An important step forward for the future development of an easy and fast procedure for identifying the most dangerous wine spoilage yeast,Dekkera bruxellensis,in wine environment

Dekkera bruxellensis is the main reason for spoilage in the wine industry. It renders the products unacceptable leading to large economic losses. Fluorescence In Situ Hybridization (FISH) technique has the potential for allowing its specific detection. Nevertheless, some experimental difficulties can be encountered when FISH technique is applied in the wine environment (e.g. matrix and cells’ autofluorescence, fluorophore inadequate selection and probes’ low specificity to the target organisms). An easy and fast in-suspension RNA-FISH procedure was applied for the first time for identifying D. bruxellensis in wine. A previously designed RNA-FISH probe to detect D. bruxellensis (26S D. brux.5.1) was used, and the matrix and cells’ fluorescence interferences, the influence of three fluorophores in FISH performance and the probe specificity were evaluated. The results revealed that to apply RNA-FISH technique in the wine environment, a red-emitting fluorophore should be used. Good probe performance and specificity were achieved with 25% of formamide. The resulting RNA-FISH protocol was applied in wine samples artificially inoculated with D. bruxellensis. This spoilage microorganism was detected in wine at cell densities lower than those associated with phenolic off-flavours. Thus, the RNA-FISH procedure described in this work represents an advancement to facilitate early detection of the most dangerous wine spoilage yeast and, consequently, to reduce the economic losses caused by this yeast to the wine industry.     

Inactivation of wine spoilage yeasts Dekkera bruxellensis using low electric current treatment (LEC)

Aims: The objective of this study was to investigate the inactivation of a selected yeast Dekkera bruxellensis strain 4481 in red wine by application of low electric current treatment (LEC). Methods and Results: LEC (200 mA) was applied for 60 days to a red wine, Montepulciano d’Abruzzo, in an alternative strategy to the SO₂ addition during wine storage. The LEC effect on both cell activity and microflora viability was assessed. LEC decreased significantly the survival viable cells and increased the death rate of D. bruxellensis strain 4481 yeast. A final comparison was made of the main physico‐chemical parameters of the wine after the different treatments. The study suggests the importance of an appropriate LEC treatment which limits wine deterioration in terms of off‐flavours synthesis. Conclusions: The results demonstrate that the growth of undesirable Dekkera can be inhibited by low voltage treatment; LEC was shown to be useful to prevent wine spoilage and has the potential of being a concrete alternative method for controlling wine spoilage. Significance and Impact of the Study: Wine spoilage can be avoided by preventing the growth of undesirable Dekkera yeasts, through the effective use of LEC in the winemaking process.     

Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.     

Brettanomyces bruxellensis evolution and volatile phenols production in red wines during storage in bottles

Aims: The presence of Brettanomyces bruxellensis is an important issue during winemaking because of its volatile phenols production capacities. The aim of this study is to provide information on the ability of residual B. bruxellensis populations to multiply and spoil finished wines during storage in bottles. Methods and Results: Several finished wines were studied. Brettanomyces bruxellensis populations were monitored during two and a half months, and volatile phenols as well as chemical parameters regularly determined. Variable growth and volatile phenols synthesis capacities were evidenced, in particularly when cells are in a noncultivable state. In addition, the volatile phenol production was clearly shown to be a two‐step procedure that could strongly be correlated to the physiological state of the yeast population. Conclusions: This study underlines the importance of minimizing B. bruxellensis populations at the end of wine ageing to reduce volatile phenols production risk once the wine in bottle. Moreover, the physiological state of the yeast seems to have an important impact on ethyl‐phenols production, hence demonstrating the importance of taking into account this parameter when analysing wine spoilage risks. Significance and Impact of the Study: Little data exist about the survival of B. bruxellensis once the wine in bottle. This study provides information on the alteration risks encountered during wine storage in bottle and reveals the importance of carrying on further studies to increase the knowledge on B. bruxellensis physiology.     

Identification of Dekkera bruxellensis (Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.     

