A simple procedure for detecting Dekkera bruxellensis in wine environment by RNA-FISH using a novel probe.

Dekkera bruxellensis, considered the major microbial contaminant in wine production, produces 4-ethylphenol, a cause of unpleasant odors. Thus, identification of this yeast before wine spoilage is crucial. Although challenging, it could be achieved using a simple technique: RNA-FISH. To reach it is necessary to design probes that allow specific detection/identification of D. bruxellensis among the wine microorganisms and in the wine environment and, if possible, using low formamide concentrations. Therefore, this study was focused on: a) designing a DNA-FISH probe to identify D. bruxellensis that matches these requirements and b) determining the applicability of the RNA-FISH procedure after the end of the alcoholic fermentation and in wine. A novel DNA-FISH D. bruxellensis probe with good performance and specificity was designed. The application of this probe using an in-suspension RNA-FISH protocol (applying only 5% of formamide) allowed the early detection/identification of D. bruxellensis at low cell densities (5 × 102 cell/mL). This was possible by flow cytometry independently of the growth stage of the target cells, both at the end of the alcoholic fermentation and in wine even in the presence of high S. cerevisiae cell densities. Thus, this study aims to contribute to facilitate the identification of D. bruxellensis before wine spoilage occurs, preventing economic losses to the wine industry.

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.

Identifying yeasts using surface enhanced Raman spectroscopy.

The molecular fingerprints of yeasts Saccharomyces cerevisiae, Dekkera bruxellensis, and Wickerhamomyces anomalus (former name Pichia anomala) have been examined using surface-enhanced Raman spectroscopy (SERS) and helium ion microscopy (HIM). The SERS spectra obtained from cell cultures (lysate and non-treated cells) distinguish between these very closely related fungal species. Highly SERS active silver nano-particles suitable for detecting complex biomolecules were fabricated using a simple synthesis route. The yeast samples mixed with aggregated Ag nanoparticles yielded highly enhanced and reproducible Raman signal owing to the high density of the hot spots at the junctions of two or more Ag nanoparticles and enabled to differentiate the three species based on their unique features (spectral fingerprint). We also collected SERS spectra of the three yeast species in beer medium to demonstrate the potential of the method for industrial application. These findings demonstrate the great potential of SERS for detection and identification of fungi species based on the biochemical compositions, even in a chemically complex sample.

Osmotolerance of Dekkera bruxellensis and the role of two Stl glycerol-proton symporters.

Dekkera bruxellensis is important for lambic beer fermentation but is considered a spoilage yeast in wine fermentation. We compared two D. bruxellensis strains isolated from wine and found that they differ in some basic properties, including osmotolerance. The genomes of both strains contain two highly similar copies of genes encoding putative glycerol-proton symporters from the STL family that are important for yeast osmotolerance. Cloning of the two DbSTL genes and their expression in suitable osmosensitive Saccharomyces cerevisiae mutants revealed that both identified genes encode functional glycerol uptake systems, but only DbStl2 has the capacity to improve the osmotolerance of S. cerevisiae cells.

Unraveling microbial ecology of industrial-scale Kombucha fermentations by metabarcoding and culture-based methods.

Kombucha, historically an Asian tea-based fermented drink, has recently become trendy in Western countries. Producers claim it bears health-enhancing properties that may come from the tea or metabolites produced by its microbiome. Despite its long history of production, microbial richness and dynamics have not been fully unraveled, especially at an industrial scale. Moreover, the impact of tea type (green or black) on microbial ecology was not studied. Here, we compared microbial communities from industrial-scale black and green tea fermentations, still traditionally carried out by a microbial biofilm, using culture-dependent and metabarcoding approaches. Dominant bacterial species belonged to Acetobacteraceae and to a lesser extent Lactobacteriaceae, while the main identified yeasts corresponded to Dekkera, Hanseniaspora and Zygosaccharomyces during all fermentations. Species richness decreased over the 8-day fermentation. Among acetic acid bacteria, Gluconacetobacter europaeus, Gluconobacter oxydans, G. saccharivorans and Acetobacter peroxydans emerged as dominant species. The main lactic acid bacteria, Oenococcus oeni, was strongly associated with green tea fermentations. Tea type did not influence yeast community, with Dekkera bruxellensis, D. anomala, Zygosaccharomyces bailii and Hanseniaspora valbyensis as most dominant. This study unraveled a distinctive core microbial community which is essential for fermentation control and could lead to Kombucha quality standardization.

