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Development of an enrichment medium to detect Dekkera/Brettanomyces bruxellensis, a spoilage wine yeast, on the surface of grape berries
Abstract
Brettanomyces bruxellensis spoilage is a serious problem for the wine industry. Mainly, by producing 4-ethylphenol and 4-ethylguaiacol, it confers off-odors to the wine and changes its aromatic quality. The presence of B. bruxellensis cells on the berry was speculated but it had never been clearly demonstrated. On grape berries, the microbial ecosystem is highly diverse and the population of B. bruxellensis can be very small. The aim of our study was to reveal and confirm the presence of B. bruxellensis on the surface of grape berries. We developed an enrichment medium for B. bruxellensis in order to overcome the detection limit of the molecular methods (species-specific PCR, ITS-RFLP PCR, PCR-DGGE). This medium, named EBB medium, made it possible to detect B. bruxellensis after 10 days of culture. For the first time, the presence of B. bruxellensis has been clearly established in several vineyards and at different stages of the grape development after the veraison. This work led to the conclusion that the grape berry is the primary source of B. bruxellensis. Grape growers and winemakers should take these results into account when deciding on the treatment to apply in the vineyards and the must. With the information provided here, B. bruxellensis prevention could start in the vineyard.
... D. bruxellensis is more frequently found on ripe grape berries, i.e. at harvest time, than during the berry growing stage, while it is still green and immature (Renouf and Lonvaud-Funel, 2007). Besides, Barbin et al. (2007) reported that D. bruxellensis allocation is related to the land's topography (physical configuration) or its surrounding. ...
... populations even in wines that were contaminated by fast-growing moulds and yeasts. Besides, these formulated novel enrichment mediums enabled its separation from grape berry surface (Renouf and Lonvaud-Funel, 2007). ...
... It has also been reported that the spoilage and formation of VP can happen in stainless steel or concrete vats, and even in bottles (Rodrigues et al., 2001;Pérez-Prieto et al., 2003). On the other hand, several strains of D. bruxellensis have been found and isolated from the vineyards (Renouf and Lonvaud-Funel, 2007). Even though all wines could be contaminated, there are some factors that can increase the risk of spoilage and which require more attention in order to avoid possible losses. ...
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
... En l'absence de croissance, il n'y aurait pas production de phénols volatils Coulon et al., 2009). Il est à noter que Serpaggi et al. (2012) ont observé la production de 4EP par les cellules non cultivables (en état VNC), ce qui n'a pas été retrouvé par Longin et al. (2016). Dans le cas de cultures « actives » (cellules en croissance), les vinyls phénols sont produits dès la phase de latence et les éthyls phénols pendant la phase exponentielle et la phase stationnaire en milieu synthétique (Dias et al., 2003a ;Harris et al., 2008 ;Coulon et al., 2009) et dans le vin . ...
... Systematic error and comparison of four methods for assessing the viability of Saccharomyces cerevisiae suspensions. BiotechnolTech 7,[223][224][225][226][227][228] Larcher, R., Puecher, C., Rohregger, S., Malacarne, M., and Nicolini, G.(2012). depletion in wine using esterified cellulose. ...
... Development of a reliable and easy method for screening Oenococcus carbohydrate consumption profile. OENO One 44,[31][32][33][34][35][36][37] Vigentini, I., Lucy Joseph, C. m., Picozzi, C., Foschino, R., and Bisson, L.F. (2013) (1); 261 (7); 264 (7) 1990,2003,2010,2012,2013,2014 France, Afrique du Sud Bordeaux * le numéro de génotype a été attribué aléatoirement. 7 114 1926, 1948, 1967, 1970 France Bordeaux 8 11 1961, 2012, 2013, 2014 France Bordeaux 9 12 2003France Languedoc, Bourgogne, Bordeaux 9 115 1909, 1926, 1948, 1970, 2001France Bordeaux 10 110 1911, 1939, 1961 France Bordeaux 12 29 1972, 2003, 2013, 2014 Etats-Unis, France 18 47 1985France Bordeaux 18 196 2014 France Languedoc 45 30 1959, 1961, 1968, 1996, 2003, 2013, 2014 France Bordeaux 51 52 1949, 1999, 2004, 2012, 2013, 2014 Australie, Italie, Allemagne, Brésil, Afrique du Sud, France ...
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.
... Since the 1980's, numerous studies have speculated on the occurrence and diffusion of Brettanomyces spp. in winemaking (Pretorius, 2000;Suárez et al., 2007;Agnolucci et al., 2009;Campolongo et al., 2010;Di Toro et al., 2015), although their origin is not well defined yet (Aguilar-Uscanga et al., 2000;Curtin and Pretorius, 2014;Avramova et al., 2018). Many studies have associated this spoilage yeast with poor hygienic practices in the cellar, presence of cellobiose and micro-oxygenation related to the use of wooden barrel, together with high ethanol content (Chatonnet et al., 1999;Renouf and Lonvaud-Funel, 2007;Boulton et al., 2013). It was only in 2007 that the presence of B. bruxellensis on the grape berry surface was clearly demonstrated, using an enrichment medium (Renouf and Lonvaud-Funel, 2007). ...
... Many studies have associated this spoilage yeast with poor hygienic practices in the cellar, presence of cellobiose and micro-oxygenation related to the use of wooden barrel, together with high ethanol content (Chatonnet et al., 1999;Renouf and Lonvaud-Funel, 2007;Boulton et al., 2013). It was only in 2007 that the presence of B. bruxellensis on the grape berry surface was clearly demonstrated, using an enrichment medium (Renouf and Lonvaud-Funel, 2007). The use of this formulated medium represents a useful tool to detect B. bruxellensis, as this yeast appears to have low presence and to lack competitive ability in ecological niches such as the grape berry surface. ...
... The refrigerated bags that contained the harvested grape berries were opened in the laboratory under sterile conditions, and 45 berries per sample were taken and placed into a 250 mL sterile flask containing 150 mL of the enrichment B. bruxellensis (EBB) medium (Renouf and Lonvaud-Funel, 2007). The EBB medium had the following composition: 200 mL/L red grape juice (commercial grape juice; Folicello, Modena, Italia with 19.1% sugar, nitrogen content YAN 115 mgN/L and pH 3.4), 40 mL/L ethanol (VWR International, Milan, Italy), 1.5 g/L malt extract (Sigma-Aldrich, Milan, Italy), 1.5 g/L yeast extract, 0.5 g/L (NH 4 ) 2 SO 4 (Sigma-Aldrich, Milan, Italy), 0.2 g/L MgSO 4 , and 0.5 mL/L Tween 80 (Merck, Hohenbrunn, Germany). ...
The spoilage yeasts belonging to the genus Dekkera (anamorph Brettanomyces) are associated with the fermentation process and can be responsible for off-flavors in wine. Brettanomyces bruxellensis is difficult to isolate from natural environments because of its low diffusion, low presence on the grape surface and low competition capacity, slow growth, and VBNC (viable but not culturable) state, even when selective media are used. In this study, to investigate the origins and occurrence of B. bruxellensis in winemaking, a total of 62 samples from grapes, winery environment, and fermenting musts were taken through direct isolation with a selective medium. B. bruxellensis was not directly detected in the grape samples but was instead widely isolated from the winery environment samples. However, using a combination of enrichment and selective media, eight of fifteen grape samples were positive for B. bruxellensis. Analysis of the genetic traits of the isolates indicated a strict relationship among the strains from the vineyard and the winery. Isolates from the vineyard and the winery were both part of the more common and dominant biotypes suggesting that the vineyard may be the contamination source of B. bruxellensis in the winery environment. For this, grapes may represent the possible primary origin source from which a flow toward the winery environment originates. On the other hand, the wide occurrence of B. bruxellensis in winery indicates that this environment can be considered as the favorable ecological niche for colonization and diffusion of these yeast.
... These latter reports suggested that a low population of the yeast on grapes could lead to a transfer of the yeast into wineries, leading to subsequent infection during ageing (Guerzoni andMarchetti 1987, Arojo et al. 1998). Though primary sources of B. bruxellensis are contaminated barrels (Chatonnet et al. 1992) and unfiltered wines (Fugelsang and Edwards 2007), Renouf and Lonvaud-Funel (2007) suggested that the grape berry can be a primary vector towards winery infections. ...
... As an example, Barbin et al. (2007) isolated the yeast from grape berries having highly diverse microbial populations using enrichment over a 2 month incubation period. Likewise, Renouf and Lonvaud-Funel (2007) developed an Brettanomyces enrichment medium (EBB) which included (i) Tween 80 to help remove yeasts from berry surfaces; (ii) ethanol as a selective agent which inhibits sensitive yeasts (Medawar et al. 2003); and (iii) chloramphenicol and biphenyl to limit growth of bacteria and moulds, respectively. An alternative approach has been the application of molecular techniques such as polymerase chain reaction, which can detect B. bruxellensis among diverse microbial populations (Cocolin et al. 2004, Agnolucci et al. 2007, Portugal and Ruiz-Larrea 2013. ...
