate was produced as expected, which increased carbon assimilation, although less galactose was uptaken from the medium. This scenario was not affected by the Pdh bypass inhibition. The confirmation that acetate production was crucial for carbon assimilation was brought by cultivations in pyruvate. All physiological data were connected to the expression patterns of PFK1, PDC1, ADH1, ALD3, ALD5 and ATP1 genes. Other respiring carbon sources could only be properly used by the cells when some external acetate was supplied. Therefore, the results reported herein helped in providing valuable contributions to the understanding of the oxidative metabolism in this potential industrial yeast.
... The first co-culture studies were mainly established to understand the natural or synthetic interactions between human microbiota and pathogens as well as to improve biochemical processes in the food (Islam et al., 2017), solvent and oil industries (Kumari and Naraian, 2016;Tao et al., 2017). Successful examples of industrially applied co-cultures can be found in wastewater treatment, soil remediation, gas production (Chaudhry and Chapalamadugu, 1991), and food products such as dairy products (Sodini et al., 2000;Martin et al., 2001;Narvhus and Gadaga, 2003;Kariluoto et al., 2006), salami (Dicks et al., 2004) and alcoholic beverages (Cort et al., 1994;Beek et al., 2002;Fleet, 2003;Clemente-Jimenez et al., 2005;Renouf et al., 2006). However, it is now well accepted that mixed cultures can also be successfully applied to improve limiting steps of a biosynthetic pathway (Zhang and Stephanopoulos, 2016), prevent enzymes from byproducts biosynthesis, increase single-cell protein production (Tesfaw and Assefa, 2014), induce or increase bioactivity (Chanos and Mygind, 2016;Sung et al., 2017), suppress virulence (Minerdi et al., 2009) and improve the bioactive metabolite production by the presence of a microorganism that alters media composition (Shimizu et al., 1999;Liu et al., 2006;Ariana and Hamedi, 2017). ...
In natural product research, microbial metabolites have tremendous potential to provide new therapeutic agents since extremely diverse chemical structures can be found in the nearly infinite microbial population. Conventionally, these specialized metabolites are screened by single-strain cultures. However, owing to the lack of biotic and abiotic interactions in monocultures, the growth conditions are significantly different from those encountered in a natural environment and result in less diversity and the frequent re-isolation of known compounds. In the last decade, several methods have been developed to eventually understand the physiological conditions under which cryptic microbial genes are activated in an attempt to stimulate their biosynthesis and elicit the production of hitherto unexpressed chemical diversity. Among those, co-cultivation is one of the most efficient ways to induce silenced pathways, mimicking the competitive microbial environment for the production and holistic regulation of metabolites, and has become a golden methodology for metabolome expansion. It does not require previous knowledge of the signaling mechanism and genome nor any special equipment for cultivation and data interpretation. Several reviews have shown the potential of co-cultivation to produce new biologically active leads. However, only a few studies have detailed experimental, analytical, and microbiological strategies for efficiently inducing bioactive molecules by co-culture. Therefore, we reviewed studies applying co-culture to induce secondary metabolite pathways to provide insights into experimental variables compatible with high-throughput analytical procedures. Mixed-fermentation publications from 1978 to 2022 were assessed regarding types of co-culture set-ups, metabolic induction, and interaction effects.
... This process can take from 5-15 days depending on the temperatures and duration of the cycles used. Cold skin contact prior to fermentation has been speculated to reinforce the capacity of Brettanomyces to adapt to the medium rather than inhibiting it [45]. When combined with typical fermentation durations of 4-21 days or more, the overall time required for effective colour extraction is considerable. ...
... For example, β-glucan seems to increase the resistance of the producing bacteria to acidic pH [18,27,48]. In France, the prevalence of ropy O. oeni strains is particularly high in regions producing naturally more acidic wines (Bourgogne or Champagne) [18,73,74]. The increase in viscosity induced by β-glucan may slow down the diffusion of abiotic compounds, such as phenolic compounds, sulfites, and ethanol, and protect the bacteria. ...
Lactic acid bacteria (LAB) from fermented beverages such as wine, cider and beer produce a wide range of exopolysaccharides (EPS) through multiple biosynthetic pathways. These extracellular polysaccharides constitute key elements for bacterial species adaptation to such anthropic processes. In the food industry, LAB polysaccharides have been widely studied for their rheological, functional and nutritional properties; however, these have been poorly studied in wine, beer and cider until recently. In this review, we have gathered the information available on these specific polysaccharide structure and, biosynthetic pathways, as well as the physiology of their production. The genes associated with EPS synthesis are also presented and compared. Finally, the possible role of EPS for bacterial survival and spread, as well as the risks or possible benefits for the winemaker and the wine lover, are discussed.
... Grape indigenous yeasts produce diverse compounds that later positively or negatively impact wine quality [20,29]. The presence or absence of particular yeasts species can promote growth of plant pathogens or spoilage microorganisms [15,19,26,30,47,48]. Knowing which endophytic and epiphytic fungal communities are present in the vineyard can be valuable for grape growers and winemakers who can produce premium wines with specific characteristics related to terroir [8,29,49]. ...
Wine grape berries (Vitis spp.) harbor a wide variety of yeasts and filamentous fungi that impact grapevine health and the winemaking process. Identification of these fungi could be important for controlling and improving wine production. The use of high-throughput sequencing (HTS) strategies has enabled identification and quantification of bacterial and fungal species in vineyards. The aims of this study were to identify mycobiota from Cabernet Sauvignon and Zinfandel (V. vinifera), Carlos and Noble muscadines (V. rotundifolia), Cynthiana (V. aestivalis), and Vignoles hybrid (cross of different Vitis spp.) grapes, and investigate the effect of grape variety, location, and year on grape fungal communities. Grape berries were collected in 2016 and 2017 from four vineyards located in Arkansas. The HTS of the Internal Transcribed Spacer 1 region was used to identify grape indigenous epiphytic and endophytic fungal communities. The predominant genera identified on the Arkansas wine grapes were Uwebraunia, Zymoseptoria, Papiliotrema, Meyerozyma, Filobasidium, and Curvibasidium. Overall, the data suggested that grape fungal community distribution and relative abundance were influenced by grape variety, year, and location, but each was influenced to a different extent. Not only were grape mycobiota influenced by year, variety, and location but also it appeared that communities from the previous year impacted microbial communities the following year. For example, an increase of the mycoparasite Ampelomyces quisqualis was noticed in 2017 on grapes that carried the causal agent of powdery mildew, Erysiphe necator, in 2016, thus, amplifying the importance of vineyard microbiota knowledge for disease management and winemaking.
... This yeast is responsible for the production of volatile phenols and most importantly 4-ethylphenol, which contributes to undesirable aromas described as "Brett character" (Chatonnet et al., 1992;Oelofse et al., 2008;Wedral et al., 2010), leading to rejection by consumers and to heavy economic losses (Fugelsang, 1997;Lattey et al., 2010). This yeast can be found at several steps in the winemaking process (Chatonnet et al., 1992;Renouf et al., 2006Renouf et al., , 2009Renouf and Lonvaud-Funel, 2007;Rubio et al., 2015;Suárez et al., 2007) due to its resistance to multiple stress conditions (Avramova et al., 2018b;Conterno et al., 2006;Longin et al., 2016;Schifferdecker et al., 2014;Serpaggi et al., 2012;Smith and Divol, 2016). The ability to form biofilm is another potential resistance strategy (Tek et al., 2018;Verstrepen and Klis, 2006), although in the case of B. bruxellensis it has been given only little attention so far. ...
Ability to form biofilms is a potential resistance strategy, although it has not been much explored so far for the spoilage yeast Brettanomyces bruxellensis. The capacity of two strains to adhere and form biofilms on stainless steel chips in wine was studied. Using electronic microscopy, some particular structures, such as filamentous cells or chlamydospore-like structure, potentially involved in B. bruxellensis resistance were revealed. Some detachment phenomenon was identified and may be at the origin of the wine recurrent contamination.
... Ces résultats laissent à penser que le vin pourrait être également un environnement transitoire de contamination et/ou à l'origine de la présence de B. bruxellensis dans l'environnement de la cave. Licker et al., 1998 ;Renouf et al., 2006 ;. . L'analyse des profils alléliques a permet l'identification de 27 groupes clonaux différents à l'intérieur de ces 3 groupes génétiques ce qui confirme la grande diversité génétique de cette espèce, comme déjà suggérée par plusieurs auteurs Vigentini et al., 2012 ;Albertin et al., 2014 ;Avramova et al., 2018). ...
La biodiversité fongique interspécifique (Illumina Mi-Seq) et la dynamique des espèces Saccharomyces cerevisiae et Brettanomyces bruxellensis ont été étudiées au sein d’une nouvelle cuverie et/ou dans 3 caves d’élevage, plus particulièrement sur le sol, les murs, le matériel vinaire et l’extérieur des fûts. Dans la nouvelle cuverie, un consortium fongique (levures et moisissures) de départ est déjà présent sur tous les environnments étudiés avant l’arrivée de la première vendange. Ce consortium est constitué de genres tels que Aureobasidium, Alternaria, Didymella et Filobasidium. Ces genres qui persistent pendant deux millésimes, ne sont pas spécifiques de l’environnement de la cuverie et semblent être adaptés à tous les environnements naturels ou anthropiques au regard de leur caractère ubiquiste. Le consortium de départ est enrichi par des genres œnologiques (exemple : Hanseniaspora, Saccharomyces) qui sont introduits dans la cuverie soit par les vendanges, soit par des transferts potentiels entre les différents environnements de la cuverie. Cependant, ces genres ne semblent pas persister ou s’implanter probablement dû à leur faible adaptation aux conditions stressantes de l’environnement de la cuverie. La dynamique de la flore indigène S. cerevisiae dans la nouvelle cuverie a été également étudiée. Aucun isolat appartenant à cette espèce n’a été retrouvé avant l'arrivée de la première vendange confirmant que cette espèce n’est pas spécifique de l'environnement de la cuverie et que sa présence est en lien avec l'activité des fermentations alcooliques. Cependant, les résultats obtenus suggèrent une colonisation potentielle de l’environnement de la nouvelle cuverie par certaines souches de S. cerevisiae. Ces souches dites « colonisatrices » ont présenté une capacité plus élevée à former des biofilms comparée à celle de souches non implantées. Cette étude met en évidence l’importance de l’environnement de la cuverie qui constitue une véritable niche écologique pour les populations fongiques capables de s’implanter au cours du processus de vinification. Dans l’environnement des 3 caves d’élevage, le matériel vinaire et l’extérieur des fûts (en contact direct avec le vin) sont les environnements qui semblent favorables au développement et à l’installation des populations microbiennes cultivables (levures totales et bactéries lactiques) et des microorganismes d’altération (bactéries acétiques et B. bruxellensis), contrairement au sol et aux murs où des populations faibles ont été trouvées. Des souches récurrentes de B. bruxellensis ont été retrouvées sur le matériel et sur l’extérieur des fûts et pourraient être à l’origine de la contamination de vins au cours de l’élevage. Ces souches récurrentes présentent des capacités de formation de biofilms et de résistance plus importantes qui pourraient expliquer la persistance de B. bruxellensis dans des caves d’élevage. Ces résultats soulignent l’importance du nettoyage du matériel vinaire et du suivi microbien régulier des vins au cours de l’élevage afin de limiter les contaminations.
... La fermentación alcohólica espontánea, está determinada por el crecimiento de las levaduras inoculadas, la cepa aislada y denominada c, y las cepas en conjunto denominadas m, las interacciones entre las especies impacta en la producción de etanol y en el consumo de nutrientes(Fleet, 2003). En los cultivos realizados en un volumen de 100 mL, al calcular el peso seco celular de las levaduras inoculadas en el medio enriquecido con glucosa, se determinó que la fase de adaptación concluyó a la tercera hora después de su inoculación, la fase exponencial inicio llegada la hora seis.En la evaluación del consumo de azúcares, la concentración de glucosa presente en el matraz disminuyó conforme la cepa aumentaba su crecimiento, el consumo de azúcares fue de 0.48 g a 0.03 g de la cepa c, y de 0.52 g a 0.06 g en la cepa m a lo largo de las 32 h de fermentación, aunque no se obtuvieron diferencias significativas entre el consumo de ambas; sin embargo, la producción del alcohol si fue diferente, siendo la cepa m la de mayor producción de etanol con 10.33% de alcohol, que es estadísticamente significativo con la cepa c, cuyo contenido de alcohol fue de 12.57% (Figura 3).Renouf et al. (2006) evaluaron los niveles de fermentación entre S. cerevisiae y un cultivo mixto, el tiempo de consumo de azúcares fue de 20 d para S. cerevisiae, y para el cultivo mixto se redujo a un total de 14 d, en cuanto al porcentaje de alcohol de las muestras, S. cerevisiae tuvo un porcentaje de alcohol de 9%(v/v) que no resultó ser estadísticamente significativo al 8%(v/v) del cultivo mixto. Este fenómeno observado ha sido tratado porOzilgen et al. (1991), quienes propusieron modelos matemáticos simples para simular el crecimiento bacteriano, reducir la utilización de azúcar y aumentar la producción de etanol en un proceso de fermentación espontánea. ...