Analysis of 100-Year-Old Beer Originated from the Czech Republic.

Three bottles of different beers were found in 2015 during a reconstruction of the brewery of the Raven Trading s.r.o. company in Záhlinice, Czech Republic. Thanks to good storage conditions, it was possible to analyze their original characteristics. All three bottles contained most probably lager type beer. One beer had sulfuric and fecal off-flavors; it was bright with the original extract of 10.3° Plato. The second beer, with an original extract of 7.6° Plato, was dark and very acidic, resembling Lambic. DNA analysis proved the presence of Dekkera bruxellensis, which corresponded to its chemical profile (total acidity, FAN, ethyl acetate, total esters). The third beer contained traces of carbon dioxide bubbles, was light brown and slightly bitter, with an original extract 10.4° Plato. Because it obviously underwent a natural aging process, sweetness, honey, and fruity off-flavors were detected and transformation products of iso-α-acids were found.

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.

Draft genome sequence and transcriptome analysis of the wine spoilage yeast Dekkera bruxellensis LAMAP2480 provides insights into genetic diversity, metabolism and survival.

Dekkera bruxellensis is the major contaminant yeast in the wine industry worldwide. Here, we present the draft genome sequence of D. bruxellensis LAMAP2480 isolated from a Chilean wine. Genomic evidence reveals shared and exclusive genes potentially involved in colonization and survival during alcoholic fermentation.

Rapid identification of the genus Dekkera/Brettanomyces, the Dekkera subgroup and all individual species.

The genus Dekkera/Brettanomyces comprises five described species: Dekkera bruxellensis, D. anomala, Brettanomyces custersianus, B. naardenensis and B. nanus. Some of them, especially D. bruxellensis, are important spoilage organisms, particularly in the wine and beverage industries. Because of their economic importance many different methods have been developed to identify members of the genus in general and D. bruxellensis in particular. These methods vary in their rapidity, complexity and cost but, partly because of confidentiality issues, it is unclear which methods are used, or how widely, in the relevant industries. Building on previous work with the genera Saccharomyces and Zygosaccharomyces, a suite of eight PCR primer pairs has been designed either on the D1-D2 region of the 26S rRNA gene or translation elongation factor TEF1-α. These primers can specifically identify the genus as a whole, only Dekkera species, each one of the five recognised species as well as a significant subgroup of D. bruxellensis represented by NCYC 3426. Multiplexing has also been tried and it has been shown to be possible with some combinations of genus or Dekkera-level and species-specific primers. Using direct colony PCR amplification followed by gel electrophoresis, a clear positive result can be obtained in less than 3h, thus providing a quick, reliable and inexpensive way to identify target species.

Impact of Australian Dekkera bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma.