... An alternative approach has been the application of molecular techniques such as polymerase chain reaction, which can detect B. bruxellensis among diverse microbial populations (Cocolin et al. 2004, Agnolucci et al. 2007, Portugal and Ruiz-Larrea 2013. Using selective media and molecular methods, B. bruxellensis has been detected on grapes from vineyards located in Chile (Barbin et al. 2007), France (Renouf and Lonvaud-Funel 2007) and Italy (Agnolucci et al. 2007). ...
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.
... Prior to the work of Renouf and Lovaud-Funel, 37 the origin of these yeasts in the winery was not completely understood, but through the development of an enrichment media specifically for growth of B. bruxellensis, detection on grape berries and the vineyard origin was inferred. These wild yeasts encounter the grapes by becoming airborne and settling on them in the vineyard, or can be spread by fruit fly and bees with traces of these yeasts having being found in the feeding and breeding areas of both insects, as well as on their legs, bodies and wings. ...
... 15 Current techniques for detection revolve around additions of compounds inhibitory to the growth of other organisms, allowing Dekkera to be the sole occupant and more easily identified, 59 in a similar manner to that previously described for detection on grapes. 37 If able to grow in wine, D. bruxellensis is associated with formation of several "spoilage" ...
... conditions. To test the efficacy of the dechloroacetylation, the p-coumaroyl analogue (37) was dissolved in 1:1 pyridine/benzene and stirred at room temperature for 24 hours. ...
The presence of esterified hydroxycinnamate conjugates in grapes and wine is well established and they account for a large proportion of the hydroxycinnamate content. There exists the possibility that these conjugates could also provide the potential for spoilage, though they have never been linked to the direct formation of ethylphenols. This research highlighted the potential role of a number of esterified conjugates in the production of ethylphenols by D. bruxellensis. Two classes of berry derived esters, the tartaric acid and glucose bound hydroxycinnamates, as well as the vinification formed ethyl esters, were synthesised and used for model fermentation experiments.
... 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. ...
... As B. bruxellensis is frequently associated with barrel-red wines, wood used in storage and aging may be a common vector for the introduction of this species in wine in red wine (Suarez et al., 2007). However, some strains have been isolated from the vineyard (Renouf & Lonvaud-Funel, 2007). In particular, Renouf & Lonvaud-Funel (2007) where able to isolate Brettanomyces spp. ...
... However, some strains have been isolated from the vineyard (Renouf & Lonvaud-Funel, 2007). In particular, Renouf & Lonvaud-Funel (2007) where able to isolate Brettanomyces spp. from grape berries by using an optimized enrichment broth, able to recover their populations in a culture-dependent manner indicating that grapes may act as a possible vector for the introduction of this yeast species into the wine. ...
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.
... Interestingly, microbiome descriptive studies on multiple fermented food and beverages highlighted presence of B. bruxellensis. Isolates belonging to the species were found on grapes (Renouf and Lonvaud-Funel, 2007), in cider (Coton et al., 2006;Morrissey et al., 2004), kombucha tea (Coton et al., 2017;Teoh et al., 2004), kefir (Laureys and De Vuyst, 2014), olives (Coton et al., 2006), bioethanol production plants (Basílio et al., 2008;Beckner et al., 2011;de Souza Liberal et al., 2007;Passoth et al., 2007;Souza et al., 2012), agave fermentation for tequila production (Lachance, 1995;Lappe-Oliveras et al., 2008), soft drinks (Deak and Beuchat, 1995;Put et al., 1976;Yarrow and Ahearn, 1971), sourdough (Hammes et al., 2005;Meroth et al., 2003), yoghourt (Kosse et al., 1997), etc. Particularly interesting case is the one of B. bruxellensis occurrence in bioethanol production plants (Basílio et al., 2008;Beckner et al., 2011;de Souza Liberal et al., 2007;Passoth et al., 2007;Souza et al., 2012). Bioethanol production through microorganism fermentation allows ethanol synthesis from organic matter, thus presenting an eco-friendly process for the production of ethanol which can be used as fuel. ...
... Brettanomyces bruxellensis (teleomorph Dekkera bruxellensis) that is typically detected during wine aging but also at lower frequency during the early stages of the winemaking process (grapes and must) (Renouf, 2009;Renouf and Lonvaud-Funel, 2007).When it grows in wine, B. bruxellensis produces odorant molecules (namely volatile phenols), which are associated with unpleasant aromas described as barnyard, horse sweat, Band-aid ® (Chatonnet et al., 1995a(Chatonnet et al., , 1992Heresztyn, 1986). Therefore, the presence of B. bruxellensis in wine often provokes rejection by consumers and serious economic losses for winemakers (Wedral et al., 2010). ...
Brettanomyces bruxellensis is a microorganism described as the first cause of microbial spoilage of wine. Its industrial relevance is highlighted by the fact that this yeast is isolated from different substrates such as beer, kombucha, bioethanol fermentation molasses and others. This project aims to explore the genetic diversity of the species by studying a large population of isolates from various geographical and ecological niches. For this purpose, a robust genotyping method (microsatellite analysis) was optimized and applied on the population, thus highlighting the coexistence of diploid and triploid populations worldwide. Further, the relation between genotypic clustering and physiological traits was studied. Namely, sulphite tolerance assay was performed on a subset of strains representative of the total population. The results reveal a link between genetic group and growth profile in the presence of sulphur dioxide. Competition experiments in presence of sulphites highlight a selective advantage of sulphite tolerant strains compared to sulphite sensitive ones, thus suggesting a specific adaptation to the main antimicrobial used in winemaking. This work contributes to a deeper understanding of this wine spoilage microorganism in means of genetic and phenotypic diversity and sheds light on putative evolutionary strategies for adaptation to human related environment of this non-conventional model yeast species.
... The dissemination of B. bruxellensis in wine related environments is hard to evidence from sources contaminated by other yeasts due to its low growth rate. As a consequence, the use of selective media and long incubation periods are essential to its recovery [29], although even when using selective media, it has been rarely isolated from grapes [30] and clean winery environments. Rotten grapes appear to bear higher populations, which most likely explains the higher incidence of phenolic wines in vintages affected by this problem [31]. ...
... It is not as tolerant to ethanol or sulfite as S. cerevisiae or Zygosaccharomyces bailii [35] but it has the ability to remain viable for long periods. The increase in their predominance appears to be the result of an exceptional resistance to minimal nutrient conditions, which is seen as the determinant in their survival during the production and storage steps and in their development once the environment becomes favorable (e.g., reduction in free sulfite during ageing) [11,30]. For instance, viable Brettanomyces have been reported in red wine bottled for more than 50 years [33]. ...
The unwanted modification of wine sensory attributes by yeasts of the species Brettanomyces bruxellensis due to the production of volatile phenols is presently the main microbiological threat to red wine quality. The effects of ethylphenols and other metabolites on wine flavor is now recognized worldwide and the object of lively debate. The focus of this review is to provide an update of the present knowledge and practice on the prevention of this problem in the wine industry. Brettanomyces bruxellensis, or its teleomorph, Dekkera bruxellensis, are rarely found in the natural environment and, although frequently isolated from fermenting substrates, their numbers are relatively low when compared with other fermenting species. Despite this rarity, they have long been studied for their unusual metabolical features (e.g., the Custers effect). Rising interest over the last decades is mostly due to volatile phenol production affecting high quality red wines worldwide. The challenges have been dealt with together by researchers and winemakers in an effective way and this has enabled a state where, presently, knowledge and prevention of the problem at the winery level is readily accessible. Today, the main issues have shifted from technological to sensory science concerning the effects of metabolites other than ethylphenols and the over estimation of the detrimental impact by ethylphenols on flavor. Hopefully, these questions will continue to be tackled together by science and industry for the benefit of wine enjoyment.
... 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]. ...
... 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]. A recent study with wineries of the D.O.Ca. ...
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.
... Its presence is synonymous with the production of volatile phenols and more particularly 4-ethylphenol (4 EP) characterized by stable odors, horse sweat which cause consumer rejection (Chatonnet et al., 1992;Lattey et al., 2010Agnolucci et al., 2017. The presence of B. bruxellensis is reported throughout the winemaking process, from grapes to the finished wine, due to mechanisms allowing it to grow under stressful conditions (Renouf and Lonvaud-Funel., 2007;Rubio et al., 2015;Avramova et al., 2018a). Genetic typing of 1488 isolates via the analysis of microsatellite markers revealed 6 different clusters according to the ploidy, the niche of origin of the isolates and geographical origin of the strains (Albertin et al., 2014;Avramova et al., 2018b). ...