Objetivo. aislar y caracterizar el sinergismo de microorganismos nativos de tinas de fermentación de maguey (Agave cupreata Trel. & Berger) para destilar mezcal.Materiales y métodos se recolectaron raspados de tina donde se fermenta el Agave molido y formulado para generar la fermentación y obtener mezcal, se procesaron en medios selectivos y se realizó primero un tamiz y posteriormente la eficiencia de conversión de azúcares,Resultados: se encontraron convenientemente dos levaduras, que mostraron indicadores prometedores para la conversión de azúcares a alcohol se les denomino c a la cepa uno y m a la unión de las dos elegidas, adicionalmente se sometieron a laevaluación de parámetros como peso seco celular, cantidad de etanol producido (10.33±0.57 (c) y 12.57 ±0.57 (m) respectivamente y la determinación de azúcares reductores totales (se partió de 500 mg L -1 ).Limitaciones del estudio/ implicaciones: se sometió a un trabajo in vitro quedando pendiente la eficiencia en azúcares de agave y el gradiente de concentración.Conclusiones: el consorcio en comparación de una cepa aislada produjo mayor cantidad de alcohol respecto su crecimiento en el medio a 32 h de fermentación a unatemperatura constante de 37 °C y 160 rpm.
... A maturité, la population de levures peut ainsi atteindre entre 10 4 et 10 6 UFC/g (Prakitchaiwattana et al., 2004;Renouf, 2006;Rosini et al., 1982) et même dépasser les 10 6 UFC/g dans le cas de baies endommagées (Mortimer et Polsinelli, 1999). Les principales levures que l'on peut retrouver à maturité font partie des genres Hanseniaspora, Candida, Metschnikowia, Zygosaccharomyces et Saccharomyces (Barata et al., 2008;Fleet, 2003;Martini et al., 1996;Renouf et al., 2006). D'autres espèces de levures peuvent cependant être présentes avec des fréquences moins importantes, c'est le cas de l'espèce Torulaspora delbrueckii récemment identifiée à la surface des baies de raisin (Martins et Masneuf-Pommarède, communication personnelle). ...
La caractérisation phénotypique de l’espèce Torulaspora delbrueckii en conditions œnologiques, à partir de l’étude d’un grand nombre de souches, a permis de mettre en évidence une grande variabilité au sein de cette espèce. En effet, les souches de T.delbrueckii présentent des différences au niveau des durées de phase de latence et de fermentation, des capacités biotiques mais aussi des productions d’éthanol (maximum 12% vol.). Cette variabilité se retrouve également pour la production d’acidité volatile, de glycérol et de certains arômes. Ce travail confirme les faibles productions d’acidité volatile et de glycérol de cette espèce et met en évidence une réponse au stress osmotique différente de celle de l’espèce Saccharomyces cerevisiae. Au final, l’espèce T. delbrueckii présente une grande « pureté » de fermentation et produit peu de composés indésirables comme le sulfure d’hydrogène, les phénols volatils, l’acetoïne, l’acétaldéhyde et le diacétyle. La réalisation de co-inoculations T. delbrueckii / S. cerevisiae sur moûts liquoreux, permet une réduction systématique de l’acidité volatile des vins, en comparaison à une fermentation pure de l’espèce S. cerevisiae, quelque soit la souche de T. delbrueckii utilisée. De plus, la souche T. delbrueckii OXT1 1//2 a permis de complexifier la composition aromatique d’un moût sec issu du cépage Sauvignon blanc (esters fermentaires +25%, phényl-2-éthanol +51% et thiols volatils +31%). Enfin, la mise au point d’un fermenteur à double compartiment, avec une séparation physique des levures tout en conservant l’homogénéité du milieu de culture, a permis d’aborder l’étude des interactions entre ces 2 espèces. Des inhibitions de type « cell-cell contact » ont ainsi été mises en évidence.
... An important bibliography is dedicated to the B. bruxellensis species, with 100 to 200 papers published each year over the last decade (source: Google Scholar). Many papers investigate volatile phenol production [7][8][9][10], the biotic and abiotic factors impacting B. bruxellensis growth [11][12][13][14][15] and some peculiarities of the species like the ability to survive in the VNC (Viable Non Culturable) state [7,16,17] or the specific oxygen needs during fermentation [18]. Moreover, different detection and quantification methods for Brettanomyces, ranging from direct plating methods through molecular detection and flow cytometry analysis, were examined (see Tubia et al., 2018 for review [19]). ...
Brettanomyces bruxellensis is the main wine spoiler yeast all over the world, yet the structure of the populations associated with winemaking remains elusive. In this work, we considered 1411 wine isolates from 21 countries that were genotyped using twelve microsatellite markers. We confirmed that B. bruxellensis isolates from wine environments show high genetic diversity, with 58 and 42% of putative triploid and diploid individuals respectively distributed in 5 main genetic groups. The distribution in the genetic groups varied greatly depending on the country and/or the wine-producing region. However, the two possible triploid wine groups showing sulfite resistance/tolerance were identified in almost all regions/countries. Genetically identical isolates were also identified. The analysis of these clone groups revealed that a given genotype could be isolated repeatedly in the same winery over decades, demonstrating unsuspected persistence ability. Besides cellar residency, a great geographic dispersal was also evidenced, with some genotypes isolated in wines from different continents. Finally, the study of old isolates and/or isolates from old vintages revealed that only the diploid groups were identified prior 1990 vintages. The putative triploid groups were identified in subsequent vintages, and their proportion has increased steadily these last decades, suggesting adaptation to winemaking practices such as sulfite use. A possible evolutionary scenario explaining these results is discussed.
... This yeast is responsible for the production of volatile phenols and most importantly 4-ethylphenol, which contributes to undesirable aromas described as "Brett character" (Chatonnet et al., 1992;Oelofse et al., 2008;Wedral et al., 2010), leading to rejection by consumers and to heavy economic losses (Fugelsang, 1997;Lattey et al., 2010). This yeast can be found at several steps in the winemaking process (Chatonnet et al., 1992;Renouf et al., 2006Renouf et al., , 2009Renouf and Lonvaud-Funel, 2007;Rubio et al., 2015;Suárez et al., 2007) due to its resistance to multiple stress conditions (Avramova et al., 2018b;Conterno et al., 2006;Longin et al., 2016;Schifferdecker et al., 2014;Serpaggi et al., 2012;Smith and Divol, 2016). The ability to form biofilm is another potential resistance strategy (Tek et al., 2018;Verstrepen and Klis, 2006), although in the case of B. bruxellensis it has been given only little attention so far. ...
The wine spoilage yeast Brettanomyces bruxellensis can be found at several steps in the winemaking process due to its resistance to multiple stress conditions. The ability to form biofilm is a potential resistance strategy, although it has been given little attention so far for this yeast. In this work, the capacity to form biofilm and its structure were explored in YPD medium and in wine. Using microsatellite analysis, 65 isolates were discriminated into 5 different genetic groups from which 12 strains were selected. All 12 strains were able to form biofilm in YPD medium on a polystyrene surface. The presence of microcolonies, filamentous cells and extracellular polymeric substances, constituting the structure of the biofilm despite a small thickness, were highlighted using confocal and electronic microscopy. Moreover, different cell morphologies according to genetic groups were highlighted. The capacity to form biofilm in wine was also revealed for two selected strains. The impact of wine on biofilms was demonstrated with firstly considerable biofilm cell release and secondly growth of these released biofilm cells, both in a strain dependent manner. Finally, B. bruxellensis has been newly described as a producer of chlamydospore-like structures in wine, for both planktonic and biofilm lifestyles.
... This yeast is able to survive and multiply in wine, especially in case of sluggish fermentation and difficulties of other species to monopolize the wine ecosystem (Renouf et al. 2006;Romano et al. 2008). Even if B. bruxellensis was detected at low level on grape berry, repetitive infections were observed in wine, suggesting that the cellar, rather than the vineyard, could be the main source of contamination (Garde-Cerdán and Ancín-Azpilicueta 2006; Barata et al. 2008;González-Arenzana et al. 2013;Rubio et al. 2015). ...
Purpose
Brettanomyces bruxellensis is a serious source of concern for winemakers. The production of volatile phenols by the yeast species confers to wine unpleasant sensory characteristics which are unacceptable by the consumers and inevitably provoke economic loss for the wine industry. This ubiquitous yeast is able to adapt to all winemaking steps and to withstand various environmental conditions. Moreover, the ability of B. bruxellensis to adhere and colonize inert materials can be the cause of the yeast persistence in the cellars and thus recurrent wine spoilage. We therefore investigated the surface properties, biofilm formation capacity, and the factors which may affect the attachment of the yeast cells to surfaces with eight strains representative of the genetic diversity of the species.
Methods
The eight strains of B. bruxellensis were isolated from different geographical and industrial fermentation origins. The cells were grown in synthetic YPD medium containing 1% (w/v) yeast extract (Difco Laboratories, Detroit), 2% (w/v) bacto peptone (Difco), and 1% (w/v) glucose. Surface physicochemical properties as electrophoretic mobility and adhesion to hydrocarbon of the cells were studied. The ability of the strains to form biofilm was quantified using a colorimetric microtiter 96-well polystyrene plate. Biochemical characteristics were examined by colorimetric methods as well as by chemical analysis.
Result
Our results show that the biofilm formation ability is strain-dependent and suggest a possible link between the physicochemical properties of the studied strains and their corresponding genetic group.
Conclusion
The capacity to detect and identify the strains of the spoilage yeast based on their biofilm formation abilities may help to develop more efficient cleaning procedures and preventing methods.
... This yeast is able to survive and multiply in wine, especially in case of sluggish fermentation and difficulties of other species to monopolize the wine ecosystem (Renouf et al. 2006;Romano et al. 2008). Even if B. bruxellensis was detected at low level on grape berry, repetitive infections were observed in wine, suggesting that the cellar, rather than the vineyard, could be the main source of contamination (Garde-Cerdán and Ancín-Azpilicueta 2006; Barata et al. 2008;González-Arenzana et al. 2013;Rubio et al. 2015). ...
Brettanomyces bruxellensis is a serious source of concern for winemakers. The production of volatile phenols by the yeast species confers to wine unpleasant sensory characteristics which are unacceptable by the consumers and inevitably provoke economic loss for the wine industry. This ubiquitous yeast is able to adapt to all winemaking steps and to withstand various environmental conditions. Moreover, the ability of B. bruxellensis to adhere and colonize inert materials can be the cause of the yeast persistence in the cellars and thus recurrent wine spoilage. We therefore investigated the surface properties, biofilm formation capacity and the factors which may affect the attachment of the yeast cells to surfaces with eight strains representative of the genetic diversity of the species. Our results show that the biofilm formation ability is strain-dependent and suggest a possible link between the physicochemical properties of the studied strains and their corresponding genetic group.
... To prevent wine spoilage and the subsequent economic losses, there is a need for a fast and accurate method to detect and quantify Brettanomyces cells in wine. To date, a number of culture-independent methods, such as PCR, nested PCR and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) (Campolongo, Rantsiou, Giordano, Gerbi, & Cocolin, 2010;Cocolin, Rantsiou, Iacumin, Zironi, & Comi, 2004;Ibeas, Lozano, Perdigones, & Jimenez, 1996;Martorell et al., 2006;Renouf et al., 2006) have been used to overcome the difficulties described above. However, these methods are often characterized by a low detection limit (ࣙ10 4 CFU/mL) of the spoilage yeasts . ...