Fermentation of lignocellulosic hydrolysate by the alternative industrial ethanol yeast Dekkera bruxellensis

Aim: Testing the ability of the alternative ethanol production yeast Dekkera bruxellensis to produce ethanol from lignocellulose hydrolysate and comparing it to Saccharomyces cerevisiae. Methods and Results: Industrial isolates of D. bruxellensis and S. cerevisiae were cultivated in small‐scale batch fermentations of enzymatically hydrolysed steam exploded aspen sawdust. Different dilutions of hydrolysate were tested. None of the yeasts grew in undiluted or 1 : 2 diluted hydrolysate [final glucose concentration always adjusted to 40 g l^?1 (0·22 mol l^?1)]. This was most likely due to the presence of inhibitors such as acetate or furfural. In 1 : 5 hydrolysate, S. cerevisiae grew, but not D. bruxellensis, and in 1 : 10 hydrolysate, both yeasts grew. An external vitamin source (e.g. yeast extract) was essential for growth of D. bruxellensis in this lignocellulosic hydrolysate and strongly stimulated S. cerevisiae growth and ethanol production. Ethanol yields of 0·42 ± 0·01 g ethanol (g glucose)^?1 were observed for both yeasts in 1 : 10 hydrolysate. In small‐scale continuous cultures with cell recirculation, with a gradual increase in the hydrolysate concentration, D. bruxellensis was able to grow in 1 : 5 hydrolysate. In bioreactor experiments with cell recirculation, hydrolysate contents were increased up to 1 : 2 hydrolysate, without significant losses in ethanol yields for both yeasts and only slight differences in viable cell counts, indicating an ability of both yeasts to adapt to toxic compounds in the hydrolysate. Conclusions: Dekkera bruxellensis and S. cerevisiae have a similar potential to ferment lignocellulose hydrolysate to ethanol and to adapt to fermentation inhibitors in the hydrolysate. Significance and Impact of the study: This is the first study investigating the potential of D. bruxellensis to ferment lignocellulosic hydrolysate. Its high competitiveness in industrial fermentations makes D. bruxellensis an interesting alternative for ethanol production from those substrates.     

Starter cultures as biocontrol strategy to prevent Brettanomyces bruxellensis proliferation in wine

Brettanomyces bruxellensis is a common and significant wine spoilage microorganism. B. bruxellensis strains generally detain the molecular basis to produce compounds that are detrimental for the organoleptic quality of the wine, including some classes of volatile phenols that derive from the sequential bioconversion of specific hydroxycinnamic acids such as ferulate and p-coumarate. Although B. bruxellensis can be detected at any stage of the winemaking process, it is typically isolated at the end of the alcoholic fermentation (AF), before the staring of the spontaneous malolactic fermentation (MLF) or during barrel aging. For this reason, the endemic diffusion of B. bruxellensis leads to consistent economic losses in the wine industry. Considering the interest in reducing sulfur dioxide use during winemaking, in recent years, biological alternatives, such as the use of tailored selected yeast and bacterial strains inoculated to promote AF and MLF, are actively sought as biocontrol agents to avoid the “Bretta” character in wines. Here, we review the importance of dedicated characterization and selection of starter cultures for AF and MLF in wine, in order to reduce or prevent both growth of B. bruxellensis and its production of volatile phenols in the matrix.

     

Enhanced volatile phenols in wine fermented with Saccharomyces cerevisiae and spoiled with Pichia guilliermondii and Dekkera bruxellensis

Aims: To investigate whether the presence of Pichia guilliermondii impacts on the production of volatile phenols from mixed wine fermentations with Dekkera bruxellensis and Saccharomyces cerevisiae. Methods and Results: Four inoculation strategies were performed in small‐scale fermentations involving P. guilliermondii, D. bruxellensis and S. cerevisiae using Syrah grape juice supplemented with 100 mg l^?1 of p‐coumaric acid. High pressure liquid chromatography was used for the quantification or volatile phenols. Significant high levels of 4‐ethylphenol and 4‐ethylguaicol (720 and 545 μg l^?1, respectively), as well as the highest levels of 4‐vinylphenol (>4500 μg l^?1), were observed when P. guilliermondii species was inoculated from the beginning of the fermentation. Conclusions: The metabolic interaction occurring between the high vinylphenol producer species P. guilliermondii and D. bruxellensis exhibiting a high vinylphenol reductase activity resulted in an increased production of volatile phenols in wine. Significance and Impact of the Study: Pichia guilliermondii must be considered a very important spoilage yeast in the wine industry capable of producing large amounts of volatile phenols.     