Brettanomyces bruxellensis is the most damaging spoilage yeast in the wine industry because of its negative impact on the wine organoleptic qualities. The strain persistence in cellars over several years associated with recurrent wine contamination suggest specific properties to persist and survive in the environment through bioadhesion phenomena. In this work, the physico-chemical surface properties, morphology and ability to adhere to stainless steel were studied both on synthetic medium and on wine. More than 50 strains representative of the genetic diversity of the species were considered. Microscopy techniques made it possible to highlight a high morphological diversity of the cells with the presence of pseudohyphae forms for some genetic groups. Analysis of the physico-chemical properties of the cell surface reveals contrasting behaviors: most of the strains display a negative surface charge and hydrophilic behavior while the Beer 1 genetic group has a hydrophobic behavior. All strains showed bioadhesion abilities on stainless steel after only 3 h with differences in the concentration of bioadhered cells ranging from 2.2 × 102 cell/cm2 to 7.6 × 106 cell/cm2. Finally, our results show high variability of the bioadhesion properties, the first step in the biofilm formation, according to the genetic group with the most marked bioadhesion capacity for the beer group.
... Brettanomyces spp. were counted on modified EBB medium (Renouf and Lonvaud-Funel, 2007), herein named DK, containing ethanol 96 % (63 mL/L), glucose (5. g/L), (NH 4 ) 2 HPO 4 (2,5 g/L), (NH 4 ) 2 SO 4 (2.5 g/ L), yeast extract (2.0 g/L), malt extract (2.0 g/L), tween 80 (1.0 g/L), vinyl-phenol (0.1 g/L), p-coumaric acid (0.1 g/L), cysteine-HCl (0.5 g/ L), green bromocresol (22 mg/L), chloramphenicol (100 mg/L), cycloheximide (100 mg/L), and the vitamin complex previously described (pH 5.5). ...
In the last several years, the popularity of homebrewed beers has skyrocketed. However, this type of product is extremely vulnerable to microbial deterioration. Twelve homemade beers, some characterized by defects or stuck fermentation, were analysed by using a polyphasic approach encompassing culturomics and culture-independent techniques to better understand mechanisms that drive microbiota evolution throughout production and to highlight determinants responsible for crowning with success. Two sour beers, one apple-flavoured ale, two Italian grape ales, and seven standard ales were sampled.
Microbiological characterization was obtained by plating on nine different media coupled with High-throughput sequencing analysis of fungal and bacterial communities by targeting ITS1–2 and the V3–V4 regions of the 16S rRNA, respectively.
Total microflora on PCA largely varied among samples, ranging from <10² CFU/mL up to around 10⁷ CFU/mL often reflecting yeast counts on WL and LM. LAB population's levels on MRS and SDBm did not overlap, with the counts on the latter being even 5 Log CFU/mL greater.
Acetic Acid bacteria were retrieved in Sour beers, as well as in one IGA, even though acetic acid was not detectable by HPLC in this last sample. Brettanomyces spp. were only found in sour beers, as expected, whereas Enterobacteriaceae were never counted.
A total of 63 yeasts were randomly isolated from countable plates. Saccharomyces cerevisiae and Wickerhamomyces anomalus were the most frequently isolated species. In many cases, Interdelta analysis biotyping of S. cerevisiae isolates consistently allowed the detection of the starter strain.
By HST S. cerevisiae dominated the mycobiota in four samples, even if in one of them residual maltose and ethanol contents suggested a stuck fermentation. W. anomalus was found to be the dominant species in two beers.
Fifty-five LAB cultures were isolated and identified. Pediococcus damnosus was the only species retrieved in sour beers and two Ales, while Levilactobacillus brevis was found in two Ale samples. HTS did not confirm this result in one Ale sample since the genus Panotea spp. accounted for over 90 % of the microbiota. Enterobacteriaceae which were never counted dominated the microbiome of two Ale beers. Biogenic amines content largely varied with three Ale samples greatly contaminated.
Based on chemical and microbiological outcomes only one beer ASAle out of 12 could be considered acceptable. Furthermore, the widespread presence of LAB by culturomics and Enterobacteriaceae by HTS raises concerns about the final products' safety.
... Specific strains of B. bruxellensis are also used in the production of Orval, a Belgian trappist beer (1). Finally, this yeast species has been isolated from many other food fermentation processes, such as dairy products, sourdough, kombucha, cider, olives, wine, and water kefir, as well as from grapes (35,(51)(52)(53)(54)(55)(56)(57). However, in most of these fermentation processes, B. bruxellensis is seen as a spoilage microorganism, since it has an unwanted impact on the flavor profile of these products. ...
Lambic beers are beers produced through spontaneous fermentation and maturation in wooden barrels. The production process of lambic beers differs from the production processes of lagers and ales in process technology, environmental parameters, and the use of specific raw materials. Moreover, every lambic beer production process is unique in terms of microbiology and flavour formation because of its dependence on the spontaneous inoculation of microorganisms coming from the environmental air (contacting the open coolship and other brewery equipment) and the inner surfaces of the barrels. Several factors influence the inter- and intraspecies microbial successions during lambic beer wort fermentation and maturation and determine the final quality of the end-products. The possibility to manually acidify the wort, the presence of species-specific metabolic traits, the environmental temperature, the co-occurrence of lactic acid bacteria and acetic acid bacteria, as well as yeasts, and the quality of the wooden barrels all determine the progress and outcome of the lambic beer production process. Further alterations in quality and flavour of lambic beers can be achieved by blending practices and additional bottle re-fermentations. This results in a vast array of lambic-derived beer products (e.g., gueuze) with complex taste and aroma profiles and specific characteristics, which separates them from most other commercially available beers.
... B. bruxellensis (teleomorph Dekkera bruxellensis) is considered the most important spoilage microorganism in the wine industry (Loureiro & Malfeito-Ferreira, 2003). This yeast has been detected at low frequency in grapes or must (Renouf & Lonvaud-Funel, 2007) but is typically found during wine ageing in barrels and in bottles due to its tolerance for both high ethanol and low oxygen concentrations (Wedral et al., 2010). ...
Brettanomyces bruxellensis is the most reported spoilage yeast in aged wines mainly due to the production of phenolic off-flavors. In the present study, 64 B. bruxellensis strains isolated from Catalonian ageing wines were genetically and physiologically evaluated. The B. bruxellensis strains had high intraspecific diversity and were distributed genetically using the polymerase chain reaction of the intron splice site (ISS-PCR) into 8 clusters, mostly depending on their origin, and into 22 clones using microsatellite analysis. Wine-like conditions resulted in poor growth of several strains but, those growing, increased their tolerance to sulfur dioxide (SO2) with incubation time. However, tolerance to SO2 was not related to the genetic clusters as defined using ISS-PCR. Furthermore, some of the strains were resistant and grew in 60 mg/L of total SO2 (2.67 mg/L molecular SO2) in wine-like conditions. Additionally, the spoilage potential of the isolated strains was evaluated using precursors of 4-ethylphenol and 4-ethylguaiacol. All the strains were able to produce these compounds above their detection threshold even if the cells were losing culturability with incubation time. Thus, both the resistance to SO2 and the production of volatile phenols varied within the strains. This complexity must be taken into account to optimize both monitoring and corrective interventions, suggesting the importance of an early detection.
... 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.
... Saccharomyces cerevisiae, Debaryomyces hansenii, Wickerhamomyces anomalus (Pichia anomala), Pichia membranifaciens, Rhodotorula glutinis, Rhodotorula mucilaginosa, Torulaspora delbrueckii, Kluyveromyces marxianus, Issatchenkia orientalis, Zygosaccharomyces bailii parapsilosis, Pichia fermentans and Hanseniaspora uvarum are frequent contaminants of wines as well [27,28]. However, Renous [29] did not describe associations between wine and Pichia manshurica, Kregervanrija fluxuum (Candida vini), Candida inconspicua and Zygotorulaspora florentina. Saez [30] found that S. cerevisiae (13.93%), ...
The aim of the present study was to identify yeasts in grape, new wine “federweisser” and unfiltered wine samples. A total amount of 30 grapes, 30 new wine samples and 30 wine samples (15 white and 15 red) were collected from August until September, 2018, from a local Slovak winemaker, including Green Veltliner (3), Mūller Thurgau (3), Palava (3), Rhein Riesling (3), Sauvignon Blanc (3), Alibernet (3), André (3), Blue Frankish (3), Cabernet Sauvignon (3), and Dornfelder (3) grapes; federweisser and unfiltered wine samples were also used in our study. Wort agar (WA), yeast extract peptone dextrose agar (YPDA), malt extract agar (MEA) and Sabouraud dextrose agar (SDA) were used for microbiological testing of yeasts. MALDI-TOF Mass Spectrometry (Microflex LT/SH) (Bruker Daltonics, Germany) was used for the identification of yeasts. A total of 1668 isolates were identified with mass spectrometry. The most isolated species from the grapes was Hanseniaspora uvarum, and from federweisser and the wine—Saccharomyces cerevisiae.
... In the wine industry, most of these genetic analyses revealed high diversity within the species, at the vineyard, in the winery or at sample levels [8,23,[27][28][29]. However, in most cases, only a small subset of isolates (a few dozens) were included, and these markers, although discriminant, were not appropriate for population genetic studies. ...
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.