In the Albanian winemaking industry, there is little awareness of the potential detrimental effect of Brettanomyces in wines. The aim of this study was to detect and quantify Brettanomyces cells in 22 Albanian bottled wines, representing all the viticultural areas of Albania. A combined approach, including culture‐dependent (viable plate counting) and culture‐independent (qPCR) methods, was applied. Spoilage indicators (ethylphenols and total and volatile acidity), as well as the primary factors known to influence the growth of Brettanomyces in wine (pH, SO2, and ethanol concentration), were also investigated. Brettanomyces was detected in only five (one Merlot, four Sheshi i Zi) out of 22 samples analyzed using viable counting, with loads ranging from 1.30 ± 0.03 log CFU/mL to 3.99 ± 0.00 log CFU/mL, whereas it was never detected in the Kallmet samples. When qPCR was applied, Brettanomyces cells were detected and quantified in all of the samples with a generally low load ranging from 0.47 ± 0.13 to 3.99 ± 0.01 log cells/mL. As a general trend, the loads of spoilage by this yeast were low (≤1.92 log cells/mL), with the exception of five samples that were also positive by plate counting. A positive correlation between the growth of this spoilage yeast on Dekkera/Brettanomyces differential media and its detection at high levels by qPCR was observed. A significant positive correlation between Brettanomyces and the concentration of ethylphenols and volatile acidity was also found. In summary, the results of this study demonstrated the low incidence of Brettanomyces spoilage yeasts in Albanian red wines.
Practical Application
The awareness of Brettanomyces spoilage in the Albanian winemaking industry is very low. This study represents the first contribution to understand the extent of this spoilage yeast in Albanian autochthonous cultivars, which tend to have high economic value, to ensure product quality and safety. qPCR is confirmed to be a very sensitive method to rapidly detect Brettanomyces spoilage in wine samples.
... se desarrolló como una alternativa de identificación de levaduras de manera rápida para su utilización en la industria. Esta técnica ha sido utilizada por diferentes autores para identificar levaduras aisladas de diferentes fuentes como mostos de vinos [1,[5][6][7][8][9][10], cidra [11], jugo de naranja [12], miel [13], cerveza de sorgo [14], masa [15], productos lácteos [16][17][18] y recientemente en muestras clínicas para la identificación de levaduras y hongos patógenos [19][20][21]. ...
RESUMEN. Las levaduras juegan un papel muy importante en los procesos de fermentación de bebidas y alimentos, por lo que es necesario identificarlas. A finales de los años 90 se propuso la técnica de análisis del polimorfismo en la longitud de los fragmentos de restricción (RFLP) de las regiones ITS-5.8S como una herramienta rápida y confiable para identificarlas. En este trabajo se analizaron 104 cepas de Saccharomyces cerevisiae de la colección de CIATEJ. Además se analizaron los patrones de bandas publicados hasta el momento para levaduras, lo que nos permitió establecer el parámetro de error de dicha técnica. A partir de este análisis se comprobó que esta técnica es rápida y confiable en la gran mayoría de los casos, sin embargo para algunas especies de levaduras existen ambigüedades en los patrones publicados o no permite diferenciar especies cercanas. Palabras clave: Levadura, Saccharomyces cerevisiae, identificación, técnica molecular, alimentos y bebidas fermentados. ABSTRACT. Yeasts play an important role in foods and beverages fermentation process, so it is necessary to identify them. In the late 90's a technique based on the restriction fragments length polymorphism (RFLP) of the ITS-5.8S regions was proposed as a fast and reliable tool to identify them. In this study 104 strains of Saccharomyces cerevisiae were analyzed from the CIATEJ collection. Also all the polymorphism patterns published in yeast were analyzed too which allowed us to set the parameter error of this technique. From this analysis it was found that this technique is fast and reliable in most cases, however for some yeast species there are ambiguities in the published standards or does not differentiate closely related species.
... The counts were reported as average value ± standard deviation (n = 2). during conditions of low sugar concentration, as previously demonstrated (Renouf et al. 2006). It might also be possible to explain the increase in B. brettanomyces cells during the last stages of the fermentation by the increased S. cerevisiae autolysis (Berbegal et al. 2017). ...
The negative effect of this yeast on wine quality, due to the production of phenolic off-flavors, and potentially large associated economical losses, require the application of specific control measures. In this study we investigated the ability of ozone to control B. bruxellensis population on Barbera wine grape berries and its impact on yeasts growth dynamics during subsequent fermentation and on the chemical composition of resulting wines. To further explore the ability of the ozone treatments to reduce B. bruxellensis population, a mix of three different strains were artificially inoculated on the surface of grape berries. Grape berries were ozone-treated either in aqueous (6 and 12 min) or gaseous form (12 and 24 h), crushed and then fermented to evaluate the effect of these treatments on B. bruxellensis and S. cerevisiae growth dynamics and wine composition. Microbiological analysis revealed a significant reduction of B. bruxellensis of about 2.2 Log colony forming units (CFU)/mL after treatments with gaseous ozone for 24 h. The wines produced from grape berries previously exposed to gaseous ozone for 24 h contained the lowest levels of acetic acid. Moreover, 4-ethylphenols were detected in wines produced from grape berries treated with water (6 and 12 min), in which B. bruxellensis population reached 5.0 Log CFU/mL at the end of fermentation. Molecular analysis results suggest that the three different strains tested had similar sensitivity to the ozone treatments applied. This study shows the first results about the ability of ozone to control the population of different B. bruxellensis strains within the same species in the same manner.
... Pomace sterilised prior to inoculation with the yeast always exhibited recovery of a population greater than that from non-sterilised pomace. Although the growth rate of B. bruxellensis compared to S. cerevisiae (Abbott et al. 2005, Blomqvist et al. 2010) is low, this yeast can outcompete some non-Saccharomyces yeasts and lactic acid bacteria under specific conditions (Renouf et al. 2006, Passoth et al. 2007. For instance, De Pina and Hogg (1999) observed that B. bruxellensis became the dominant microbe in pomace samples stored at ambient temperature for 24 weeks even though a wide range of microbes were present. ...
Background and Aims
Spreading grape pomace in vineyards could promote dispersal of the spoilage yeast, Brettanomyces bruxellensis. Thus, we evaluated the ability of this yeast to survive in inoculated pomace stored at variable temperature as well as in regional vineyards.
Methods and Results
Three strains of B. bruxellensis were inoculated into Syrah or Merlot pomace which was stored at 21, 10, 0, or –18°C. All strains maintained ≥10⁴ colony‐forming units (CFU)/mL at 21 or 10°C for 10 weeks but declined to ≤10³ CFU/mL when incubated at 0 or –18°C. Additional Syrah pomace, half sterilised by gamma irradiation, was inoculated with B. bruxellensis, placed into sterilised bottles capped with 0.22 μm filters, and placed into three vineyards. Overall, the yeast population increased or decreased with varying seasonal temperature but remained viable (~10³ CFU/mL) for up to 130 weeks regardless of vineyard location. Non‐sterilised pomace generally had a population lower than that of sterilised samples and yielded wide variations in population corresponding to seasons. The recovery of Brettanomyces from infected pomace was reduced, if not prevented, by heating to ≥50°C and adding 45 mg/L total SO2.
Conclusions
As B. bruxellensis can survive at least 130 weeks in pomace, infected winery waste dispersed in vineyards may serve as a reservoir for the continued contamination of grape harvests. Such contamination can be minimised by heating potentially infected pomace to ≥50°C for at least 10 min and adding sulfites is recommended.
Significance of the Study
Long‐term survival of B. bruxellensis in pomace was established and microbial control strategies using heat and sulfites demonstrated.
... The high ethanol levels and low sugar, pH, and oxygenation levels cause other yeast cells, such as Saccharomyces cerevisiae, to undergo autolysis. However, Brettanomyces has exceptional resistance to alcoholic conditions within a minimal nutrient environment and slow growth characteristics (Renouf et al., 2006). Also, when the wine is stored for aging in wooden barrels, the porous microstructure of the oak barrels allows the entrance of oxygen which promotes the growth of Brettanomyces (du Toit et al., 2006). ...
Spoilage yeasts detection is the key to improve the quality of alcoholic fermentation beverages such as wine and cider. The metabolic activity of the spoilage yeast causes irreparable damage to many liters of final products every year. Therefore, winemakers and cider-house companies suffer a substantial economic impact. Thus, over the years, many detection techniques have been proposed to control the occurrence of spoilage yeast. Out of the many spoilage yeast genera, Brettanomyces is one of the most commonly encountered in the beverage industry. Leveraging its ability to thrive in wine and cider conditions (low pH, high levels of ethanol, and low oxygenation levels), Brettanomyces can proliferate inside beverage production tanks. Moreover, their resultant by products reduce the quality of the beverage. While the beverage industry has made great strides in detecting harmful organisms, gaps remain. Traditional methods such as microscopy, cell plating, gas chromatography–mass spectrometry, etc. are often imprecise, expensive, and/or complicated. New emerging spoilage yeast detection platforms, such as biosensors and microfluidic devices, aim to alleviate these constraints. Novel platforms have already demonstrated great promise to be a real alternative for in situ and fast detection in the beverage industry. Finally, the review discusses the potential of emerging spoilage yeast detection and treatment methods.
... Usually, the concentration of B. bruxellensis is low during the first steps of winemaking, due to slow growth [14]. In addition, during alcoholic fermentation (AF), the metabolic activity of S. cerevisiae inhibits its development, and its population remains low. ...
Biocontrol strategies for the limitation of undesired microbial developments in foods and beverages represent a keystone toward the goal of more sustainable food systems. Brettanomyces bruxellensis is a wine spoilage microorganism that produces several compounds that are detrimental for the organoleptic quality of the wine, including some classes of volatile phenols. To control the proliferation of this yeast, sulfur dioxide is commonly employed, but the efficiency of this compound depends on the B. bruxellensis strain; and it is subject to wine composition and may induce the entrance in a viable, but nonculturable state of yeasts. Moreover, it can also elicit allergic reactions in humans. In recent years, biological alternatives to sulfur dioxide such as the use of yeasts and lactic acid bacteria starter cultures as biocontrol agents are being investigated. The controlled inoculation of starter cultures allows secure, fast and complete alcoholic and malolactic fermentations, limiting the residual nutrients that B. bruxellensis utilizes to survive and grow in wine. The current study is focused on the assessment of the effect of autochthonous yeasts and bacterial strains from the Apulia Region on the development of B. bruxellensis in wine, in terms of both growth and volatile phenols' production. The investigation evidences the positive role of indigenous mixed cultures in the control of this spoilage yeast, either co-inoculating different strains of Saccharomyces cerevisiae, S. cerevisiae/non-Saccharomyces or co-inoculating S. cerevisiae/Oenococcus oeni. Our findings expand the existing knowledge of the application of protechnological microbial diversity and of non-Saccharomyces as a biocontrol agent in oenology. We report a further demonstration of the interest in selecting indigenous strains as a strategic tool for winemakers interested in the improvement of regional wines.
... Interestingly, aerobic fermentation has also been observed in some evolutionarily independent yeast lineages. Among them, some Dekkera species, such as D. bruxellensis, often occupy rich glucose environments similar to Saccharomyces, and tend to accumulate ethanol during growth (Renouf et al. 2006). On the other hand, Kluyveromyces lactis, a relative of Saccharomyces that did not undergo WGD, performs respiratory metabolism in rich glucose conditions, as does Brettanomyces custersianus, a close relative of D. bruxellensis. ...
Parallel evolution occurs when a similar trait emerges in independent evolutionary lineages. Although changes in protein coding and gene transcription have been investigated as underlying mechanisms for parallel evolution, parallel changes in chromatin structure have never been reported. Here Saccharomyces cerevisiae and a distantly related yeast species, Dekkera bruxellensis, are investigated because both species have independently evolved the capacity of aerobic fermentation. By profiling and comparing genome sequences, transcriptomic landscapes, and chromatin structures, we revealed that parallel changes in nucleosome occupancy in the promoter regions of mitochondria-localized genes led to concerted suppression of mitochondrial functions by glucose, which can explain the metabolic convergence in these two independent yeast species. Further investigation indicated that similar mutational processes in the promoter regions of these genes in the two independent evolutionary lineages underlay the parallel changes in chromatin structure. Our results indicate that, despite several hundred million years of separation, parallel changes in chromatin structure, can be an important adaptation mechanism for different organisms. Due to the important role of chromatin structure changes in regulating gene expression and organism phenotypes, the novel mechanism revealed in this study could be a general phenomenon contributing to parallel adaptation in nature.