Spoilage of red wine by the yeast species Dekkera bruxellensis is a common problem for the global wine industry. When conditions are conducive for growth of these yeasts in wine, they efficiently convert non-volatile hydroxycinnamic acids into aroma-active ethylphenols, thereby reducing the quality of the wine. It has been demonstrated previously that dissolved oxygen is a key factor which stimulates D. bruxellensis growth in wine. We demonstrate that whereas the presence of oxygen accelerates the growth of this species, oxygen-limited conditions favour 4-ethylphenol production. Consequently, we evaluated wine spoilage potential of three D. bruxellensis strains (AWRI1499, AWRI1608 and AWRI1613) under oxygen-limited conditions. Each strain was cultured in a chemically-defined wine medium and the fermentation products were analysed using HPLC and HS-SPME-GC/MS. The strains displayed different growth characteristics but were equally capable of producing ethylphenols. On the other hand, significant differences were observed for 18 of the remaining 33 metabolites analysed and duo-trio sensory analysis indicated significant aroma differences between wines inoculated with AWRI1499 and AWRI1613. When these wines were spiked with low concentrations of 4-ethylphenol and 4-ethylguaiacol, no sensorial differences could be perceived. Together these data suggest that the three predominant D. bruxellensis strains previously isolated during a large survey of Australian wineries do not differ substantively in their capacity to grow in, and spoil, a model wine medium.

Advances in the control of wine spoilage by Zygosaccharomyces and Dekkera/Brettanomyces.

Understanding the characteristics of yeast spoilage, as well as the available control technologies, is vital to producing consistent, high-quality wine. Zygosaccharomyces bailii contamination may result in refermentation and CO2 production in sweet wines or grape juice concentrate, whereas Brettanomyces bruxellensis spoilage often contributes off-odors and flavors to red wines. Early detection of these yeasts by selective/differential media or genetic methods is important to minimize potential spoilage. More established methods of microbial control include sulfur dioxide, dimethyl dicarbonate, and filtration. Current research is focused on the use of chitosan, pulsed electric fields, low electric current, and ultrasonics as means to protect wine quality.

Detection of Brettanomyces spp. in red wines using real-time PCR.

UNLABELLED: The question if the "Brett character" is a favorable wine attribute is one of the most controversial issues and it is currently addressed by many researches. Actually, the presence of Brettanomyces/Dekkera in wine during barrel aging is often associated to detrimental organoleptic characteristics depending on the release of volatile phenols (for example, 4-ethylphenol and 4-ethylguaiacol); for that reason the possibility to rapidly detect the yeast at the early stage of wine production could allow preventive actions to reduce wine spoilage. In this work, 25 and 5 samples from conventional and organic vineyards, respectively, all suspected to be spoiled by Brettanomyces/Dekkera spp., were analyzed using both culture-dependent and culture-independent techniques. In particular, a DNA extraction protocol and a real-time quantitative PCR (qPCR) assay to directly detect and quantify B. bruxellensis were optimized. Results showed that B. bruxellensis was present in 22 of 30 samples, ranging from 10 to 10(4) CFU/mL, lower values being found in organic wines (10 to 10(2) CFU/mL). Overall, qPCR was proved to be a useful and valuable wine control system, since 12 samples were recorded as positive for yeast presence when analyzed by qPCR and negative in case of plate count analyses. PRACTICAL APPLICATION: Brettanomyces cells were detected using a qPCR method, optimized in this study, which allows to obtain results quickly.

The consequences of Lactobacillus vini and Dekkera bruxellensis as contaminants of the sugarcane-based ethanol fermentation.

This work describes the effects of the presence of the yeast Dekkera bruxellensis and the bacterium Lactobacillus vini on the industrial production of ethanol from sugarcane fermentation. Both contaminants were quantified in industrial samples, and their presence was correlated to a decrease in ethanol concentration and accumulation of sugar. Then, laboratory mixed-cell fermentations were carried out to evaluate the effects of these presumed contaminants on the viability of Saccharomyces cerevisiae and the overall ethanol yield. The results showed that high residual sugar seemed the most significant factor arising from the presence of D. bruxellensis in the industrial process when compared to pure S. cerevisiae cultures. Moreover, when L. vini was added to S. cerevisiae cultures it did not appear to affect the yeast cells by any kind of antagonistic effect under stable fermentations. In addition, when L. vini was added to D. bruxellensis cultures, it showed signs of being able to stimulate the fermentative activity of the yeast cells in a way that led to an increase in the ethanol yield.