... B. bruxellensis yeasts usually do not require nutrition-rich environments for their growth. It has been reported that this genus of yeast is the only microorganism that can survive in wine after bottling, due to its ability to resist in the anaerobic conditions (Renouf and Lonvaud-Funel, 2007). The hydroxycinnamate decarboxylase and vinylphenol reductase activities of these yeasts enable them to produce 4-ethylphenol and 4-ethylguaiacol from p-coumaric acid ( Kheir et al., 2013). ...
... During storage wines are subjected to several operations directed to the improvement of storage and aging conditions (Renouf and Lonvaud-Funel, 2004). Clarification by settling or centrifugation leads to the reduction of suspended material including microorganisms. ...
Wine is obtained by fermentation of grape juice where the yeast species Saccharomyces cerevisiae plays the major role. The process is subjected to hazards of microbial origin from grape harvesting to wine bottling. Contamination yeasts have different spoiling potential and incidence. The most dangerous and frequent species include Dekkera bruxellensis, Zygosaccharomyces bailii and S. cerevisiae, which are able to grow in wine produced according to Good Manufacturing Practices. The former species is responsible for the “horse sweat” taint due to production of volatile phenols in bulk or bottled red wine. The other two species may proliferate in bottled wines producing visible sediments or turbidity. Other species producing off-flavours or films in bulk wine (e.g. Kloeckera apiculata, Pichia spp., Candida spp.) may be easily prevented by proper technological measures.
... Dekkera/Brettanomyces can grow during the wine aging and even after their bottling; on the contrary, these yeasts are rarely found during the alcoholic fermentation of grape must. A few studies have reported their presence on grapes due to the difficult cultivation while in winery, in particular in vats, pumps or equipments difficult to sanitizes, Brettanomyces yeasts are more easily found (Fugelsang and Zoecklein 2003;Pretorius 2000;Renouf and Lonvaud-Funel 2007). ...
The ecological distribution of yeasts through the various stages of winemaking process have an important role on the characteristics of wines influencing both the analytical composition and the sensory profile of product. The yeast community of grapes, affected by several biotic and abiotic factors, is directly involved in grape juice fermentation taking part more or less actively in both spontaneous or inoculated fermentations. The yeast community of winery also take part in winemaking process influencing positively or negatively the final wine composition. Selected starter cultures of Saccharomycs cerevisiae are usually added to control the winemaking process and to achieve specific desired enological traits. The aim is that to dominate indigenous yeasts belonging to the vineyard environment, winery facilities and cellar equipment obtaining wines tailored to consumer requests. More recently, the revaluation of the role of non-Saccharomyces yeasts allowed to enologists to obtain some peculiar characters and more complex aroma to the wines. In this regard, the use of controlled mixed fermentation with selected non-Saccharomyces and S. cerevisiae wine yeasts represents a novelty in oenological field.
... and grapes and ending with the winery equipment, barrels, and bottling rooms (Oelofse, Pretorius, & Toit, 2008). Brettanomyces is rarely detected on grapes, and, to our knowledge, this spoilage yeast has only been isolated in a sole study (Renouf & Lonvaud-Funel, 2007) after an enrichment step. This result could explain the dominance of the highly abundant species over the minority species, such as B. bruxellensis, which may remain undetected on grapes. ...
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.
... During storage wines are subjected to several operations directed to the improvement of storage and aging conditions (Renouf and Lonvaud-Funel, 2004). Clarification by settling or centrifugation leads to the reduction of suspended material including microorganisms. ...
... These yeasts are distributed worldwide, have been isolated from vineyards, and are found in grape musts and bottled wines with or without residual sugars (Renouf et al. 2008). B. bruxellensis has been isolated from several vineyards at different stages of grape berry development, using mainly enrichment media, so grape berries are a possible vector for introduction of this yeast species into wine (Agnolucci et al. 2007, Barbin et al. 2007, Renouf andLonvaud-Funel 2007). ...
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.
... In contrast, there are a few studies which describe the presence of this strain in grape products. Renouf et al., 2005, Renouf andLonvaud-Funel, 2007) reported that the Pediococcus genera, particularly Pediococcus parvulus, P. damnosus, and P. acidilactici, was isolated from grape. Crowley et al. (2013) reported that P. pentosaceous was shown to have protective properties against P. expansum spoilage when applied in pear, plum, and grape models. ...
... In contrast, there are a few studies which describe the presence of this strain in grape products. Renouf et al., 2005, Renouf andLonvaud-Funel, 2007) reported that the Pediococcus genera, particularly Pediococcus parvulus, P. damnosus, and P. acidilactici, was isolated from grape. Crowley et al. (2013) reported that P. pentosaceous was shown to have protective properties against P. expansum spoilage when applied in pear, plum, and grape models. ...
The aim of this study was to isolate lactic acid bacteria from two varieties of grape bunch cultivated in Tunisia (cardinal and red-globe) in order to assess their ability to inhibit the growth of their most widespread contaminant, Aspergillus niger aggrégats and Aspergillus carbonarius. Antifungal activity of 18 isolates was investigated using overlay technique; selected isolates were than identified using 16s rDNA sequence analysis. Isolates with antifungal activities were screened out and studied for their probiotic properties using in vitro tests (tolerance to simulated gastric jus, bile salts, hydrophobicity properties).The most efficient strain was also investigated for its ability to reduce the concentration of ochratoxin A (OTA) on liquid medium. Determination of OTA content in media was released using HPLC analysis. Selected strains (RG7B) (C11C) and (RG8A) showing a good antifungal activities against Aspergillus niger aggrégats and Aspergillus carbonarius were identified as Pediococcus pentosaceus and Lactobacillus plantarum, respectively. Pediococcus pentosaceus (RG7B) showed promising potential probiotic characteristics and had a high ability for OTA removal after 48 h of incubation in both MRS and PBS media. The OTA removal percentage was significantly higher in MRS than in PBS media (84 and 25%, respectively). This study provides evidence for the control of black Aspergillus growth in grape and OTA detoxifying by the use of autochthones LAB strains having antifungal effect and probiotic potential.
... Final pH, 7.4 ± 0.2 (25 °C). All products were purchased from Biokar Diagnostics [11][12][13][14][15]. ...
... these stressful conditions, some opportunistic microorganisms manage to survive and multiply during and after alcoholic fermentation. A striking example is the wine spoilage yeast Brettanomyces bruxellensis (teleomorph Dekkera bruxellensis) that is typically detected during wine aging but also at lower frequency during the early stages of the winemaking process (grapes and must) 1,2 . When it grows in wine, B. bruxellensis produces odorant molecules (namely volatile phenols), which are associated with unpleasant aromas described as barnyard, horse sweat, Band-aid ® [3][4][5] . ...
Brettanomyces bruxellensis is a unicellular fungus of increasing industrial and scientific interest over the past 15 years. Previous studies revealed high genotypic diversity amongst B. bruxellensis strains as well as strain-dependent phenotypic characteristics. Genomic assemblies revealed that some strains harbour triploid genomes and based upon prior genotyping it was inferred that a triploid population was widely dispersed across Australian wine regions. We performed an intraspecific diversity genotypic survey of 1488 B. bruxellensis isolates from 29 countries, 5 continents and 9 different fermentation niches. Using microsatellite analysis in combination with different statistical approaches, we demonstrate that the studied population is structured according to ploidy level, substrate of isolation and geographical origin of the strains, underlying the relative importance of each factor. We found that geographical origin has a different contribution to the population structure according to the substrate of origin, suggesting an anthropic influence on the spatial biodiversity of this microorganism of industrial interest. The observed clustering was correlated to variable stress response, as strains from different groups displayed variation in tolerance to the wine preservative sulfur dioxide (SO2). The potential contribution of the triploid state for adaptation to industrial fermentations and dissemination of the species B. bruxellensis is discussed.
... This phenomenon probably takes place because hydroxycinnamic acids are detrimental for yeasts, and their transformation is necessary to detoxify the extracellular environment (Oelofse et al., 2008;Suárez, Suárez-Lepe, Morata, & Calderón, 2007). In red wines, accumulation of volatile phenols over the sensorial threshold (about 400 μg/L) causes the organolepetic fault known as "Brett character", with the consequential loss of varietal flavours and an increase in unpleasant odours with descriptors such as "animal, ink, chemical, and unnaturally spicy" (Curtin, Varela, & Borneman, 2016;Malfeito-Ferreira, 2011;Renouf & Lonvaud-Funel, 2007). Biogenic amines represent additional undesirable bio-products resulting from of winemaking, insufficient SO 2 concentrations and inefficient filtration before bottling (Curtin, Kennedy, & Henschke, & P. A., 2012;Oelofse et al., 2008). ...
... Selective media have been developed for the isolation of B. bruxellensis, such as the Differential Brettanomyces Dekkera Medium, DBDM, (Couto et al. 2005a). Moreover, the formulation of novel enrichment media, allowed its isolation from grape surfaces (Renouf and Lonvaud-Funel 2007). ...
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.
... 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.