... In our study, we also incorporated several Brettanomyces strains that are used in the brewing of specific beers, such as lambic beers [56,57]. Species within this genus have been reported to display high ethanol tolerance and for appearing and even dominating in wine and industrial bioethanol fermentations as contaminant [58][59][60]. However, in our study most of the yeast species within this genus, such as Brettanomyces anomalus, Brettanomyces (Dekkera) bruxellensis, and Brettanomyces naardenensis did not grow on test plates containing more than 12% v/v ethanol. ...
Background
Non-conventional yeasts present a huge, yet barely exploited, resource of yeast biodiversity for industrial applications. This presents a great opportunity to explore alternative ethanol-fermenting yeasts that are more adapted to some of the stress factors present in the harsh environmental conditions in second-generation (2G) bioethanol fermentation. Extremely tolerant yeast species are interesting candidates to investigate the underlying tolerance mechanisms and to identify genes that when transferred to existing industrial strains could help to design more stress-tolerant cell factories. For this purpose, we performed a high-throughput phenotypic evaluation of a large collection of non-conventional yeast species to identify the tolerance limits of the different yeast species for desirable stress tolerance traits in 2G bioethanol production. Next, 12 multi-tolerant strains were selected and used in fermentations under different stressful conditions. Five strains out of which, showing desirable fermentation characteristics, were then evaluated in small-scale, semi-anaerobic fermentations with lignocellulose hydrolysates. ResultsOur results revealed the phenotypic landscape of many non-conventional yeast species which have not been previously characterized for tolerance to stress conditions relevant for bioethanol production. This has identified for each stress condition evaluated several extremely tolerant non-Saccharomyces yeasts. It also revealed multi-tolerance in several yeast species, which makes those species good candidates to investigate the molecular basis of a robust general stress tolerance. The results showed that some non-conventional yeast species have similar or even better fermentation efficiency compared to S. cerevisiae in the presence of certain stressful conditions. Conclusion
Prior to this study, our knowledge on extreme stress-tolerant phenotypes in non-conventional yeasts was limited to only few species. Our work has now revealed in a systematic way the potential of non-Saccharomyces species to emerge either as alternative host species or as a source of valuable genetic information for construction of more robust industrial S. serevisiae bioethanol production yeasts. Striking examples include yeast species like Pichia kudriavzevii and Wickerhamomyces anomalus that show very high tolerance to diverse stress factors. This large-scale phenotypic analysis has yielded a detailed database useful as a resource for future studies to understand and benefit from the molecular mechanisms underlying the extreme phenotypes of non-conventional yeast species.
... Non-conventional yeast, which include non-Saccharomyces and noncerevisiae yeast, are part of the natural microbiota present on grapes, and harvesting and winemaking equipment, and are present at least during the early stages of fermentation (Fleet and Heard, 1993;Renouf et al., 2006;Renouf and Lonvaud-Funel, 2007). The use of nonconventional yeast is increasingly popular particularly for their effects on wine composition, flavour, aroma and colour (Jolly et al., 2014;Varela, 2016) and for their potential to produce reduced-alcohol wines (Ciani et al., 2016;Varela, 2016). ...
Strategies for production of wines containing lower alcohol concentrations are in strong demand, for reasons of quality, health, and taxation. Development and application of wine yeasts that are less efficient at transforming grape sugars into ethanol has the potential to allow winemakers the freedom to make lower alcohol wines from grapes harvested at optimal ripeness, without the need for post-fermentation processes aimed at removing ethanol. We have recently shown that two non-conventional wine yeast species Metschnikowia pulcherrima and Saccharomyces uvarum were both able to produce wine with reduced alcohol concentration. Both species produced laboratory-scale wines with markedly different volatile aroma compound composition relative to Saccharomyces cerevisiae. This work describes the volatile composition and sensory profiles of reduced-alcohol pilot-scale Merlot wines produced with M. pulcherrima and S. uvarum. Wines fermented with M. pulcherrima contained 1.0% v/v less ethanol than S. cerevisiae fermented wines, while those fermented with S. uvarum showed a 1.7% v/v reduction in ethanol. Compared to S. cerevisiae ferments, wines produced with M. pulcherrima showed higher concentrations of ethyl acetate, total esters, total higher alcohols and total sulfur compounds, while wines fermented with S. uvarum were characterised by the highest total concentration of higher alcohols. Sensorially, M. pulcherrima wines received relatively high scores for sensory descriptors such as red fruit and fruit flavour and overall exhibited a sensory profile similar to that of wine made with S. cerevisiae, whereas the main sensory descriptors associated with wines fermented with S. uvarum were barnyard and meat. This work demonstrates the successful application of M. pulcherrima AWRI3050 for the production of pilot-scale red wines with reduced alcohol concentration and highlights the need for rigorous evaluation of non-conventional yeasts with regard to their sensory impacts.
... The isolation of yeast on Wallerstein Laboratory Nutrient (WLN)-based medium is routinely used as a simple test in the wine industry (Rodrigues et al., 2001) as recommended by the Oenological Codex 2016 Edition. In addition, several DNA-based techniques are used to detect or quantify this yeast by culture dependent or independent methods, such as RNA-FISH (Röder et al., 2007;Serpaggi et al., 2010;Stender et al., 2001), PNA-FISH (Stender et al., 2001), ITS PCR coupled to RFLP (Esteve-Zarzoso et al., 1999), DGGE (Cocolin et al., 2004;Prakitchaiwattana et al., 2004;Renouf et al., 2006), TGGE (Hernán-Gómez et al., 2000), loop mediated isothermal amplification (Hayashi et al., 2007), and quantitative PCR (qPCR) (Delaherche et al., 2004;Phister and Mills, 2003;Tessonnière et al., 2009;Willenburg and Divol, 2012). Several specific qPCR-based quantification kits have been developed based on previous studies reporting the efficiency of qPCR to specifically quantify B. bruxellensis and are commercially available. ...
Brettanomyces bruxellensis is well adapted to high ethanol concentrations and low pH which allows it to develop in difficult environments, such as wine. B. bruxellensis is mainly found in red wine and is regarded as a spoilage yeast due to its production of ethylphenols and other compounds responsible for organoleptic defects. The detection and quantification of this yeast is essential to preventing wine spoilage. Several specific detection and quantification kits based on real time quantitative PCR are commercially available. Although these kits are frequently used by private enological and research laboratories, no scientific report on the reliability and performance of these kits, including inter-laboratory and inter-assay comparisons have been published. The aim of this work was to compare available kits to quantify B . bruxellensis in red wine to classical method (plate counting on selective medium) in an interlaboratory study. Three different commercial kits were tested on three different wines from Bordeaux, Côtes du Rhône, and Burgundy inoculated with B . bruxellensis at four different concentrations. Five naturally contaminated wines from different French wine regions were also tested. Our results suggest that all the kits tested probably over or underestimate the quantity of B . bruxellensis in red wine and, under specific conditions, give false positives. Quantification may be very heterogeneous depending on the wine, laboratory, or population level. Underestimations or false negative results may have serious consequences for winemakers. Overestimation may be partly due to the quantification of dead cells qPCR.
This study highlights that quantification of B . bruxellensis in red wine using commercial kits requires a high level of expertise in molecular biology. We recommend that all users use a microbiological internal control to validate DNA extraction yield.
... They are only produced by a handful of organisms, such as Brettanomyces bruxellensis [4], Brettanomyces anomalus [8] and Pichia guilliermondii [10]. However, because of B. bruxellensis' tolerance to low pH and to higher concentrations of alcohol [9,11], it is the only relevant organism to winemaking that produces significant amounts of these particular volatile phenols, which can cause considerable financial losses to a winery. It is much rarer for lactic and acetic bacteria isolated from wine to synthesize significant quantities of 4-EP [4]. ...
Brettanomyces/Dekkera bruxellensis is a cause of major concern for the winemaking industry worldwide. If a slight presence of this spoilage yeast in red wine adds a Brett character, a strong contamination has irreversible and detrimental effects on the organoleptic qualities due to the production of volatile phenols such as 4-ethylphenol. Time is a key factor in the treatment of B. bruxellensis contaminations. Nowadays, the diagnostic and quantification resources available are time consuming and too expensive, making them either inadequate or inaccessible to most of the winemakers. This study was focused on a new, easy to use, inexpensive method that could allow winemakers to directly detect B. bruxellensis contamination in red wine at an early stage, hence, reducing wine spoilage. In this work, the ability of Pseudomonas putida 4-ethylphenol methylene hydroxylase was tested in order to catabolize the 4-ethylphenol and to elaborate an enzymatic assay with the purpose of detecting early contaminations by B. bruxellensis in red wine. We have developed a colorimetric enzymatic assay, based on the redox state of the 4-ethylphenol methylene hydroxylase co-factor, cytochrome C, that can detect and quantify low concentrations of 4-ethylphenol. The range of concentrations detected is well below the level detectable by the human nose. Combined to an enrichment step, this method allows the detection of B. bruxellensis at an initial concentration of less than 10 cells per ml.
... I microrganismi inquinanti possono appartenere a gruppi differenti. (Renouf et al., 2006). ...
... Although these yeasts have rarely been reported in grapes [29] or in fermenting musts [28], they are commonly detected in red wine during ageing in oak barrels [6,12,21]. Brettanomyces reaches the winery via the vineyard, and can colonize particular surfaces and equipment that could be difficult to clean and sterilize [2,19,26,29,30,31]. ...
Two hundred young wines from the D.O.Ca. Rioja (Spain) coming from two consecutive vintages, 100 wines from 2012 and 100 wines from 2013, were analysed and compared with respect to physicochemical composition and Brettanomyces presence. This microorganism was tested using two techniques: qPCR and an odorimetric test. In 2013, the harvest was characterized by late ripening, health problems with the grapes, and difficulties during vinification. Differences between 2012 and 2013 wines were found both for analytical parameters and as for the presence of the spoilage yeast, Brettanomyces, which was much more prevalent in the 2013 vintage. Moreover, the results showed that for some wines in which Brettanomyces was detected by qPCR at concentration higher than 102 cells/ml, no “Brett” character was found in the odorimetric test. This would indicate that the contamination of a wine by this yeast is not the only factor involved in spoilage. The strains of Brettanomyces, their physiological state, and viability, besides the specific composition of wine, could be important in the “Brett” character appearance. In this study, the wines which developed spoilage in the odorimetric test had slightly lower pH and alcoholic strength values, but higher levels of volatile acidity, reducing sugars and density in comparison with those wines than were not affected. Most of these factors are favourable for the development of Brettanomyces.
... In our experience, many "wild" Saccharomyces strains will grow slowly on lysine and some other non-Saccharomyces yeast may not grow well on lysine. One can also use a direct selection, such as plating a wine on media containing cycloheximide, which is a standard selection for Brettanomyces yeast (Boulton et al. 1996), but has also been used to select for other non-Saccharomyces yeast (Renouf et al. 2006b). Researchers attempt to select for a wide range of organisms by plating on non-selective media such as Wallersteins Nutrient agar (WL) and identifying yeast by colony morphology and dye uptake (Pallmann et al. 2001). ...
Hanseniaspora species (anamorph Kloeckera sp.) are common yeast constituents on grapes and often dominate in the early stages of wine fermentations. Growth of Hanseniaspora sp. in must has been linked to changes in sensory attributes of wine and is proposed as a factor in stuck and sluggish fermentations. A real-time quantitative polymerase chain reaction (PCR) assay was developed to rapidly profile Hanseniaspora sp. populations. The assay allows direct enumeration of Hanseniaspora sp. populations in either must or wine and is not impacted by high concentrations of Saccharomyces cerevisiae DNA. The development of this assay will enable high throughput surveys of must samples to better explore the relationship between early Hanseniaspora sp. populations and the ensuing wine fermentation or sensory changes. Copyright © 2007 by the American Society for Enology and Viticulture. All rights reserved.
... In our experience, many "wild" Saccharomyces strains will grow slowly on lysine and some other non-Saccharomyces yeast may not grow well on lysine. One can also use a direct selection, such as plating a wine on media containing cycloheximide, which is a standard selection for Brettanomyces yeast (Boulton et al. 1996), but has also been used to select for other non-Saccharomyces yeast (Renouf et al. 2006b). Researchers attempt to select for a wide range of organisms by plating on non-selective media such as Wallersteins Nutrient agar (WL) and identifying yeast by colony morphology and dye uptake (Pallmann et al. 2001). ...