Inventory and monitoring of wine microbial consortia

The evolution of the wine microbial ecosystem is generally restricted to Saccharomyces cerevisiae and Oenococcus oeni, which are the two main agents in the transformation of grape must into wine by acting during alcoholic and malolactic fermentation, respectively. But others species like the yeast Brettanomyces bruxellensis and certain ropy strains of Pediococcus parvulus can spoil the wine. The aim of this study was to address the composition of the system more precisely, identifying other components. The advantages of the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) approach to wine microbial ecology studies are illustrated by bacteria and yeast species identification and their monitoring at each stage of wine production. After direct DNA extraction, PCR-DGGE was used to make the most exhaustive possible inventory of bacteria and yeast species found in a wine environment. Phylogenetic neighbor-joining trees were built to illustrate microbial diversity. PCR-DGGE was also combined with population enumeration in selective media to monitor microbial changes at all stages of production. Moreover, enrichment media helped to detect the appearance of spoilage species. The genetic diversity of the wine microbial community and its dynamics during winemaking were also described. Most importantly, our study provides a better understanding of the complexity and diversity of the wine microbial consortium at all stages of the winemaking process: on grape berries, in must during fermentation, and in wine during aging. On grapes, 52 different yeast species and 40 bacteria could be identified. The diversity was dramatically reduced during winemaking then during aging. Yeast and lactic acid bacteria were also isolated from very old vintages. B. bruxellensis and O. oeni were the most frequent.     

Microbiological,biochemical, physicochemical surface properties and biofilm forming ability of Brettanomyces bruxellensis

Purpose

Brettanomyces bruxellensis is a serious source of concern for winemakers. The production of volatile phenols by the yeast species confers to wine unpleasant sensory characteristics which are unacceptable by the consumers and inevitably provoke economic loss for the wine industry. This ubiquitous yeast is able to adapt to all winemaking steps and to withstand various environmental conditions. Moreover, the ability of B. bruxellensis to adhere and colonize inert materials can be the cause of the yeast persistence in the cellars and thus recurrent wine spoilage. We therefore investigated the surface properties, biofilm formation capacity, and the factors which may affect the attachment of the yeast cells to surfaces with eight strains representative of the genetic diversity of the species.

Methods

The eight strains of B. bruxellensis were isolated from different geographical and industrial fermentation origins. The cells were grown in synthetic YPD medium containing 1% (w/v) yeast extract (Difco Laboratories, Detroit), 2% (w/v) bacto peptone (Difco), and 1% (w/v) glucose. Surface physicochemical properties as electrophoretic mobility and adhesion to hydrocarbon of the cells were studied. The ability of the strains to form biofilm was quantified using a colorimetric microtiter 96-well polystyrene plate. Biochemical characteristics were examined by colorimetric methods as well as by chemical analysis.

Result

Our results show that the biofilm formation ability is strain-dependent and suggest a possible link between the physicochemical properties of the studied strains and their corresponding genetic group.

Conclusion

The capacity to detect and identify the strains of the spoilage yeast based on their biofilm formation abilities may help to develop more efficient cleaning procedures and preventing methods.

     

South Brazilian wines: culturable yeasts associated to bottled wines produced in Rio Grande do Sul and Santa Catarina

A comprehensive understanding of the presence and role of yeasts in bottled wines helps to know and control the organoleptic quality of the final product. The South Region of Brazil is an important wine producer, and the state of “Rio Grande do Sul” (RS) accounts for 90% of Brazilian wines. The state of “Santa Catarina” (SC) started the production in 1975, and is currently the fifth Brazilian producer. As there is little information about yeasts present in Brazilian wines, our main objective was to assess the composition of culturable yeasts associated to bottled wines produced in RS and SC, South of Brazil. We sampled 20 RS and 29 SC bottled wines produced between 2003 and 2011, and we isolated culturable yeasts in non-selective agar plates. We identified all isolates by sequencing of the D1/D2 domain of LSU rDNA or ITS1-5.8 S-ITS2 region, and comparison with type strain sequences deposited in GenBank database. Six yeast species were shared in the final product in both regions. We obtained two spoilage yeast profiles: RS with Zygosaccharomyces bailii and Pichia membranifaciens (Dekkera bruxellensis was found only in specific table wines); and SC with Dekkera bruxellensis and Pichia manshurica. Knowledge concerning the different spoilage profiles is important for winemaking practices in both regions.     