... Despite that it is mainly known for its spoilage capacity in wine, it has been isolated from various man-made ecological niches, including (spontaneous) alcoholic fermentation processes (wine, beer, cider, bioethanol, etc.), soft drinks, dairy products, kombucha tea and sourdough (Crauwels et al. 2015a;Steensels et al. 2015;Smith and Divol 2016). The only source from which B. bruxellensis has been isolated that is not associated with industrial settings is grape berries (Renouf and Lonvaud-Funel 2007), illustrating its close association with man-made ecological niches. A common thread in these niches is the harsh environmental conditions that are detrimental for many microbes, such as high ethanol concentrations, low pH, the absence of readily fermentable nitrogen and carbon sources, and low oxygen (Smith and Divol 2016). ...
Brettanomyces (Dekkera) bruxellensis is an ascomycetous yeast of major importance in the food, beverage and biofuel industry. It has been isolated from various man-made ecological niches that are typically characterized by harsh environmental conditions such as wine, beer, soft drink etc. Recent comparative genomics studies revealed an immense intraspecific diversity, but it is still unclear whether this genetic diversity also leads to systematic differences in fermentation performance and (off-)flavor production, and to what extent strains have evolved to match their ecological niche. Here, we present an evaluation of the fermentation properties of eight genetically diverse B. bruxellensis strains originating from beer, wine and soft drinks. We show that sugar consumption and aroma production during fermentation are determined by both the yeast strain and composition of the medium. Furthermore, our results indicate a strong niche adaptation of B. bruxellensis, most clearly for wine strains. For example, only strains originally isolated from wine were able to thrive well and produce the typical Brettanomyces-related phenolic off-flavors 4-ethylguaiacol and 4-ethylphenol when inoculated in red wine. Sulfite tolerance was found as a key factor explaining the observed differences in fermentation performance and off-flavor production. Sequence analysis of genes related to phenolic off-flavor production, however, revealed only marginal differences between the isolates tested, especially at the amino acid level. Altogether, our study provides novel insights in the Brettanomyces metabolism of flavor production, and is highly relevant for both the wine and beer industry.
... The spoilage yeast Brettanomyces bruxellensis and its teleomorph, Dekkera bruxellensis, currently represent a major issue for winemakers around the world [1]. These yeasts are naturally present on the skin of grapes [2], but at low levels that would not be sufficient to cause organoleptic defects [3]. They can be easily transferred into the winemaking process and have detrimental effects on the wine quality by altering its odour, flavour and colour [4,5]. ...
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.
... Nonetheless, with increasing demands for innovative products, other non-Saccharomyces species have been isolated and characterized for the development of specialty beers, with distinctive aromatic and flavor components (Vanderhaegen et al., 2003). They generally present low fermentation yields, and are more sensitive to ethanol stress (Di Maro, Ercolini, & Coppola, 2007), but may display a great range of possibilities for beer fermentations in order to provide distinctive aroma and flavor (Renouf & Lonvaud-Funel, 2007). It has been shown, for example, that Dekkera bruxellensis and Hanseniaspora uvarum (anamorph, Kloeckera apiculata) have the ability to produce many esters with fruity character, besides promoting the enzymatic breakdown of some proteins (De Keersmaecker, 1996;Kumara & Verachtert, 1991). ...
Brettanomyces bruxellensis is considered the main source of spoilage in red wine. This yeast, by producing volatile phenols, is responsible for the development of unpleasant aromas affecting the quality of final products and resulting in substantial economic losses for wine producers. This work therefore describes the development of an easy to-use colorimetric molecular diagnostic test for the rapid and specific detection of B. bruxellensis in wine. Detection was achieved using a sandwich hybridization format in which the target RNA was recognized by an immobilized DNA capture probe and a labelled DNA signal probe. The proposed device was highly specific to B. bruxellensis and showed a linear relationship between measured signal and target RNA concentration in the range 0.1-5 ng μL-1, with a limit of detection value of 0.1 ng μL-1 of total RNA. The colorimetric assay was validated on red wine samples, with a detection limit of 102 CFU mL-1. This study suggests that the reported method could be used for early detection of spoilage yeasts in wine and other alcoholic beverages.
This is the highly anticipated third edition of a book written by the Working Party on Culture Media of the International Committee on Food Microbiology and Hygiene. It is a handy reference for microbiologists wanting to know which media to use for the detection of various groups of microbes in foods and how to check the performance of the media. The book is divided into two parts and concentrates on media for water as well as food microbes - selecting those which have been evaluated and shown to function optimally. The first part consists of a series of chapters written by various experts from all over the world, reviewing the media designed to detect the major groups of microbes important in food spoilage, food fermentations and food-borne disease. The history and rationale of the selective agents and indicator systems used, as well as the relative merits of the various media are surveyed by reference to the scientific literature. The second part contains monographs on almost 100 of the media considered most useful. Each monograph, written in the style of a pharmacopoeia, includes: a short section on the history and selective principle of the medium; a method for its preparation from basic ingredients; its appearance and physical properties, including pH; its shelf-life; instructions concerning method of inoculation, incubation and interpretation; the recommended method(s) and a list of test strains suitable for assessing the quality (productivity and selectivity) of the medium and a description of the typical appearance of the target organism.
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.
Human-associated microorganisms are ideal models to study the impact of environmental changes on species evolution and adaptation because of their small genome, short generation time, and their colonization of contrasting and ever-changing ecological niches. The yeast Brettanomyces bruxellensis is a good example of organism facing anthropogenic-driven selective pressures. It is associated with fermentation processes in which it can be considered either as a spoiler (e.g. winemaking, bioethanol production) or as a beneficial microorganism (e.g. production of specific beers, kombucha). Besides its industrial interests, noteworthy parallels and dichotomies with Saccharomyces cerevisiae propelled B. bruxellensis as a valuable complementary yeast model. In this review, we emphasize that the broad genetic and phenotypic diversity of this species is only beginning to be uncovered. Population genomic studies have revealed the co-existence of auto- and allotriploidization events with different evolutionary outcomes. The different diploid, autotriploid and allotriploid subpopulations are associated with specific fermented processes, suggesting independent adaptation events to anthropized environments. Phenotypically, B. bruxellensis is renowned for its ability to metabolize a wide variety of carbon and nitrogen sources, which may explain its ability to colonize already fermented environments showing low-nutrient contents. Several traits of interest could be related to adaptation to human activities (e.g. nitrate metabolization in bioethanol production, resistance to sulphite treatments in winemaking). However, phenotypic traits are insufficiently studied in view of the great genomic diversity of the species. Future work will have to take into account strains of varied substrates, geographical origins as well as displaying different ploidy levels to improve our understanding of an anthropized yeast's phenotypic landscape.
Microbial spoilage of beer and related products results in high economic loss. Undesired microbes can impact the quality of the end product at any stage of the production process. Brettanomyces and Saccharomyces wild strains, including B. bruxellensis and S. cerevisae diastaticus (S. diastaticus), are commonly isolated as spoilage yeast. Knowledge of the taxonomy, ecology, and mechanisms of resistance against antimicrobial activity of beer (products) and preservation methods is now emerging, which can be used to develop spoilage prevention strategies.
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.
During the winemaking process the quality of a wine can quickly be affected by microbial spoilage due to a number of yeast and bacterial species present in wine. In order to prevent the microbial spoilage of wine, it is essential to be able to understand where it is most likely to occur, recognize it when it occurs, and which wine microorganisms are involved. To do this, traditional microbial detection methods such as plating and microscopy are beginning to be combined with powerful new detection methods based upon molecular biology techniques. The control of microbial growth in wine is still largely achieved through the use of SO2. However, new options such as the use of lysozyme, dimethyldicarbonate, bacteriocins, and chitosan may allow a reduction in the amount of SO2 required. The design and use of a quality control program such as Hazard Analysis and Critical Control Point (HACCP) can be effective in integrating all the information regarding wine microbial spoilage that is now available to a modern winemaker. This systematic approach allows the identification of key spoilage problems, corrective actions that will be undertaken if a problem is found, verification of the impact of the corrective action, and documentation of the entire process.
Brettanomyces is a genus of ‘rogue’ yeasts that may grow in red wines and, very occasionally, white and sparkling wines. By far the most prevalent species is Brettanomyces bruxellensis. The compounds metabolised by the yeast give off‐odours including those of antiseptic, sweat, stables, and vomit. These compounds include 4‐ethyphenol, 4‐ethylguaiacol, and isovaleric acid. The yeast may grow in wine during production, maturation, and/or storage, and off‐odours may emerge years after the wine has been bottled. Growth often occurs during the barrel maturation of wines. Prevention may be effected by the formulation of a control strategy and implementation of appropriate winemaking procedures. Treatment of affected wine prior to bottling is a two‐stage process, involving removal of viable yeast cells, and reduction in the level of volatile phenols.