The biodiversity of grape and wine flora has been extensively examined using an array of technologies. Although there are some regional differences in the major genera found, a remarkably consistent progression of yeast species is seen. Several key factors have been shown to influence the types of yeasts present and their persistence during fermentation. Generally, the major species identified on the surface of grapes are the major species found at the onset of the alcoholic fermentation. These non-Saccharomyces yeasts impact the aroma and flavor composition of the finished wine, as well as determine its potential for microbial instability during aging and post-bottling. Significant diversity among wine isolates of Saccharomyces has also been documented and this diversity likewise impacts the composition of the finished wine. A thorough understanding of the flora present during the production of wine is important in determining the appropriate processing strategies to achieve the desired composition of the finished product.
... Using a combination of culture-based and direct detection methods, B. bruxellensis has been described across all continents, in niches mostly related to industrial fermentation (Table 1), typically recovered from equipment and difficult-to-sanitise storage vessels such as oak barrels and/or fermented products exposed to them. Indeed, during wine production, the most common period during which B. bruxellensis proliferates and spoils wine is during barrel maturation prior to completion of malolactic fermentation (Renouf et al. 2006), although postbottling spoilage has been reported (Renouf et al. 2007b, Coulon et al. 2010. The points of entry into industrial fermentation niches are of great interest, but remain largely obscure, with no clear association between B. bruxellensis and nonhuman-influenced environs. ...
In the competitive global wine industry, production of wines reflective of their place of origin is critical. While the evidence is mounting that microbial populations in vineyards and wineries differ geographically and represent an important component of 'terroir', there are some microbial influences on wine style that are generally considered undesirable, regardless of whether the offending species is part of the natural winemaking microflora. Brettanomyces spoilage of wine remains one of the most important microbiological issues facing winemakers. While most prevalent in premium, barrel-aged reds, 'Brett' also affects sparkling wines and fortified wines. This review will cover recent advances in knowledge of the biology of this species alongside practical implications for risk management in the winery. For example, insights into genome evolution and population genetics will be discussed in the context of potential for emergence of more sulfite-tolerant strains. Strategies to reduce the risk of Brett spoilage will be updated, with discussion of alternative approaches drawn from other food and beverage sectors that may allow winemakers to reduce their reliance upon the common preservative sulfur dioxide.
Wine is a complex matrix resulting from a fermentation process carried out by specific microbial communities. These communities can be in competition and the development of some microorganisms, as the yeast Brettanomyces bruxellensis , can impact the fermentation process and lead to organoleptic alterations of wine. To manage this risk, microbiological diagnostic methods as microscopic observations, qPCR or flow cytometry are already used in oenology, but remain either not specific enough, or tedious. In this context, IAGE (Ingénierie et Analyses en Génétique Environnementale) has developed the first digital-PCR system enabling the detection and quantification of B. bruxellensis during the whole winemaking process. Furthermore, wine DNA extraction was optimized to enable a representative and sensitive analysis of B. bruxellensis intact cells, as well as an easy-to-implement protocol to cope with the increasing number of samples to analyze. The IAGE workflow for B. bruxellensis quantification has been proven to be successful when analyzing naturally-contaminated samples during the different steps of the winemaking process and offers a robust method to oenologists for appropriate treatments and risk management in wine cellars.
Highlights
Development of a dPCR method led to a highly-specific analysis of B. bruxellensis intact cells in different steps of the winemaking process.
DNA extraction method has been optimized to be robust across various types of wine with varying concentrations of inhibitors, as well as throughout different stages of the wine making process.
The complete process was proven successful in analyzing a large number of naturally-contaminated samples, giving results in less than 48 hours.
This chapter briefly summarizes the history and the worldwide impact of Brettanomyces off-flavor in wines. The production of volatile compounds by Brettanomyces bruxellensis, defined metabolic pathways, and the relation of these compounds to wine off-flavors are described. Brettanomyces/Dekkera physiology is discussed based on factors affecting yeast growth, nutritional requirements, and metabolites not solely related to wine “Brett” off-flavor. Other faults attributable to Brettanomyces/Dekkera presence are described, as mousy taint and the relationships of B. bruxellensis to biogenic amines production. Other microbially related wine spoilage problems (e.g., excess acetic acid; excess diacetyl, geranium taint, mannitol) associated with microbial species other than Brettanomyces are reviewed. Detection and identification of microorganisms in the winery and in wine with specific reference to Brettanomyces is described. This chapter also provides a definition of hygiene principles, and a description of specific technologies which can be used to guarantee wine microbial stability (filtration, preservatives, ozone, pasteurization, DMDC), Brief descriptions of modern technologies are included.
Most alcoholic fermentations in winemaking are nowadays performed by commercially produced yeast strains that are inoculated into the fermentation vessel. Although many yeast strains are available on the global market, the generation of new strains that display different and/or new traits continues to attract significant industrial interest. Many traditional breeding and selection, as well as genetic engineering, approaches have been used to achieve specific targets. These approaches can now be complemented with powerful systems biology tools which provide new insights and opportunities for the generation of new yeast strains while holistically improving our knowledge of wine microbial ecology.
Yeast have a fundamental role in winemaking. They carry out alcoholic fermentation and they contribute to the quality of the wine, although they can also cause spoilage during grape must transformation and in the final product. To detect and identify wine yeast and control their activities, a plethora of different methods can be utilized. As reported in the present chapter, these methods have different degrees of complexity and vary in terms of cost, rapidity and sensitivity. Those based on yeast isolation, namely culture-dependent methods, are widely utilized to define the composition of the microflora associated with wine-related environments and for yeast identification at the strain level, besides providing a means for ex-situ preservation of wine yeast biodiversity. Culture-independent methods bypass microorganisms cultivation, thus avoiding any bias introduced by their isolation and uncovering cell populations undetected by culture-dependent methods. These methods can be utilized to evaluate the impact of all of the components of the wine microbiota on the quality of the final product, to implement a quality control system based on real-time detection and quantification of specific targets, such as the inoculated starter (s) or the spoilage yeast, or to provide further insights into the composition of the microbial communities involved in the grape must transformation.
Enzymes of microbial origin are of immense importance for organic material decomposition leading to bioremediation of organic waste, bioenergy generation, large-scale industrial bioprocesses, etc. The market demand for microbial cellulase enzyme is growing more rapidly which ultimately becomes the driving force towards research on this biocatalyst, widely used in various industrial activities. The use of novel cellulase genes obtained from various thermophiles through metagenomics and genetic engineering as well as following metabolic engineering pathways would be able to enhance the production of thermophilic cellulase at industrial scale. The present review is mainly focused on thermophilic cellulolytic bacteria, discoveries on cellulase gene, genetically modified cellulase, metabolic engineering, and their various industrial applications. A lot of lacunae are yet to overcome for thermophiles such as metagenome analysis, metabolic pathway modification study, search of heterologous hosts in gene expression system, and improved recombinant strain for better cellulase yield as well as value-added product formation.
Microbial cellulases, a major group of industrial enzymes, have a wide range of applications as far as biotechnological, environmental issues, industrial products, and processes are concerned. Thermophilic bacteria act as a good source for industrial cellulase because of its compatibility during industrial harsh processes, for example, deinking of paper, softening of fabric material, pulp and paper, biostoning and biopolishing of fabric materials, juice, and animal feed, etc. Genome mediated application of thermophilic bacteria in various fields has become a rising fantasy among biotechnologists in the current scenario. The genetic manipulation of thermophilic bacteria may lead to enhanced cellulase production through recombinant technology, an emerging technology towards cellulase gene isolation of thermophilic bacteria and their expression in suitable hosts for enhanced cellulase production. Cellulase gene isolation from various thermophilic bacteria could be done through various approaches, such as a classical approach, whole genome isolation, whole metagenome isolation, and bioinformatics. In this chapter, various methods toward the isolation of cellulase genes from thermophilic bacteria are described which may lead to novel cellulase gene discovery as well as enhanced cellulase enzyme production.
The wine matrix contains limited carbon compounds to sustain microbial life. Brettanomyces bruxellensis is one of very few yeast species that has adapted to this environment. Indeed, the presence of growth-inhibiting compounds and conditions do not prevent its proliferation. Literature regarding the nutritional requirements of this yeast is surprisingly poor, given the observation that B. bruxellensis produces biomass with apparently less nutrients than other yeasts. In this study, various carbon sources were screened in a synthetic wine medium, under anaerobic and semi-aerobic growth conditions, in order to determine which compounds B. bruxellensis assimilates. Slight differences were observed between strains but overall, B. bruxellensis produced biomass from limited nutrients consumed in a specific order regardless of the oxygen conditions. Upon initial consumption of the simple sugars, B. bruxellensis was able to remain viable, by concurrently utilising ethanol (only in the presence of oxygen) and malic acid. Although initially beneficial, oxygen was found detrimental in the long term. Formation of volatile phenols occurred during the consumption of the sugars but not as a mechanism to help correct the redox imbalance. The study confirms that B. bruxellensis is able to survive using limited amount of nutrients, making this yeast a challenge for winemakers.
Brettanomyces bruxellensis is a common red wine spoilage yeast. Yet, in addition to wine, it has been isolated from other ecological niches that are just as nutritionally deficient as wine. B. bruxellensis can therefore be regarded as a survivor, well adapted to colonise harsh environments not often inhabited by other yeasts. This review is focused on the nutritional requirements of B. bruxellensis and the relevance thereof for its adaptation to the different matrices within which it occurs. Furthermore, the environmental conditions necessary (e.g. aerobic or anaerobic conditions) for the assimilation of the carbon or nitrogenous sources are discussed in this review. From literature, several confusing inconsistencies, regarding nutritional sources necessary for B. bruxellensis survival, in these specialist ecological niches are evidenced. The main focus of this review is wine but other products and niches that B. bruxellensis inhabits namely beer, cider, fruit juices and bioethanol production plants are also considered. This review highlights the lack of knowledge regarding B. bruxellensis when considering its nutritional requirements in comparison to S. cerevisiae. However, there is a large enough body of evidence showing that the nutritional needs of B. bruxellensis are meagre, explaining its ability to colonise harsh environments.
This chapter presents the taxonomy, genomic analysis, and general description of the Brettanomyces/Dekkera genera. Physiological properties include regulation of respiration–fermentation metabolism (Custers effects), production of volatile phenols, esters, tetrahydropyridines and acetic acid, α-amylase activity. Also covered are the ecological distribution and implications of fermented food and beverages; spoilage activity and strategies for their control; methods of detection and enumeration by culture-dependent and -independent approaches; and interactions with other microorganisms.
Aims: This work reviews the latest knowledge concerning the role of Brettanomyces bruxellensis in red wine alteration.
Results and conclusion: The origin of this yeast species and its place in the wine microbial consortium are discussed as well as microbial equilibriums with the other species, notably Saccharomyces cerevisiae and lactic acid bacteria. As a consequence, fermentations are described as key steps in Brettanomyces development management. Furthermore, the influence of ageing through the use of traditional winemaking practices is explained.
Significance and impact of study: Finally, this paper emphases the need for a better understanding of chemical and microbial analysis together in order to better control this undesirable yeast and prevent the production of volatile phenols.
CITATION: Pretorius, I. S., Van Der Westhuizen, T. J. & Augustyn, O. P. H. 1999. Yeast biodiversity in vineyards and wineries and its importance to the South African wine industry. a review. South African Journal of Enology & Viticulture, 20(2):61-70, doi:10.21548/20-2-2234.
This study aims to understand the effect on micro-organism of racking when the wine is aged in barrels. According to the kind of micro-organism, the effects are different. Bacteria are stimulated by oxygen and their population increases. Yeasts are concentrated to the bottom of the barrel. Between two successive racking a yeast population gradient was established. Yeast cells which are larger and heavier than bacteria cells and they are deposited on the barrel bottom with other wine micro-particles. In some cases, the yeast population at the bottom was more than thousand times than at the wine surface. Moreover, the species identified at different heights in the barrel were different. Saccharomyces cerevisiae was the main yeast detected at the surface, whereas Brettanomyces bruxellensis was the main yeast lees. After racking yeast population decreases because they are eliminated with the lees during the operation. Among them, Brettanomyces bruxellensis was the majority. Since they are able to produce volatile phenols, their preservation in the barrel can lead to the alteration of the wine. Indeed, the ability of the lees to produce volatile phenols was clearly established. The importance of regular racking for microbial wine stabilization is evident. The risks of «sur lies» wine aging and sticking's operations are underlined.