Intraspecific variations of Dekkera/Brettanomyces bruxellensis genome studied by capillary electrophoresis separation of the intron splice site profiles.

In enology, "Brett" character refers to the wine spoilage caused by the yeast Dekkera/Brettanomyces bruxellensis and its production of volatile phenolic off-flavours. However, the spoilage potential of this yeast is strain-dependent. Therefore, a rapid and reliable recognition at the strain level is a key point to avoid serious economic losses. The present work provides an operative tool to assess the genetic intraspecific variation in this species through the use of introns as molecular targets. Firstly, the available partial D./B. bruxellensis genome sequence was investigated in order to build primers annealing to introns 5' splice site sequence (ISS). This analysis allowed the detection of a non-random vocabulary flanking the site and, exploiting this feature, the creation of specific probes for strain discrimination. Secondly, the separation of the intron splice site PCR fragments was obtained throughout the set up of a capillary electrophoresis protocol, giving a 94% repeatability threshold in our experimental conditions. The comparison of results obtained with ISS-PCR/CE versus the ones performed by mtDNA RFLP revealed that the former protocol is more discriminating and allowed a reliable identification at strain level. Actually sixty D./B. bruxellensis isolates were recognised as unique strains, showing a level of similarity below 79% and confirming the high genetic polymorphism existing within the species. Two main clusters were grouped at similarity levels of about 46% and 47%, respectively, showing a poor correlation with the geographic area of isolation. Moreover, from the evolutionary point of view, the proposed technique could determine the frequency of the genome rearrangements that can occur in D./B. bruxellesis populations.

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.

Implications of Lactobacillus collinoides and Brettanomyces/Dekkera anomala in phenolic off-flavour defects of ciders.

Different Lactobacillus collinoides and Brettanomyces/Dekkera anomala cider strains were studied for their ability to produce volatile phenols in synthetic medium. All strains were able to produce 4-ethylcatechol (4-EC), 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) from caffeic, p-coumaric and ferulic acids, respectively. Interestingly, D. anomala and L. collinoides were also able to produce 4-EC, 4-EP and 4-EG in cider conditions. The quantities of ethylphenols produced by these two species were similar in both tested ciders. The impact of precursor quantities was studied and it showed that the addition of caffeic and p-coumaric acids in ciders allowed for higher 4-EC and 4-EP production by D. anomala and L. collinoides. In parallel, D. anomala and L. collinoides strains were isolated from a phenolic off-flavour defective bottled cider after ethylphenol production hence confirming the implication of these two species in this cider spoilage. Finally, detection thresholds of the main ethylphenols were determined in ciders by orthonasal and retronasal sampling. The 4-EC and 4-EP detection thresholds (close to 20-25mg/l and 1.5-2.0mg/l, respectively) were matrix dependant.

Detection and enumeration of Dekkera anomala in beer, cola, and cider using real-time PCR.

AIMS: In this article, a quantitative real-time PCR assay for detection and enumeration of the spoilage yeast Dekkera anomala in beer, cola, apple cider, and brewing wort is presented as an improvement upon existing detection methods, which are very time-consuming and not always accurate. METHODS AND RESULTS: Primers were designed to exclude other organisms common in these beverages, and the assay was linear over 6 log units of cell concentrations. The addition of large amounts of non-target yeast DNA did not affect the efficiency of this assay. A standard curve of known DNA was established by plotting the C(t) values obtained from the QPCR against the log of plate counts on yeast peptone dextrose medium and unknowns showed exceptional correlation when tested against this standard curve. The assay was found to detect D. anomala at levels of 10-14 CFU ml⁻¹ in either cola or beer and at levels of 9·4-25·0 CFU ml⁻¹ in apple cider. The assay was also used to follow the growth of D. anomala in brewing wort. CONCLUSIONS: The results indicate that real-time PCR is an effective tool for rapid, accurate detection and quantitation of D. anomala in beer, cola and apple cider. SIGNIFICANCE AND IMPACT OF THE STUDY: This method gives a faster and more efficient technique to screen beer, cola, and cider samples and reduce spoilage by D. anomala. Faster screening may allow for significant reduction in economic loss because of reduced spoilage.

beta-Glucanases as a tool for the control of wine spoilage yeasts.