The study aimed to identify the filamentous fungi and yeast mycobiota found on the surface and in grapes, grape must, and wine obtained from four red grape varieties: Alibernet, Dornfelder, Blue Frankish, Cabernet Sauvignon, and four white grape varieties: Green Veltliner, Rheinriesling, Pinot Blanc, Sauvignon. Grapes from vineyard Vrbové located in southwestern Slovakia were used for the research in 2020. The identification of filamentous fungi was performed using the macroscopic and microscopic observations and yeasts were identified by MALDI-TOF Mass Spectrometer. A total of 642 isolates were obtained. Grapes were rich in diversity of filamentous fungi (13 genera) and must on yeasts (8 genera). Penicillium, Botrytis, and Hanseniaspora uvarum were identified in both grapes and must. Three of the fungal genera identified by conventional or molecular techniques from the surface of red grape varieties were predominant: Alternaria (26%), Botrytis (21%), and Issatchenkia terricola (13%), two from endogenous mycobiota: Hanseniaspora uvarum (45%) and Botrytis (17%), four from the surface of white grape varieties: Penicillium (25%), Botrytis (21%), Alternaria (16%) and Hanseniaspora uvarum (15%), and three from endogenous mycobiota: Botrytis (44%), Hanseniaspora uvarum (23%) and Alternaria (20%). Saccharomyces cerevisiae, Candida krusei, C. utilis, and Cryptococcus neoformans were identified only in wine.
Depuis 2006, mes travaux ont été centrés sur l’étude de la diversité génétique et phénotypique des levures du raisin et du vin, qu’il s’agisse d’espèces bénéfiques utilisées comme auxiliaires technologiques (Saccharomyces sp., non-Saccharomyces d’intérêt oenologique) ou qu’il s’agisse d’espèces d’altération (Brettanomyces bruxellensis). Nous avons développé des approches multi-échelles, combinant des outils classiques (microbiologie traditionnelle, génétique des populations), des approches spécifiques de l’oenologie (suivi des cinétiques fermentaires, analyse sensorielle), et des analyses –omic à haut-débit couplées à des outils bio-informatiques. Ces différents projets ont été financés sur fonds publiques (ANR, région, Bordeaux INP, etc.) ou privés (industriels de l’oenologie, interprofession, etc.), et ont nécessité l’engagement de nombreux collègues, collaborateurs et étudiants – nationaux ou internationaux. Sur le plan fondamental, nos travaux ont permis d’améliorer nos connaissances des levures d’oenologie à travers la publication de >40 articles scientifiques dans des revues avec comité de lecture. Quelques applications finalisées ont également vu le jour (sélection de souches de levure pour l’oenologie, développement de marqueurs moléculaires, etc.). Nous avons également contribué au transfert de connaissance vers la profession sous la forme d’articles ou de communications techniques.
Les projets que j’aimerai développer à l’avenir se déclinent en trois grands thèmes :
1- mieux comprendre l’adaptation des espèces et sous-populations de levure à des environnements variés, anthropisés ou non ;
2- améliorer nos connaissances des mécanismes d’interactions entre espèces microbiennes, notamment dans l’environnement vitivinicole ;
3- approfondir l’étude de certains phénotypes d’intérêt oenologique.
Comme pour nos travaux passés, les projets futurs comporteront lorsque cela est possible un volet fondamental et appliqué afin de mieux maitriser la qualité et l’identité des vins d’aujourd’hui et demain.
L’étude des communautés microbiennes de la baie de raisin dans des conditions de production à l’échelle de la parcelle montre une dynamique temporelle des populations cultivables, qui se traduit par une augmentation des niveaux de population à partir des stades de début véraison et début maturité. Concernant la communauté bactérienne cultivable, 44 espèces appartenant à 21 genres ont été identifiées. Parmi les huit genres identifiés pour la population fongique, les espèces appartenant au genre Aureobasidium sont les plus abondantes, contrairement aux espèces fermentaires qui restent minoritaires. L’incidence des facteurs biotiques et abiotiques sur différents paramètres de population microbienne tels que la structure, la densité et l’activité métabolique a été analysée. Nous avons observé que les zones climatiques plus fraîches et humides, favorisent le développement des microorganismes. Ces travaux mettent en évidence l’impact écotoxique du cuivre sur la communauté microbienne, en particulier dans sa fraction bactérienne. Le développement de Botrytis cinerea sur la grappe modifie la communauté microbienne des baies de raisin sain : le nombre d’espèces bactériennes augmente ainsi que leur diversité. La communauté bactérienne de la baie de raisin est proche de celle des feuilles d’un point de vue de sa structure, et mais éloignée de celles des écorces et du sol, avec des indices de diversité et de richesse plus faibles.
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.
The assembly of a novel disposable amperometric immunosensor for the detection of the red wine spoilage yeast Brettanomyces bruxellensis is reported. The nanostructured sensing interface was prepared by first coating carbon screen printed electrodes with a gold nanoparticles-reduced graphene oxide hybrid nanomaterial, which was then modified with 3-mercaptopropionic acid to further immobilize specific antibodies for B. bruxellensis via a carbodiimide-coupling reaction. The functionalized electrode allowed the amperometric detection of B. bruxellensis in buffered solutions and red wine samples in the range of 10–10⁶ CFU/mL and 10²–10⁶ CFU/mL, with low detection limits of 8 CFU/mL and 56 CFU/mL, respectively. The electrochemical immunosensor also exhibited high reproducibility, selectivity, and storage stability.
Les microorganismes rencontrés dans les moûts et les vins : levures, bactéries lactiques et acétiques, sont représentés par un nombre limité d'espèces. Les auteurs proposent une technique rapide de dénombrement sélectif des trois populations en mélange et l'application de l'Api-system pour identifier les levures et les bactéries lactiques du vin.
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The number of microorganisms species occuring in musts and wines (yeasts, lactic and acetic bacteria) is limited. It is presented a quick, selective technique to count these miscellaneous microorganisms and application of Api-system for identification of yeasts and lactic bacteria of wine.
CITATION: Pretorius, I. S. 2000. Geographical distribution of indigenous saccharomyces cerevisiae strains isolated from vineyards in the coastal regions of the Western Cape in South Africa. South African Journal of Enology & Viticulture, 21(1):3-9, doi:10.21548/21-1-2179.
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.
Brettanomyces/Dekkera yeasts grow in wine mainly during barrel aging. Their presence is often associated with formation of off-flavors. This potential spoilage generates a strong demand for a sensitive, rapid, and reliable identification procedure. Ribosomal DNA restriction fragment length polymorphism and comparative sequence analysis of the two internal transcribed spacer (ITS) regions located between the ribosomal RNA genes was carried out using Brettanomyces/Dekkera yeast reference strains and wine isolates. ITS1 and ITS2 were found to contain distinct regions with sufficient sequence divergence to make them suitable as specific identification target sites. Specific oligonucleotides were designed for each Brettanomyces/Dekkera species and evaluated for specificity and reliability. No cross-reaction products were detected when the specific primers were assayed in a PCR reaction with Brettanomyces/Dekkera strains of different species or other wine-related non-Brettanomyces/Dekkera yeasts. Thus, PCR using a combination of all four specific primers gave a specific and reproducible detection assay for the genus Brettanomyces/Dekkera. Use of these specific primers allowed for species-specific discrimination. Brettanomyces/Dekkera yeast isolates from wine were shown to uniquely belong to the species B. bruxellensis.
A new filt e r-based chemiluminescent in s itu hybridiza tion method us ing peptide nucle ic a cid probes for rapid and si mult a neous d e tectio n , identifi cati o n, a nd enumeration of Brettanomyces from a ir s a mples was develope d. Th e me thod a ls o a llows fo r d e tection of the yeas t prior to vi s ual a ppearance of large colonies. Us ing thi s tec hnique, a ir samples fr o m various lo cations within a winery (c rush, ta nk , barrel , and bottling rooms) were a na ly zed. Brettanomyces was id e ntified in samples ta ken from all fou r rooms, confirming that thi s s poil age yeast c a n be s pread through a winery by air currents.
Microbial species present on the surface of grape berries at harvest play an important role in winemaking, thus counting and identifying them is of great importance. The use of conventional microbial techniques and molecular methods allowed a quantitative and qualitative inventory of the different microbial species present on the grape berries. These experiments were carried out in several areas of the Bordeaux region on the red grape varieties Merlot, Cabernet Sauvignon and Cabernet Franc. Populations and species clearly varied according to berry development stage. The most widespread yeast species at berry set, Aureobasidium pullulans was never detected at harvest. Fermentative yeasts were detected at harvest and not in the first stage of grape growth. Oenoccocus oeni was detected on immature as well as on mature berries. Gluconobacter oxydans was detected mainly at harvest. Detection of Pediococcus parvulus, was dependent on the vineyard. Veraison appeared to be a key stage for yeast colonisation and the increase in population involved a change in the proportion of each species. The number of A. pullulans fell significantly at veraison as it was superseded by fermentative yeasts. Microbial populations peaked at harvest when the berry surface available for adhesion was largest and no agrochemical treatments had been applied for some weeks. Soil, grape variety and grapegrowing practices may also influence this microbial ecosystem. Based on these and published data, we formulated hypotheses to describe this microbial ecosystem, thus enabling us to develop the concept of a microbial biofilm.
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.
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.