L'élevage des vins rouges en barriques peut présenter certains risques microbiologiques lorsqu'il n'est pas correctement maÎtrisé. L'augmentation de la teneur du vin en acide acétique liée au développement de bactéries acétiques aérobies est facilement inhibée par le respect de règles élémentaires. En revanche, l'hydrolyse des groupements acétyles des hémicelluloses du bois de chêne conduit systématiquement à une faible augmentation de l'acidité volatile des vins conservés en barriques neuves. Ce phénomène devient négligeable avec le bois usagé lorsque l'élevage est bien contrôlé. Le développement de levures de contamination du genre Brettanomyces/Dekkera, produisant des teneurs élevées en éthyl-phénols entre le début de l'été et la fin de l'automne, est plus difficile à maÎtriser. Seul le maintien d'une teneur suffisante en anhydride sulfureux tout au long de leur élevage permet d'empêcher la multiplication de ces micro-organismes dans les vins rouges.
En plus de la dose, les conditions de sulfitage des vins et des barriques sont également importantes. L'apport d'une quantité de SO2 non inférieure à 7 g par fût sous forme gazeuse dans un fût vide permet simultanément la désinfection des douelles et le sulfitage des vins. A dosage identique, avec un sulfitage direct du vin, le développement de Brettanomyces intermedius n'est pas inhibé et les vins risquent de présenter un caractère phénolé désagréable en fin d'élevage. Par ailleurs, les sulfitages à la mèche ou à la pastille ne possèdent pas la même efficacité. Si à quantité de soufre identique le méchage à la pastille procure un sulfitage presque deux fois plus important, selon l'origine et les méthodes de fabrication employées, les pastilles de soufre possèdent une stabilité au vieillissement variable se traduisant par des sulfitages irréguliers en fonction de leur âge et de leurs conditions de stockage. La présence d'une charge non combustible hygroscopique entrant dans la composition de certaines pastilles de soufre est responsable de cette instabilité.
Brettanomyces, a contaminant yeast, is relatively common in wines and mainly in red wines during barrel aging. The results presented here relate to the effects of yeast lees autolysis on the growth of Brettanomyces. Experiments were realised in a culture medium after alcoholic fermentation, in a hydroalcoholic wine-like solution and in a red wine. Brettanomyces was inoculated at low level : 102 cfu/ml and the growth was controlled by counting on agar appropriate medium. Yeast lees from S. cerevisiae were added to these media in the presence or absence of an exogenous enzymatic preparation containing pectinase and β (1→3) glucanase activities. Brettanomyces is able to grow in the synthetic media containing yeast lees. The addition of the glucanase preparation on yeast lees was correlated with an enhance in extracellular glucose level. This higher glucose level corresponds to the acceleration of hydrolysis of cell-wall glucans but was not correlated with a higher level of Brettanomyces population. This result confirms that this yeast multiply by fermenting very small quantities of glucose. In red wine the implantation of Brettanomyces is easy. With a low level of inoculation (102 cfu/ml), contaminant yeast grows rapidly to 106 cfu/ml (in 5 days). The presence of S. cerevisiae lees in red wine enhances significantly Brettanomyces population to 107 cfu/ml. During their development, these yeast produced less than 200 μg/1 of 4-ethyl phenol and 4-ethyl gaïacol. Volatile phenol content in red wine containing yeast lees were 3-5 times lower. Yeast lees have probably the particular property to being able to adsorb these volatile phenols by complexion with the cell-walls.
Wine fermentations are conducted by naturally occurring or selected industrial wine yeast strains of Saccharomyces cerevisiae. However, non-Saccharomyces yeasts also occur naturally in fermenting grape mus ts, especially in the initial stages of the fermentatio n. It has been speculated that these yeasts can con tribute to the over- all characteristics of the wine. Generally, it is ac cepted that Kloeckera apiculata is the predominant non- Saccharomyces yeast species in grape must. However, it was shown previously that Candida pulcherrima was the predominant non-Saccharomyces yeast species in a grape must after sedimentation a nd prior to inoculation with commercial wine yeast. Subsequently, this yeast was investigated in laboratory and small-scale wine fer mentations of Chenin blanc wine. As it could not ferment grape juice to dryness on its own, it was used in combina tion with a S. cerevisiae wine yeast strain. The effect of SO 2, di-ammonium phosphate (DAP), pH and temperature on the growth of C. pulcherrima was also investigated. In combined fermentations, no change in overall fermentation rate or standard wine chemical analyses could be observed in comparison to a control S. cerevisiae fermentation. However, wine production in three consecutive years showed that the wine produced by the combined fermentation was of higher quality than that produced by the S. cerevisiae only. In South Africa the most widely planted white grape variety is Chenin blanc. However, wines produced from this cultivar tend to be neutral in aroma and taste. A number of viticult ural (pruning, ripeness at harvest) and winemaking practices (skin contact, post- fermentation lees contact, maturation practices) are currently being investigated to improve wine quality. A furth er aspect receiving attention is the role of yeast.
Research has shown that non-Saccharomyces yeast strains can be detected throughout wine fermentation. Non-Saccharomyces yeasts can therefore influence the course of fermen tation and also the character of the resultant wine. Previously it was shown that four n on-Saccharomyces species, i.e. Kloeckera apiculata, Candida stellata, Candida pulcherrima and Candida colliculosa, predominated in grape must at the start of fermentation. In this study these four yeasts were used singularly a nd in combination with an industrial wine yeast ( Saccharomyces cerevisiae strain VIN 13) to ferment must on a laboratory scale. The resultant wine was analysed for ethanol, volatile acidity, total SO 2 and glycerol. Results show that, in comparison with the industrial wine yeast, the non-Saccharomyces yeast strains could not ferment all the sugar. Furt hermore, while the individual non-Saccharomyces-fermented wines had different chemical analyses, the wines fermented by the combinations were similar to the wine produced by the industrial yeast only. In subsequent, small-scale winemaking t rials some of the wines produced by combined fermentations were judged to be of better quality than those produce d by the S. cerevisiae only. However, this quality increase could not be lin ked to increased ester levels.
The melting behaviour of a DNA fragment carrying the mouse βmajglobin promoter was investigated as a means of establishing
procedures for separating fragments differing by any single base substitution using the denaturing gradient gel electrophoresis
procedure of Fischer and Lermen (1,2). We find that attachment of a 300 base pair GC-rich DNA sequence, termed a GC-clamp.
to a 135 bp DNA fragment carrying the mouse β-globin.promoter significantly alters the pattern of melting within the promoter
When the promoter, is attathed to the clamp, the promoter sequences melt without undergoing strand dissociation. The calculated
distribution of melting domains within the promoter differs markedly according to the relative orientation of the clamp and
promoter sequences. We find that the behavior of DNA fragments containing the promoter and clamp sequences on denaturing gradient
polyacrylamide gels is in close agre with the theoretical melting calculations. These studies provide the basis for critical
evaluation of the parameters for DNA melting calculations, and they establish conditions for determining whether all single
base substitutions within the promoter can be separated on denaturing gradient gels.
Stress tolerance of Saccharomyces cerevisiae was examined after exposure to heat and salt shock in the presence or absence of the protein synthesis inhibitor cycloheximide. Cells heat-shocked (37 degrees C for 45 min) in the absence of cycloheximide demonstrated increased tolerance of heat, freezing and salt stress. For cells heat-shocked in the presence of cycloheximide, heat and salt tolerance could still be induced, although at lower levels, while induction of freezing tolerance was completely inhibited. These results indicated that while heat shock proteins (hsps) may contribute to induced heat and salt tolerance they are not essential, although induction of freezing tolerance appears to require protein synthesis. Exposure of cells to salt shock (300 mM NaCl for 45 min) induced stress protein synthesis and the accumulation of glycerol, responses analogous to induction of hsp synthesis and trehalose accumulation in cells exposed to heat shock. Cells salt-shocked in the absence of cycloheximide showed a similar pattern of induced stress tolerance as with heat, with increased tolerance of heat, salt and freezing. Cells salt-shocked in the presence of cycloheximide continued to show induced heat and salt tolerance, but freezing tolerance could not be induced. These results lend support to the hypothesis that hsp synthesis is not essential for induced tolerance of some forms of stress and that accumulated solutes such as trehalose or glycerol may contribute to induced stress tolerance.
In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation.
We have constructed restriction site maps of the 5.8S rRNA gene and two ITS regions in 60 strains of Kluyveromyces genus. We test the value of this region as a phylogenetic indicator, and its possible use as a fast and easy method to identify species of this genus. Despite some minor incongruences, our results are in good agreement with previous phylogenetic reconstructions based on the 18S rRNA gene sequencing (Cai et al., 1996; James et al., 1997). A highly significant monophyletic group was formed by K. lactis, K. marxianus, K. aestuarii, K. dobzhanskii and K. wickerhamii, which should be considered the true Kluyveromyces genus. The other species of the genus were grouped with lower bootstrap levels. Finally, the restriction map showed by three K. lactis strains, previously identified as K. marxianus var. drosophilarum, could be interpreted as indicatory of the possible existence of different species.
Yeasts play a central role in the spoilage of foods and beverages, mainly those with high acidity and reduced water activity (a(w)). A few species are capable of spoiling foods produced according to good manufacturing practices (GMPs). These can survive and grow under stress conditions where other microorganisms are not competitive. However, many of the aspects determining yeast spoilage have yet to be clarified. This critical review uses the wine industry as a case study where serious microbiological problems are caused by yeasts. First, the limitations of the available tools to assess the presence of spoilage yeasts in foods are discussed. Next, yeasts and factors promoting their colonisation in grapes and wines are discussed from the ecological perspective, demonstrating that a deeper knowledge of vineyard and winery ecosystems is essential to establish the origin of wine spoilage yeasts, their routes of contamination, critical points of yeast infection, and of course, their control. Further, zymological indicators are discussed as important tools to assess the microbiological quality of wines, although they are rarely used by the wine industry. The concepts of the susceptibility of wine to spoilage yeasts and wine stability are addressed based on scientific knowledge and industrial practices for monitoring yeast contamination. A discussion on acceptable levels of yeasts and microbiological criteria in the wine industry is supported by data obtained from wineries, wholesalers, and the scientific literature.Finally, future directions for applied research are proposed, involving collaboration between scientists and industry to improve the quality of wine and methods for monitoring the presence of yeast.
Replicated, sterile Pinot noir (Vitis vinifera L.) wines were individually inoculated with one of six strains of Brettanomyces bruxellensis (Ave, M, 216, Vin 1, Vin 4, or Vin 5) at initial numbers <50 cfu/mL. In two separate studies, population changes were monitored for 23 months, or until cell densities peaked and subsequently declined to ≤30 cfu/mL. Significant variation was noted in both growth rate and stationary phase population densities among strains. The concentration of selected volatile components was monitored using solid-phase microextraction GC/MS. Large increases in the concentration of 4-ethylphenol occurred after titers reached 2.5 x 10 5 cfu/mL. Brettanomyces-inoculated wines were found to have detectable concentrations of ethyldecanoate, isoamyl alcohol, 4-ethylguaiacol, and 4-ethylphenol, with some significant differences in their concentrations among strains. Duo-trio testing suggested sensory differences between the control and all inoculated wines and among wines inoculated with strains Ave and Vin 5, M and 216, and M and Vin 4. Consensus training analysis suggested that all Brettanomyces wines had a greater perception of earthy, smoky, spicy, and cardboard descriptors than uninoculated control wines.
A simple non-structured model is proposed here to evaluate the lag phase before yeast growth as a function of the ethanol concentration in the medium. Indeed, ethanol is the main yeast growth inhibitor acting in wine. So different cultures of Brettanomyces were run varying the initial ethanol content (0–90g/l). Its influence on yeast growth was shown. A term representing the critical ethanol concentration was introduced in the model as an indicator of the ethanol tolerance. This model fairly represented the behaviour of Brettanomyces intermedius yeast, a wine spoilage micro-organism. The convenience of the model was also observed with literature data obtained for Saccharomyces sp. yeasts. Such a model can be applied in predictive microbiology, to determine a risk-free time lag towards the development of a given yeast.
Sources of yeast, which may contaminate the curd during the manufacture of Cheddar cheese, were examined in a single cheese factory. A total of 77 yeast species present in the factory environment, manufacturing and ripening of Cheddar cheese were identified according to cellular long-chain fatty acid analysis and verified with conventional identification techniques. Product line samples were taken at critical control points in the manufacturing process and analysed after incubation at 25°C for 96 h. The progression of yeast species during cheese-making and ripening was monitored after renneting and at subsequent 48-h intervals. Dominant species wereDebaryomyces hanseniiandCryptococcus albidus, whileYarrowia lipolytica, Rhodotorula minuta, Torulaspora delbrueckii, Rhodotorula glutinisandKluyveromyces marxianuswere present at low numbers. The results obtained showed that yeasts were present in all cheese samples examined, at quantities ranging from 9×102to 1·4×107cfu g−1.