Spoilage yeasts from Patagonian cellars: characterization and potential biocontrol based on killer interactions

Indigenous yeasts associated with surfaces in three North Patagonian cellars were isolated by means of selective media developed for the isolation of Dekkera/Brettanomyces yeasts; 81 isolates were identified as belonging to Candida boidinii (16%), Hanseniaspora uvarum (38%), Pichia guilliermondii (3%), Saccharomyces cerevisiae (1%), Geotrichum silvicola (16%) and the new yeast species Candida patagonica (26%). No Dekkera/Brettanomyces isolate was obtained, however, 41 isolates (51% of the total isolates) produced some enologically undesirable features under laboratory conditions including the production of 4-ethylphenol and 4-vinylphenol, observed in the Candida boidinii and Pichia guilliermondii isolates. The sensitivity of the 41 spoilage isolates and seven Brettanomyces bruxellensis collection strains was evaluated against a panel of 55 indigenous and ten reference killer yeasts. Killer cultures belonging to Pichia anomala and Kluyveromyces lactis species showed the broadest killer spectrum against spoilage yeasts, including Dekkera bruxellensis collection strains. These killer isolates could be good candidates for use in biocontrol of regionally relevant spoilage yeasts.     

Comparison of media formulations used to selectively cultivate Dekkera/Brettanomyces

Aims: The objectives of this research were to (i) optimize the concentration of cycloheximide for use in WL media used in the wine industry and (ii) evaluate Dekkera/Brettanomyces differential medium (DBDM) as a means to detect Dekkera. Methods and Results: Dekkera bruxellensis and other yeasts were transferred into WL broths containing 0, 10, 50 or 100 mg l^?1 of cycloheximide. While several grew in 10 mg l^?1, only Hanseniaspora uvarum, Pichia guillermondii, Schizosaccharomyces pombe and D. bruxellensis tolerated ≥50 mg l^?1 of the antibiotic. On solidified WL media after 8‐days incubation, colony sizes of two strains of D. bruxellensis (B1b and ATCC 52905) decreased with increased concentrations of cycloheximide, while others (F3 and P2) were unaffected. Although D. bruxellensis B1b did not grow well on another selective medium, DBDM, colony development was improved by the addition of sterilized red wine. Conclusions: Of the concentrations tested, 50 mg l^?1 cycloheximide inhibited many grape/wine yeasts yet generally yielded countable colonies of Dekkera (1–2·5 mm diameter). Several strains of Dekkera did not grow well on DBDM, probably due to the lack of an unidentified nutrient(s). Significance and Impact of the Study: Better media formulations will improve the detection of Dekkera, thereby increasing microbiological control during winemaking.     

Dekkera and Brettanomyces growth and utilisation of hydroxycinnamic acids in synthetic media

Dekkera and Brettanomyces yeast are important spoilage organisms in a number of food and beverage products. Isolates of both genera were cultured in a defined medium and supplemented with hydroxycinnamic acids and vinylphenols to investigate their influence on growth and the formation of ethyl phenol derivatives. The growth rate of Brettanomyces species in the presence of acids was reduced, and no significant conversion to vinyl or ethyl derivatives was observed. The growth rate and substrate utilisation rates of Dekkera anomala and Dekkera bruxellensis yeast differed depending on strain and the acid precursor present. Growth of D. bruxellensis was slowed by the presence of ferulic acid with the addition of 1 mM ferulic acid completely inhibiting growth. This study provides an insight into the spoilage potential of these organisms and possible control strategies involving hydroxycinnamic acids.     

Biotechnological exploitation of Tetrapisispora phaffii killer toxin: heterologous production in Komagataella phaffii (Pichia pastoris)

The use of natural antimicrobials from plants, animals and microorganisms to inhibit the growth of pathogenic and spoilage microorganisms is becoming more frequent. This parallels the increased consumer interest towards consumption of minimally processed food and ‘greener’ food and beverage additives. Among the natural antimicrobials of microbial origin, the killer toxin produced by the yeast Tetrapisispora phaffii, known as Kpkt, appears to be a promising natural antimicrobial agent. Kpkt is a glycoprotein with β-1,3-glucanase and killer activity, which induces ultrastructural modifications to the cell wall of yeast of the genera Kloeckera/Hanseniaspora and Zygosaccharomyces. Moreover, Kpkt maintains its killer activity in grape must for at least 14 days under winemaking conditions, thus suggesting its use against spoilage yeast in wine making and the sweet beverage industry. Here, the aim was to explore the possibility of high production of Kpkt for biotechnological exploitation. Molecular tools for heterologous production of Kpkt in Komagataella phaffii GS115 were developed, and two recombinant clones that produce up to 23 mg/L recombinant Kpkt (rKpkt) were obtained. Similar to native Kpkt, rKpkt has β-glucanase and killer activities. Moreover, it shows a wider spectrum of action with respect to native Kpkt. This includes effects on Dekkera bruxellensis, a spoilage yeast of interest not only in wine making, but also for the biofuel industry, thus widening the potential applications of this rKpkt.