The identification and classification of yeasts have traditionally been based on morphological, physiological and biochemical traits. Various kits have been developed as rapid systems for yeast identification, but mostly for clinical diagnosis. In recent years, different molecular biology techniques have been developed for yeast identification, but there is no available database to identify a large number of species. In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts. In many cases, the size of the PCR products and the restriction patterns obtained with endonucleases CfoI, HaeIII and HinfI yielded a unique profile for each species. Accordingly, the use of this molecular approach is proposed as a new rapid and easy method of routine yeast identification.
The nutritional requirements of Brettanomyces bruxellensis have been investigated. Batch culture and chemostat pulse techniques were used to identify growth-limiting nutrients. The study included determination of the essential components of the culture medium and quantification of the effects of the components. Among the components tested, ammonium sulfate and yeast extract had a significant effect on glucose consumption, growth, and ethanol production. However, if the ammonium sulfate concentration is above 2 g/L, an inhibitory effect on B. bruxellensis growth is observed. The yeast extract appears to be the most important and significant component for growth. The maximum amount of synthesized biomass is proportional to the concentration of yeast extract added to the culture broth (in the tested range). Magnesium and phosphate ions are probably not essential for B. bruxellensis. These ions appear to be supplied in sufficient amounts by the yeast extract in the culture medium. Brettanomyces bruxellensis appears to have very low nutritional requirements for growth.
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.
Recent ecological surveys indicate that the wine yeast Saccharomyces cerevisiae may be isolated with extreme difficulty from natural substrates, such as vineyard soil or the surface of mature grapes, conventionally believed to be its elected habitat. Conversely, it is amply demonstrated that its preferential location as the only fermenting colonizer is the surfaces of the wineries. Non-conventional methods for the separation, isolation, and enumeration of yeast cells from mature grapes of different varieties produced additional and unequivocal evidence on the numerical inconsistency of S. cerevisiae cells in nature. The taking over of musts by the natural yeast flora was also followed in microfermentations.
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.
Laboratory and pilot scale fermentations have been carried out using worts spiked with a range of carbonyl compounds, many of which are derived from malt. Aldehydes and vinyl ketones were chemically reduced during fermentation and could not be detected in the resulting beers; on the other hand certain saturated and non-conjugated unsaturated ketones were only partially reduced. The corresponding saturated alcohols and acetate esters were usually detected as transformation products.
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.
Traditional oak wood storage vats used to ferment and mature commercial fermented alcoholic cider are being phased-out and replaced by stainless steel vessels. Each wood vat, however, despite hot-wash cleaning between cider batches, is shown to retain an extensive and distinctive microflora of yeast and bacteria. These micro-organisms could act as a reservoir of both beneficial and spoilage organisms. Over a period of 10 weeks, the progressive microbial penetration and colonization of blocks of vat wood suspended in maturing commercial cider is demonstrated by use of scanning electron microscopy. Within 2 weeks cell penetration was to a depth of 1.2 cm. Lactic acid bacteria originating from wood are also shown to be capable of sponsoring malo-lactic fermentation of freshly fermented cider and hence positively contributing to changes in the organoleptic profile of a stored beverage.
The yeast content in spoiled decorated soft cheese packed in modified atmosphere from two different dairies was examined. Spoilage was recognized as swelling of packets or as visible growth on the surface of the cheese. In total, 18 yeast species were isolated from the cheese.Torulaspora delbrueckii,Candida parapsilosis,Pichia fermentans,Pichia norvegensis,Pichia membranaefacienswere most commonly isolated in spoiled cheese from Dairy A.Yarrowia lipolytica,Debaryomyces hansenii,Pichia guilliermondii,Cryptococcussp. andRhodotorulasp. were most common on cheese from Dairy B. Yeast occurrence on cheese from Dairy A was most likely caused by recontamination from production and packing area. Yeast occurrence on cheese from Dairy B was associated with the yeasts found on the material used for decoration.
Historically, the fermentation of grape juice to wine has been carried out by indigenous yeasts found on the berry. However, in newer wine regions, e.g. the USA, inoculation with selected wine yeast strains is employed. Grape juice is high in nutritional factors and difficulties in fermentation usually arise from the inhibitory effects of the high concentration of sugar initially present and the ethanol produced. A secondary fermentation, brought about by indigenous or added lactic acid bacteria, converts malic acid to lactic acid and carbon dioxide and often occurs. This ‘malolactic’ fermentation is usually slow. For both yeast and bacterial fermentations strain selection is based more on fermentation performance than on sensory characteristics of the wine, with increased tolerance of the yeast to ethanol and of the bacteria to low pH being emphasized. Attempts to increase the malolactic fermentation rate have been made by cloning and transferring the malolactic gene from Lactobacillus to wine yeast. In early attempts to produce wines with enhanced or novel sensory characteristics a leucine-less mutant of a homothallic wine yeast has been obtained which does not produce isoamyl alcohol.
Foods and drinks with high solute concentration (fruit juices and concentrates, marzipan, salted and dry-cured meats, olives and cheeses), and foods containing preservatives (wine, beverages and sauces) were selected in order to characterise the contaminant yeast flora by rapid typing techniques. These included the testing of several types of molecular methods (e.g. RFLP, DNA-fingerprinting, PCR-based techniques), analysis of long-chain fatty acids and of isoenzymes. The PCR-based detection methods enabled a faster detection of emerging specific spoilers at earlier stages of processing. Fatty acid characterisation allowed the assessment of the most frequent types of contamination yeasts and supplied the information for the definition of relevant zymological indicators. A selected group of strains was used for further studies of mechanisms underlying the resistance/tolerance of yeasts towards preservatives (weak acids) and other stress factors (temperature, high sugar and salt concentrations). This study enabled the acquisition of data on the basic biology of yeasts used in the development of differential and selective media for Zygosaccharomyces bailii, Yarrowia lipolytica, Kluyveromyces marxianus and Dekkera sp.
Recent ecological evidence points to a circulation model for Saccharomyces cerevisiae in nature which is different from that proposed at the end of the last century. The wine yeast ‘par excellence’ is isolated with extreme difficulty from conventional habitats, such as vineyard soil or the surface of ripe grapes, while it is almost the only species colonising the surfaces of the winery equipment.
Minimum inhibitory concentrations (MIC) of various cleaner or sanitizer compounds were determined by a Spiral Gradient Endpoint agar diffusion method against four micro-organisms associated with spoilage of orange juice (Lactobacillus plantarum, Leuconostoc mesenteroides, Gluconobacter oxydans, and Saccharomyces cerevisiae) and against eight unidentified micro-organisms isolated from citrus fruit surfaces (two yeasts and six bacteria). Survivor curve testing was conducted on the four known micro-organisms. Compounds that were effective at the lowest concentrations were chlorine dioxide, the iodophor and quaternary ammonium compound. Dimethyldicarbonate, hypochlorite, peracetic acid and a phosphoric acid anionic compound also possessed substantial antimicrobial activity. Citric and lactic acids and d-limonene were less effective as antimicrobial compounds.
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 alcoholic fermentation of Botrytis-affected wines is stopped by the addition of high concentrations of sulfur dioxide (SO2). The natural microbial unstability of these wines and the binding phenomena forces winemakers to periodically add sulfur dioxide during maturation, leading to a high concentration of a maximum of 400 mg/l in the bottled wine. Dimethyldicarbonate (DMDC) is now considered as a reliable fungicide which could be partially used instead of SO2, especially just before bottling. This study investigated the use of DMDC to stop alcoholic fermentation. The experiment was carried out on pure cultures of three yeast species present in this type of wine (Saccharomyces cerevisiae, Candida stellata and Zygosaccharomyces bailii). The results were very promising and suggested that DMDC was more effective than SO2. The yeast cells died after the addition of DMDC whereas they partially entered into a viable but non-culturable (VBNC) state with SO2. However, the same experiment carried out on botrytized must, whose fermentation was carried out using indigenous microflora, was less conclusive. It pointed out that DMDC, used in a concentration of 200 mg/l, was more effective than SO2 but leading to the same results: the entering of a part of the cells into a VBNC state. DMDC could be used to stop alcoholic fermentation, but could not replace SO2. Nevertheless, the concentrations of SO2 added in this type of wine could be reduced in this way.
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.
The ability to produce 4-ethylphenol from the substrate p-coumaric acid in synthetic media was evaluated for several yeast species associated with wine production. Molar conversion rates as high as 90% were found by only Dekkera bruxellensis, D. anomala and by some unidentified strains isolated from wine-related environments. Other unidentified strains produced traces of 4-ethylphenol. All unidentified strains showed the same cultural characteristics as D. bruxellensis when grown on DBDM (Dekkera/Brettanomyces differential medium) agar. The determination of long-chain fatty acid compositions and the utilization of peptide nucleic acid (PNA) probes specific for D. bruxellensis showed that the unidentified strains did not belong to this species. Further identification, by restriction pattern generated from PCR-amplification of the 5.8S rRNA gene and the two internal transcribed spacers (ITS), assigned the unidentified strains to Candida cantarelli, C. wickerhamii, Debaryomyces hansenii, Kluyveromyces lactis and Pichia guilliermondii. However, only some strains of P. guilliermondii were capable of converting p-coumaric acid into 4-ethylphenol with efficiencies close to those observed in D. bruxellensis and D. anomala.