This article emphasises the importance of making quantitative measurements of the growth of yeast species during wine fermentations. Although such studies confirm Saccharomyces cerevisiae as the principal wine yeast, they show that indigenous species of Kloeckera and Candida make a more significant contribution to the fermentation than previously thought. Inoculation of grape juice with S. cerevisiae does not necessarily suppress growth of these indigenous species, nor does it ensure that the inoculated strain will become dominant over indigenous strains of S. cerevisiae. Factors that affect growth of yeasts during fermentation are examined along with the related aspects of stuck fermentations, killer‐yeast activity and yeast autolysis. The contribution of non‐Saccharomyces yeasts to wine quality warrants more serious consideration.
Fermentation by naturally occurring yeasts may produce wines of complex oenological properties that are unique to a specific region. The present work analyses the population dynamics of the yeasts during spontaneous fermentation of six varieties of grape must from the "Valle del Andarax" area (Spain). In this study we identified members of the genera Candida, Hanseniaspora, Issatchenkia, Metschnikowia, Pichia and Saccharomyces by PCR-RFLP of the ITS region. The capability of these yeasts to ferment grape must of the Macabeo variety was studied, and the volatile profile of the wine from each microvinification has been determined. Of all the yeasts isolated Candida stellata and Saccharomyces cerevisiae were able to consume virtually all the initial glucose, producing ethanol contents typical of table wines. The best profile of higher alcohols was given by Saccharomyces cerevisiae followed by Hanseniaspora uvarum, Issatchenkia orientalis and Candida stellata. Metschnikowia pulcherrima and Pichia fermentans showed the highest production of ethyl caprilate and 2-phenyl ethanol, compounds associated with pleasant aromas.
Aroma compounds of wines resulting from fermentation of sterile grape musts from Monastrell variety inoculated with pure and mixed cultures of apiculate and Saccharomyces yeasts, were isolated and analyzed by gas chromatography with flame ionization and mass spectrometry. Samples fermented with mixed cultures produced a higher concentration of selected compounds and higher total amounts of alcohols and acids, in contrast with wines produced with pure cultures of Saccharomyces spp. Apiculate yeasts are important in the chemical composition and quality of wine.
Ethylphenols are important aromatic compounds of red wines. These compounds are formed in wines by some yeast species belonging to the genus Brettanomyces/Dekkera in the presence of hydroxycinnamic acids. These volatile phenols are responsible for the ‘phenolic’, ‘animal’ and ‘stable’ off-odours found in certain red wines. The results presented show that the synthesis of the high quantities of ethylphenols found in the ‘phenolic’ red wines can occur during the ageing of wines having normally completed their alcoholic and malo-lactic fermentations. This olfactory fault caused by Brettanomyces/Dekkera is found more frequently than the classical ‘mousy-taint’ attributed to this yeast genus. In addition, the study of the mechanisms of biosynthesis of ethylphenols by Brettanomyces/Dekkera has shown the sequential activities of two enzymes. The first, is a cinnamate decarboxylase (CD), which assures the transformation of certain cinnamic acids into the correspondent vinylphenols; the second is a vinylphenol reductase, which catalyses the reduction of vinylphenols into ethylphenols. The CD activity of Brettanomyces/Dekkera is not inhibited by the polyphenolic compounds of red wines (procyanidins and catechins) while these compounds do inhibit the CD activity of Saccharomyces cerevisiae. On the other hand, the substrate specificities of the CD activities of Brettanomyces/Dekkera and Saccharomyces are different.
We present a method to directly characterize the yeast diversity present in wine fermentations by employing denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S ribosomal RNA (rRNA) genes. PCR-DGGE of a portion of the 26S rRNA gene was shown to distinguish most yeast genera associated with the production of wine. With this method the microbial dynamics in several model wine fermentations were profiled. PCR-DGGE provided a qualitative assessment of the yeast diversity in these fermentations accurately identifying populations as low as 1000 cells ml−1. PCR-DGGE represents an attractive alternative to traditional plating schemes for analysis of the microbial successions inherent in the fermentation of wine.
The formation of 4-ethyl and 4-vinyl derivatives of guaiacol, phenol and syringol from ferulic acid,p-coumaric acid and sinapic acid, respectively, byBrettanomyces sp. in a synthetic medium was studied by gas chromatography-mass spectrometry. Some of these metabolites possess strong spicy, smoke-like, medicinal, clove-like, woody or phenolic odours and their role as spoilage compounds in wine is discussed. Their formation appears to be characteristic of this yeast genus and its sporulating formDekkera, suggesting these yeasts are Pof+. This paper attempts to clarify the distinctive and characteristic odours which have long been attributed toBrettanomyces yeast metabolism.
Four different zones from the Friuli Venezia Giulia region, North East of Italy, were sampled for the study of the yeast bio-diversity in raw milk. Samples were analysed by traditional methods to isolate different yeast strains that were subjected to identification by sequencing the D1–D2 domains of the 26S rRNA gene. Twelve different species of yeast were identified, six of them belonging to the genera Candida and two to the genera Kluyveromyces. The identified strains were then used for the optimization of a method based on polymerase chain reaction and denaturing gradient gel electrophoresis that was used for a direct monitoring of the populations in the samples. Applying the method to the DNA extracted directly from the raw milk samples, new bands appeared in the gel underlining a different bio-diversity in respect to the traditional method. The approach described is a powerful and reliable tool to monitor directly yeast ecology in milk and milk products without the need of traditional isolation and it could be used to follow specific populations to prevent spoilage or to control contamination.
The kinetics of cell inactivation at high concentrations of ethanol (22.5% and 25% v v−1) was studied with particular reference to the influence of different cultivation conditions (aerobiosis, semi-aerobiosis, anaerobiosis, addition of survival factors) prior to ethanol challenge. The levels of fatty acids and sterols in cells grown under these different conditions were analysed in order to derive a potential relationship between the ethanol tolerance and the lipid composition of the different strains studied. Under the conditions tested, Hanseniaspora guilliermondii showed an ethanol tolerance very similar to that of Saccharomyces cerevisiae, and very different to that of the other apiculate yeast, H. uvarum. The survival of S. cerevisiae and H. guilliermondii at 25% (v v−1) ethanol was strongly influenced by the conditions of cultivation prior to the ethanol challenge. A small increase in survival was observed for H. uvarum and Torulaspora delbrueckii in the cultures grown in aerobiosis. Growth of these yeasts in anaerobiosis in the presence of added ergosterol and Tween 80 (as a source of oleic acid) did not induce a considerable tolerance to the ethanol challenge, in spite of the incorporation of these compounds by the cells. In these growth conditions Debaryomyces hansenii was not capable of incorporating the survival factors and did not increase the ethanol tolerance. It is shown in this work that, besides S. cerevisiae, the presence of oxygen and the addition of survival factors to the culture conditions and the subsequent increase in the proportion of cell sterols and unsaturated fatty acids may play an important role in the ethanol tolerance of some non-Saccharomyces yeasts. This effect was not, however, observed in all of the yeasts studied.
A study of the evolution of the monosaccharides arabinose, rhamnose, galactose, glucose, xylose, mannose, fructose and ribose in a red wine aged in oak barrels is presented. The influences of the barrel manufacture, the variety and the origin of the oak wood have also been considered. The monosaccharide variations depend on the type of wood; higher values during aging are observed for galactose, fructose and xylose. The relationships between monosaccharides explain some structural ratios and their sequence of formation can also be deduced. The oak variety and the coopery have an influence on the monosaccharide composition of the aged wine, which indicates a structural difference between the hemicelluloses of French and American oak wood.
The conversion of p-coumaric acid into 4-ethylphenol was studied in Dekkera bruxellensis ISA 1791 under defined conditions in synthetic media. The production of 4-ethylphenol occurred roughly between mid-exponential growth phase and the beginning of the stationary phase. This behaviour was observed when glucose was the only energy and carbon source, the conversion rate being close to 90%. Ethanol, as the single energy source, yielded conversion rates close to 80% while in the presence of trehalose and acetic acid conversion rates lower than 10% were obtained. The production of 4-ethylphenol was not observed when the cells were maintained in buffer solution without carbon and energy sources. The precursor of 4-ethylphenol, p-coumaric acid, was not utilized as energy and carbon source. Furthermore, it was shown that 4-vinylphenol may be used as a precursor of 4-ethylphenol in the absence of p-coumaric acid.Growth and 4-ethylphenol production were inhibited by increasing concentrations of ethanol, being fully prevented at 13% (v/v) ethanol.The cultivation of strain ISA 1791 in mixed culture with Saccharomyces cerevisiae, in synthetic medium, showed that the cell numbers of D. bruxellensis increased from 104 cfu/ml to 5×109 cfu/ml. Laboratory microvinifications of white and red juices inoculated with as low as 10 cfu/ml of D. bruxellensis and 107 cells/ml of S. cerevisiae showed growth of D. bruxellensis to levels of about 5×108 cfu/ml. In addition, 4-ethylphenol production by D. bruxellensis was observed only after complete fermentation of the grape juices.
This review considers the effect of ethanol-induced water stress on yeast metabolism and integrity. Ethanol causes water stress by lowering water activity (aw) and thereby interferes with hydrogen bonding within and between hydrated cell components, ultimately disrupting enzyme and membrane structure and function. The impact of ethanol on the energetic status of water is considered in relation to cell metabolism. Even moderate ethanol concentrations (5 to 10%, w/v) cause a sufficient reduction of aw to have metabolic consequences. When exposed to ethanol, cells synthesize compatible solutes such as glycerol and trehalose that protect against water stress and hydrogen-bond disruption. Ethanol affects the control of gene expression by the mechanism that is normally associated with (so-called) osmotic control. Furthermore, ethanol-induced water stress has ecological implications.
Because of their well-documented fermentative and claimed health-promoting properties, lactic acid bacteria (LAB) are one of the most important groups of bacteria to the food industry. The correct species identification of LAB is of paramount importance from the technological, ecological, and safety point of view. This paper provides an overview of phenotypic and genotypic techniques that are currently used for the culture-dependent and culture-independent identification of LAB. In particular, the direct identification potential of the culture-independent Denaturing Gradient Gel Electrophoresis (DGGE) technique is highlighted.
This compendium of techniques coverage of state-of-the-art developments in molecular biology, with over 600 pages of information contributed by a wide range of authorities.
Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rat, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing D-[6-3H] glucose, D-[1-14C] glucosamine, L-[U-14C] lysine or arginine, or KH232PO4 lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5-7 times greater than that in the cell-free medium.
DNA fragments 536 base pairs long differing by single base-pair substitutions were clearly separated in denaturing gradient gel electrophoresis. Transversions as well as transitions were detected. The correspondence between the gradient gel measurements and the sequence-specific statistical mechanical theory of melting shows that mutations affecting final gradient penetration lie within the first cooperatively melting sequence. Fragments carrying substitutions in domains melting at a higher temperature reach final gel positions indistinguishable from wild type. The gradient data and the sites of substitution bracket the boundary between the first domain and its neighboring higher-melting domain within eight base pairs or fewer, in agreement with the calculated boundary. The correspondence between the gradient displacement of the mutants and the calculated change in helix stability permits substantial inference as to the type of substitution. Excision of the lowest melting domain allows recognition of mutants in the next ranking domain.
Determination of the membrane lipid composition of Saccharomyces cerevisiae revealed an increase in the unsaturation index, qualitative and quantitative changes in sterol content and an alteration of the activity of the plasma membrane ATPase when cells were pre-adapted to ethanol. All these changes may constitute different adaptation mechanisms which allow the cell to cope with ethanol stress. The importance of the lipid environment on the plasma membrane ATPase activity is also discussed.