     

Raman Spectroscopy and Chemometrics for Identification and Strain Discrimination of the Wine Spoilage Yeasts Saccharomyces cerevisiae,Zygosaccharomyces bailii,and Brettanomyces bruxellensis

The yeasts Zygosaccharomyces bailii, Dekkera bruxellensis (anamorph, Brettanomyces bruxellensis), and Saccharomyces cerevisiae are the major spoilage agents of finished wine. A novel method using Raman spectroscopy in combination with a chemometric classification tool has been developed for the identification of these yeast species and for strain discrimination of these yeasts. Raman spectra were collected for six strains of each of the yeasts Z. bailii, B. bruxellensis, and S. cerevisiae. The yeasts were classified with high sensitivity at the species level: 93.8% for Z. bailii, 92.3% for B. bruxellensis, and 98.6% for S. cerevisiae. Furthermore, we have demonstrated that it is possible to discriminate between strains of these species. These yeasts were classified at the strain level with an overall accuracy of 81.8%.     

The physiological characteristics of the yeast Dekkera bruxellensis in fully fermentative conditions with cell recycling and in mixed cultures with Saccharomyces cerevisiae

The yeast Dekkera bruxellensis plays an important role in industrial fermentation processes, either as a contaminant or as a fermenting yeast. In this study, an analysis has been conducted of the fermentation characteristics of several industrial D. bruxellensis strains collected from distilleries from the Southeast and Northeast of Brazil, compared with Saccharomyces cerevisiae. It was found that all the strains of D. bruxellensis showed a lower fermentative capacity as a result of inefficient sugar assimilation, especially sucrose, under anaerobiosis, which is called the Custer effect. In addition, most of the sugar consumed by D. bruxellensis seemed to be used for biomass production, as was observed by the increase of its cell population during the fermentation recycles. In mixed populations, the surplus of D. bruxellensis over S. cerevisiae population could not be attributed to organic acid production by the first yeast, as previously suggested. Moreover, both yeast species showed similar sensitivity to lactic and acetic acids and were equally resistant to ethanol, when added exogenously to the fermentation medium. Thus, the effects that lead to the employment of D. bruxellensis in an industrial process and its effects on the production of ethanol are multivariate. The difficulty of using this yeast for ethanol production is that it requires the elimination of the Custer effect to allow an increase in the assimilation of sugar under anaerobic conditions.     

Insights into the Dekkera bruxellensis Genomic Landscape: Comparative Genomics Reveals Variations in Ploidy and Nutrient Utilisation Potential amongst Wine Isolates

The yeast Dekkera bruxellensis is a major contaminant of industrial fermentations, such as those used for the production of biofuel and wine, where it outlasts and, under some conditions, outcompetes the major industrial yeast Saccharomyces cerevisiae. In order to investigate the level of inter-strain variation that is present within this economically important species, the genomes of four diverse D. bruxellensis isolates were compared. While each of the four strains was shown to contain a core diploid genome, which is clearly sufficient for survival, two of the four isolates have a third haploid complement of chromosomes. The sequences of these additional haploid genomes were both highly divergent from those comprising the diploid core and divergent between the two triploid strains. Similar to examples in the Saccharomyces spp. clade, where some allotriploids have arisen on the basis of enhanced ability to survive a range of environmental conditions, it is likely these strains are products of two independent hybridisation events that may have involved multiple species or distinct sub-species of Dekkera. Interestingly these triploid strains represent the vast majority (92%) of isolates from across the Australian wine industry, suggesting that the additional set of chromosomes may confer a selective advantage in winery environments that has resulted in these hybrid strains all-but replacing their diploid counterparts in Australian winery settings. In addition to the apparent inter-specific hybridisation events, chromosomal aberrations such as strain-specific insertions and deletions and loss-of-heterozygosity by gene conversion were also commonplace. While these events are likely to have affected many phenotypes across these strains, we have been able to link a specific deletion to the inability to utilise nitrate by some strains of D. bruxellensis, a phenotype that may have direct impacts in the ability for these strains to compete with S. cerevisiae.