Foods and drinks with high solute concentration (fruit juices and concentrates, marzipan, salted and dry-cured meats, olives and cheeses), and foods containing preservatives (wine, beverages and sauces) were selected in order to characterise the contaminant yeast flora by rapid typing techniques. These included the testing of several types of molecular methods (e.g. RFLP, DNA-fingerprinting, PCR-based techniques), analysis of long-chain fatty acids and of isoenzymes. The PCR-based detection methods enabled a faster detection of emerging specific spoilers at earlier stages of processing. Fatty acid characterisation allowed the assessment of the most frequent types of contamination yeasts and supplied the information for the definition of relevant zymological indicators. A selected group of strains was used for further studies of mechanisms underlying the resistance/tolerance of yeasts towards preservatives (weak acids) and other stress factors (temperature, high sugar and salt concentrations). This study enabled the acquisition of data on the basic biology of yeasts used in the development of differential and selective media for Zygosaccharomyces bailii, Yarrowia lipolytica, Kluyveromyces marxianus and Dekkera sp.
Archaic speculations and firmly established legends regarding the origin of the yeast Saccharomyces cerevisiae and related species are revisited in light of past and recent ecological evidence pointing to a strict association with artificial, man-made environments such as wineries and fermentation plants. The nomenclature within this industrially important group is also discussed in view of the modifications imposed from application of molecular techniques to classification.
The growth of several yeast species in milk containing added sodium chloride (0-15%, w/v) at 25 degrees C and 10 degrees C was examined in conjunction with yeast metabolism of milk constituents. Depending on conditions, all yeasts grew to maximum populations of 10(7)-10(8) cfu/ml. Kluyveromyces marxianus gave strong utilisation of lactose and weak metabolism of citrate, protein and fat with the production of ethanol, glycerol, lactic acid and propionic acid. As measured by the production of free amino acids and free fatty acids, Candida lipolytica and Candida catenulata gave strong proteolytic and lipolytic reactions, the specificities of which appeared to be influenced by temperature and the presence of NaCl. These species also metabolised organic acids. Although giving strong growth responses, Debaryomyces hansenii and Saccharomyces cerevisiae did not metabolise lactose and gave only very weak lipolytic and proteolytic reactions. Citrate was metabolised by D. hansenii but not by S. cerevisiae. Both species produced small amounts of ethanol, glycerol and lactic acid.
The Gram positive bacteria are generally regarded as the most hazardous beer spoilage organisms in modern breweries, especially the lactobacilli: L. brevis, L. lindneri, L. curvatus, L. casei, L. buchneri, L. coryneformis, L. plantarum, L. brevisimilis, L. malefermentans and L. parabuchneri and the pediococci: P damnosus, P. inopinatus and P. dextrinicus. Micrococcus kristinae is the only species within the micrococci relevant to brewing. The Gram negative strictly anaerobic bacteria are apparently increasing in importance and include Pectinatus cerevisiiphilus, Pectinatus frisingensis and Selenomonas lacticifex, reported as obligate beer spoilage organisms: Zymophilus raffinosivorans as a potential beer spoilage organism; Megasphaera cerevisiae as an obligate spoilage organism of low alcohol beer and Zymomonas mobilis as capable of spoiling primed beer. With improved process technology the importance of aerobic bacteria has decreased and the same applies for the Gram negative aerobic bacteria Hafnia protea and Enterobacter cloacae which are capable of surviving beer fermentation. Beer spoilage organisms include several so-called wild yeasts, of which Saccharomyces species are generally considered the most important. Even though the detection of beer spoilage organisms by cultivation in laboratory media does not always provide the specificity and the sensitivity required, the use of selective media and incubation conditions still appears to be the method preferred by breweries. The media used depend on the type of sample, the specificity required and, for detection of wild yeasts, to some extent, the characteristics of the culture yeast. Among the media reported so far no single medium can be used to detect all members within a group of specific beer spoilage organisms and further work on the development of improved substrates are required both for bacteria and wild yeasts.
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.
There is still a lack of agreement concerning the relative contribution of wine yeast that may originate in the vineyard compared to that which may originate in the cellar. Part of this controversy is due to the extreme difficulty of finding Saccharomyces cerevisiae on the grapes. We estimate that only about one in one-thousand grape berries carries wine yeast. However, we have found that grape berries that are damaged (i.e. the skin is broken) are very rich depositories of microorganisms including S. cerevisiae, and that one in four such berries is S. cerevisiae-positive. These positive berries have between 100,000 and 1,000,000 wine yeast cells on them, and there is evidence that these yeasts are clonal. We believe that the yeasts are brought to the berries by insects such as bees, wasps, and Drosophila and that they multiply in the rich medium of the grape interior. Even though there are many cells of S. cerevisiae on the damaged berries, they are in a definite minority. All the other organisms that are found in wine fermentations are also present on these berries, and their total numbers are in the range of 10 million to 100 million cells per berry.
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.
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 identification, differentiation and characterization of indigenous Saccharomyces sensu stricto strains isolated from Croatian vineyards and the evaluation of their oenological potential.
A total of 47 Saccharomyces sensu stricto strains were isolated from Chardonnay grapes and identified by physiological and molecular genetic methods. By using the standard physiological and biochemical tests, six isolates were identified as Saccharomyces cerevisiae and 41 as Saccharomyces paradoxus. However, PCR-RFLP analyses of the internal transcribed spacer (ITS1) region of the 18S ribosomal DNA identified 12 of the isolates as S.cerevisiae and 35 as S. paradoxus. Fermentation trials in a grape juice medium showed that these isolates ferment vigorously at 18 degrees C and display tolerance to high levels of ethanol. None of these isolates appeared to produce either hydrogen sulphide or killer toxins.
Saccharomyces paradoxus, possessing potentially important oenological characteristics, occurs in much higher numbers than S. cerevisiae in the indigenous population of Saccharomyces sensu stricto strains in Croatian vineyards.
This study forms an essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in the Croatian grape-growing regions. The results obtained demonstrate the value of using molecular genetic methods, such as PCR-RFLP analyses, in conjunction with the traditional taxonomic methods based on phenotypic characteristics in such ecotaxonomic surveys. The results also shed some light on the ecology and oenological potential of S.paradoxus, which is considered to be the natural parent species of the domesticated species of the Saccharomyces sensu stricto group.
Yeast colonies isolated from vineyard and cellar substrates were analysed in the present study. Yeast species assessment was carried out by amplification and digestion of a region of the ribosomal RNA gene repeat unit. Saccharomyces strains were also characterised using mitochondrial DNA restriction analysis. Oxidative basidiomycetous yeasts without enological potential were predominant in the vineyard environment. Yeasts associated with grape skin depend on grape variety, vintage and degree of grape maturation. These species from grape surface constituted the predominant microbiota in must and they developed during the first stages of the process. Yeasts colonies were also isolated and identified from the walls of a fermentation vat some days before the harvest. Contray to what was expected, Saccharomyces cerevisiae was not the major species isolated as Candida sorbosa represented 76% of the species isolated. Saccharomyces strains isolated from the fermentation vat had been previously isolated in wine fermentations in this cellar. Therefore, these strains should be considered as constant residents of this winery.
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.
Wine is the product of complex interactions between fungi, yeasts and bacteria that commence in the vineyard and continue throughout the fermentation process until packaging. Although grape cultivar and cultivation provide the foundations of wine flavour, microorganisms, especially yeasts, impact on the subtlety and individuality of the flavour response. Consequently, it is important to identify and understand the ecological interactions that occur between the different microbial groups, species and strains. These interactions encompass yeast-yeast, yeast-filamentous fungi and yeast-bacteria responses. The surface of healthy grapes has a predominance of Aureobasidium pullulans, Metschnikowia, Hanseniaspora (Kloeckera), Cryptococcus and Rhodotorula species depending on stage of maturity. This microflora moderates the growth of spoilage and mycotoxigenic fungi on grapes, the species and strains of yeasts that contribute to alcoholic fermentation, and the bacteria that contribute to malolactic fermentation. Damaged grapes have increased populations of lactic and acetic acid bacteria that impact on yeasts during alcoholic fermentation. Alcoholic fermentation is characterised by the successional growth of various yeast species and strains, where yeast-yeast interactions determine the ecology. Through yeast-bacterial interactions, this ecology can determine progression of the malolactic fermentation, and potential growth of spoilage bacteria in the final product. The mechanisms by which one species/strain impacts on another in grape-wine ecosystems include: production of lytic enzymes, ethanol, sulphur dioxide and killer toxin/bacteriocin like peptides; nutrient depletion including removal of oxygen, and production of carbon dioxide; and release of cell autolytic components. Cell-cell communication through quorum sensing molecules needs investigation.
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.