The effects of water activity (aw), solutes, potassium sorbate and incubation temperature on the fatty acid methyl ester composition of the total extracted lipid, triacylglycerol, phospholipid, free fatty acid and steryl ester components of Zygosaccharomyces rouxii were determined. Oleic (C18:1) and linoleic (C18:2) acids were the major components of the total fatty acid fraction, regardless of culturing conditions. Cells grown at 35 degrees C contained a significantly higher percentage of C18:1 and a lower percentage of C18:2 than did cells grown at 21 degrees C. At both temperatures, cells cultured in low-aw (0.93) media contained a significantly higher percentage of C18:1, with a corresponding decrease in C18:2 content than did cells grown at aw 0.99, regardless of the addition of glucose, sucrose, or NaCl to suppress the aw. At 21 degrees C, cells cultured in high-aw (0.99) media supplemented with 300 micrograms sorbate per ml contained higher percentages of C18:1 than did cells cultured in media not supplemented with sorbate. Cells grown at 35 degrees C in low-aw media supplemented with sorbate contained substantially more C18:1 than did cells cultured without sorbate.
Brettanomyces sp. and its ascosporogenous sexual state, Dekkera sp., have been well documented as spoilage microorganisms, usually associated with barrel-aged red wines. In this report, we describe the genetic characterization, on the basis of DNA content per cell, electrophoretic karyotyping, and mitochondrial DNA restriction patterns, of a Dekkera yeast strain isolated from sherries and of a number of other Brettanomyces and Dekkera strains. By using a genomic DNA fragment of the isolated Dekkera strain, we developed a two-step PCR method which directs the specific amplification of target DNA from this strain and from other Brettanomyces-Dekkera strains. The method efficiently amplified the target DNA from intact cells, obviating DNA isolation, and yielded a detection limit of fewer than 10 yeast cells in contaminated samples of sherry.
Autoantibody formation is possibly integral to the development of non-respiratory manifestations of acute Mycoplasma pneumoniae infection. We sought to confirm the occurrence of smooth muscle antibodies (SMA) in humans with acute Myc. pneumoniae respiratory infection and furthermore to assess whether similar autoantibodies would develop in a hamster model of respiratory infection. Paired sera from 21 patients with acute infection were assayed for SMA by immunofluorescence on mouse kidney/stomach substrates. The frequency of SMA was then determined for 52 paediatric patients with acute Myc. pneumoniae infection and 16 controls, and for sera from a hamster model of infection. Five of 21 paired sera had an increment in SMA between acute and convalescent specimens. At a screening dilution of 1:40, 18/52 infected and 0/16 controls had positive sera (P = 0.003); positive specimens demonstrated IgG rather than IgM SMA. In the hamster model of Myc. pneumoniae respiratory infection, significant IgG SMA increases occurred in 7/19 infections but not in 11 controls (P = 0.02). Immunoblotting did not identify actin as the substrate for SMA. Smooth muscle antibody increases are found in a significant minority of Myc. pneumoniae-infected humans and hamsters. A role for SMA in the pathogenesis of Myc. pneumoniae infection remains to be defined.
Ninety-six strains of apiculate wine yeasts were studied for their ability to produce glycerol, acetaldehyde, ethyl acetate, sulphur dioxide and hydrogen sulphide in synthetic medium. Hanseniaspora guilliermondii produced smaller quantities of glycerol, acetaldehyde and hydrogen sulphide than Kloeckera apiculata, whereas the production of ethyl acetate and sulphur dioxide was found to be similar. Strains characterized by different capacities and properties were found for both species. The existence of apiculate strains differing in secondary compound production is of technological interest, as these yeasts constitute potential flavour producers. Selected strains of apiculate yeasts might favour an enhanced flavour formation and yield desirable characteristics to the final product.
Five years after the introduction of denaturing gradient gel electrophoresis(DGGE) and temperature gradient gel electrophoresis (TGGE) in environmental microbiology these techniques are now routinely used in many microbiological laboratories worldwide as molecular tools to compare the diversity of microbial communities and to monitor population dynamics. Recent advances in these techniques have demonstrated their importance in microbial ecology.
Colony counting and DEFT did not give the same results when wine micro-organisms were enumerated. Both methods were used to monitor the population of acetic acid bacteria (AAB) and lactic acid bacteria (LAB) during wine storage. Results suggest that part of the populations had reached a viable but non-culturable (VBNC) state. These cells were unable to produce colonies but could hydrolyse fluorescent esters and could be counted by DEFT. For AAB, O2 deprivation quickly induced this state. Recovery from this state was very rapid as soon as O2 was available. The response was not so clear for LAB during wine storage. However, a similar state was induced by sulfiting. Moreover, filtration of wine stored in barrels and contaminated by Brettanomyces, AAB and LAB demonstrated that cell size was not homogeneous. Cells which remained in wine after several weeks could pass through a 0.45-microm membrane. However, when they re-entered a growing phase, they were again retained by membrane filtration. During and after the decline phase, wine micro-organisms might survive as smaller cells in a VBNC state.
The heat inducibility of the yeast heat-shock response (HSR) pathway has been shown to be critically dependent on the level of unsaturated fatty acids present in the cell. Here the inducibility by heat or salt of the independently regulated general stress response (GSR) pathway is shown to be affected in the same way. An increase in the percentage of unsaturated fatty acids in heat- or salt-acclimated cells correlated with a decrease in the induction of a general stress-response-promoter-element (STRE)-driven reporter gene by either stress. Despite inducing reporter gene expression, sorbic acid treatment did not confer salt cross-tolerance on the cells. This failure correlated with a failure to increase the percentage of unsaturated fatty acids in the cells, suggesting that GSR pathway induction, in the absence of lipid changes, is insufficient for the induction of cross-tolerance. Cells grown with fatty acid supplements under anaerobic conditions provided further evidence for a potential role for lipids in the acquisition of stress resistance. These cells contained different fatty acid profiles depending on the fatty acid supplement supplied, exhibited differential sensitivity to both heat and salt stress, but had not undergone STRE induction. These results suggest that heat- and salt-stress induction of the GSR are sensitive to the level of unsaturated fatty acids present in the cell and that stress cross-tolerance may be a lipid-mediated phenomenon. Given that an increased level of unsaturated fatty acids also down-regulates heat induction of the HSR pathway, these observations lead to the provocative hypothesis that lipid modifications, rather than HSR or GSR pathway induction, are a major contributor to the induced heat and salt tolerance of yeast cells.
We present a method to directly characterize the yeast diversity present in wine fermentations by employing denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S ribosomal RNA (rRNA) genes. PCR-DGGE of a portion of the 26S rRNA gene was shown to distinguish most yeast genera associated with the production of wine. With this method the microbial dynamics in several model wine fermentations were profiled. PCR-DGGE provided a qualitative assessment of the yeast diversity in these fermentations accurately identifying populations as low as 1000 cells ml(-1). PCR-DGGE represents an attractive alternative to traditional plating schemes for analysis of the microbial successions inherent in the fermentation of wine.
Within-family selective breeding techniques have been used to create two lines of mice to be insensitive (HOT) and two lines to be sensitive (COLD) to the hypothermic effects of an acute 3.0-g/kg ethanol (EtOH) injection. Previous studies have found HOT mice to be relatively resistant to the development of tolerance to this effect, whereas COLD mice readily develop tolerance. The breeding program is currently in selected Generation 52, and the HOT and COLD mice differ by about 10 degrees C (average of both replicates) in their selected hypothermic response. Starting with selection Generation 20, separate lines of mice were inbred from the HOT-2 and COLD-2 selected lines, while selection continued for the original two replicate lines of HOT and COLD mice. To assess whether different dose treatments would produce differential tolerance development in the HOT and COLD selected lines, we administered different dose regimens across 5 days to HOT and COLD mice. The COLD mice developed tolerance while the HOT mice did not, regardless of total EtOH administered. In a separate study, we administered EtOH (3.0 g/kg) to mice for 3 days to assess a shorter tolerance paradigm. We also present here responses to the selection dose of 3.0-g/kg EtOH in the inbred HOT (IHOT-2) and COLD (ICOLD-2) mice tested after 41 generations of brother-sister mating. In addition, we report recent attempts to find doses of EtOH that would produce an equivalent initial hypothermic response in each of the six lines (HOT-1, COLD-1, HOT-2, COLD-2, ICOLD-2, and IHOT-2). When doses were selected to produce similar initial hypothermic sensitivity, tolerance was tested by giving three daily doses and examining the attenuation of the hypothermic response on the third day. All three COLD lines developed significant tolerance, while the HOT lines did not. The HOT and COLD mice provide a genetic model to study mechanisms mediating acute EtOH-induced hypothermia as well as tolerance development.
The objectives of this work were to develop a selective and/or differential medium able to efficiently recover Dekkera/Brettanomyces sp. from wine-related environments and to determine the relationship between these yeasts and the 4-ethylphenol content in a wide range of wines.
The selectivity of the developed medium was provided by the addition of ethanol, as single carbon source, and cycloheximide. The inclusion of bromocresol green evidenced acid-producing strains. The inclusion of p-coumaric acid, substrate for the production of 4-ethylphenol, enabled the differentiation by smell of Dekkera/Brettanomyces sp. from all other yeast species growing in the medium. The medium was used either by plating after membrane filtration or by the Most Probable Number (MPN) technique. In 29 white and 88 red randomly collected wines, these yeasts were found only in red wines at levels up to 2500 MPN ml-1, but constituted less than 1% of the total microbial flora. In red wines, 84% showed detectable amounts of 4-ethylphenol up to 4430 microg l-1 while 28% of the white wines showed detectable levels up to 403 microg l-1.
The use of the medium proposed in this work evidenced the presence of low relative populations of Dekkera/Brettanomyces sp. even in wines contaminated by fast-growing yeasts and moulds.
Further ecological studies on Dekkera/Brettanomyces sp. should take into account the use of highly specific culture media in order to establish their true occurrence in nature.
Yeast isolates from orange fruit and juice in a spontaneous fermentation were identified and classified by two molecular techniques. The first was analysis of the restriction pattern generated from the polymerase chain reaction (PCR)-amplified 5.8S rRNA gene and the two internal transcribed spacers (ITS) using specific primers. The second technique was sequence analysis of the ITS regions using the same two primers. Nine different restriction profiles were obtained from the size of the PCR products and the restriction analyses with three endonucleases (CfoI, HaeIII and HinfI). These groups were identified as Candida tropicalis, Clavispora lusitaniae, Hanseniaspora uvarum, Pichia anomala, Pichia fermentans, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces unisporus, and Trichosporon asahii. Checking against identification according to morphological, physiological and biochemical traits corroborated this molecular identification. A total concordance was found in the identification with PCR-restriction fragment length polymorphism of the ITS region after analysing certified yeast strains from two different culture collections. Consequently, a rapid and reliable identification of the yeast populations was achieved by using molecular techniques.
The performance of denaturing gradient gel electrophoresis (DGGE) for analysing yeasts associated with wine grapes was compared with cultural isolation on malt extract agar (MEA). After optimisation of PCR and electrophoretic conditions, the lower limit of yeast detection by PCR-DGGE was 10(2) cfuml(-1), although this value was affected by culture age and the relative populations of the species in mixed culture. In mixed yeast populations, PCR-DGGE detected species present at 10-100-fold less than other species but not when the ratio exceeded 100-fold. Aureobasidium pullulans was the main species isolated from immature, mature, and both damaged and undamaged grapes. It was not detected by PCR-DGGE when present at populations less than 10(3) cfug(-1). When approaching maturity, damaged grapes gave a predominance of Metschnikowia and Hanseniaspora species (10(5)-10(7) cfug(-1)), all detectable using PCR-DGGE. However, various species of Rhodotorula, Rhodosporidium and Cryptococcus were not detected by this method, even when populations were as high as 10(4) cfug(-1). PCR -DGGE was less sensitive than culture on MEA for determining the yeast ecology of grapes and could not reliably detect species present at populations less than 10(4) cfug(-1). However, this method detected a greater diversity of species than agar plating.
Brettanomyces bruxellensis is a well-known wine spoilage yeast that causes undesirable off-flavours. Likewise, glucan-producing strains of ropy Pediococcus damnosus are considered as spoilage micro-organisms because the synthesis of glucan leads to an unacceptable viscosity of wine.
We developed a real-time PCR method to detect and quantify these two spoilage micro-organisms in wine. It is based on specific primer pairs for amplification of target DNA, and includes a melting-curve analysis of PCR products as a confirmatory test.
The detection limit in wine was 10(4) CFU ml(-1) for B. bruxellensis and 40 CFU ml(-1) for ropy Pediococcus damnosus. The real-time PCR proved to be reliable for the early, sensitive detection and quantification of B. bruxellensis and ropy P. damnosus in wine.
The real-time PCR-based method described in this study provides a new tool for monitoring spoilage micro-organisms in wine. Time-consuming culture and colony isolation steps are no longer needed, so winemakers can intervene before spoilage occurs.