In this study, the antimicrobial activity of a commercial beta-glucanase preparation against wine spoilage yeasts such as Cryptococcus albidus, Dekkera bruxellensis, Pichia membranifaciens, Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Zygosaccharomyces bisporus has been evaluated. Yeast species tested showed different sensitivities to the enzyme preparation. In vitro assays in laboratory medium (GPY) showed inhibition by the beta-glucanase preparation of D. bruxellensis and Z. bailii growth with IC(50) and MIC values approximately 3 to 4-fold greater than the recommended dose for improving wine filtration. Wine spoilage experiments showed antimicrobial action against D. bruxellensis and Z. bailii although with reduced effectiveness to that showed in laboratory medium. Under the conditions tested, the addition of beta-glucanase did not affect wine enological parameters. Our data suggest the potential use of beta-glucanases as antimicrobial agents in wine and indicate that the application of antimicrobial enzymes for yeast spoilage control deserves further investigation.

Identification and characterization of Dekkera bruxellensis, Candida pararugosa, and Pichia guilliermondii isolated from commercial red wines.

Yeast isolates from commercial red wines were characterized with regards to tolerances to molecular SO(2), ethanol, and temperature as well as synthesis of 4-ethyl-phenol/4-ethyl-guaiacol in grape juice or wine. Based on rDNA sequencing, nine of the 11 isolates belonged to Dekkera bruxellensis (B1a, B1b, B2a, E1, F1a, F3, I1a, N2, and P2) while the other two were Candida pararugosa (Q2) and Pichia guilliermondii (Q3). Strains B1b, Q2, and Q3 were much more resistant to molecular SO(2) in comparison to the other strains of Dekkera. These strains were inoculated (10(3)-10(4)cfu/ml) along with lower populations of Saccharomyces (<500 cfu/ml) into red grape juice and red wine incubated at two temperatures, 15 degrees C and 21 degrees C. Although Saccharomyces quickly dominated fermentations in grape juice, B1b and Q2 grew and eventually reached populations >10(5)cfu/ml. In wine, Q3 never entered logarithmic growth and quickly died in contrast to Q2 which survived >40 days after inoculation. B1b grew well in wine incubated at 21 degrees C while slower growth was observed at 15 degrees C. Neither Q2 nor Q3 produced 4-ethyl-phenol or 4-ethyl-guaiacol, unlike B1b. However, lower concentrations of volatile phenols were present in wine incubated at 15 degrees C compared to 21 degrees C.

Complex nature of the genome in a wine spoilage yeast, Dekkera bruxellensis.

When the genome organizations of 30 native isolates belonging to a wine spoilage yeast, Dekkera (Brettanomyces) bruxellensis, a distant relative of Saccharomyces cerevisiae, were examined, the numbers of chromosomes varied drastically, from 4 to at least 9. When single gene probes were used in Southern analysis, the corresponding genes usually mapped to at least two chromosomal bands, excluding a simple haploid organization of the genome. When different loci were sequenced, in most cases, several different haplotypes were obtained for each single isolate, and they belonged to two subtypes. Phylogenetic reconstruction using haplotypes revealed that the sequences from different isolates belonging to one subtype were more similar to each other than to the sequences belonging to the other subtype within the isolate. Reanalysis of the genome sequence also confirmed that partially sequenced strain Y879 is not a simple haploid and that its genome contains approximately 1% polymorphic sites. The present situation could be explained by (i) a hybridization event where two similar but different genomes have recently fused together or (ii) an event where the diploid progenitor of all analyzed strains lost a regular sexual cycle, and the genome started to accumulate mutations.