Yeast Brettanomyces Bruxellensis Research Articles

No, <i>Brettanomyces bruxellensis</i> is not responsible for all woes!

In wines, rice cake and sausage skin aromas are generally associated with the alteration of mousy off-flavours and constitute an increasingly prevalent deviation in some wines. In the present study microorganisms present in 25 altered wines were isolated. The spoilage yeast Brettanomyces bruxellensis, and lactic acid bacteria of the species Oenococcus oeni and Lentilactobacillus hilgardii were identified, and their ability to produce the N-heterocycles responsible for the mousy off-aroma was deterimined. B. bruxellensis was found in a minority of the wines altered by the mousiness.

Effect of Alcoholic Extract of Syntrichia intermedia on some Yeasts Isolated from Mobile Phones and the User’s Finger

Syntrichia intermedia was isolated from the Amadiya region in northern Iraq for the first time, as it was used against the yeast Brettanomyces bruxellensis, Candida krusei and the yeast Meyerozyma caribbica isolated fore the first one and registered as a new strain in the Gen Bank of the National Center for Biotechnology Information (NCBI) with identification number LC723825.1. Three methods were used to determine the effect of the alcoholic extract of S. intermedia at the following concentrations, 1, 2, 4, 6, 8, and 10 mg/ml against yeast isolates, When using the first method-turbidity measurement the concentrations were shown starting from 1 mg/ml an inhibitory effect respectively up to the highest concentration of 10 mg/ml against yeast B. bruxellensis, C. krusei and M. caribbica. and When using the second method-disc diffusion the following concentration showed the highest inhibition percentage the concentration of 2 mg / ml with a diameter of (12.50 mm) against the yeast B. bruxellensis. and The concentrations 1 with a diameter of (11.0 mm) against the yeast C. krusei and The concentrations 4 mg / ml with a diameter of (11.50 mm) Compared with the antibiotic Nystatin which showed a higher percentage of inhibition than the alcoholic extract, While the third method-diffusion by wells was used and the concentration of 4 mg / ml showed the highest percentage of inhibition with a diameter of (23.0 mm) for yeast B. bruxellensis and a concentration of 6 mg / ml showed the highest percentage of inhibition with a diameter of (19.00 mm) against yeast C. krusei. and a concentration of 8 mg / ml showed the highest percentage of inhibition with a diameter of (15.67 mm) against yeast M. caribbica compared with the antibiotic Nystatin, which showed less inhibition than the alcoholic extract, so the yeast B. bruxellensis was more sensitive to the alcoholic extract, followed by the yeast M. caribbica. Then the yeast When using the method of diffusion by discs and digging, either by measuring the turbidity the yeast M. caribbica was the most sensitive to the alcoholic extract followed by C. krusei Then the yeast B. bruxellensis, so the turbidity measurement method is considered one of the best methods that can be used to measure the effect of the alcoholic extract because of measuring the turbidity using a spectrophotometer, followed by the diffusion method by wells, then the method of diffusion by discs.

Assessment of the impact of chitosan treatment on microorganisms in enology

As part of the move to reduce SO2 in wines, the empirical use of chitosan to eliminate the spoilage yeast Brettanomyces bruxellensis is becoming a frequent alternative. Chitosan treatment has an effect on a number of microbial species, at least temporarily, but there is wide variability in response within strains of each species. In addition, the effectiveness of chitosan also depends on the physical and chemical parameters of the wine. However, when chitosan treatment is effective, it appears to be long-lasting.

Role of p-Coumaric Acid and Micronutrients in Sulfur Dioxide Tolerance in Brettanomyces bruxellensis

Role of p-Coumaric Acid and Micronutrients in Sulfur Dioxide Tolerance in Brettanomyces bruxellensis

Grape stalks: From wastes to source of antioxidants and nutraceuticals

Wine production is one of the most significant agricultural activities worldwide. The winemaking process generates large amounts of by-products: grape marc, stalks, and exhausted grape marc. Until now, many studies have been focused on marc valorization, very few on stalks. The aim of this research was to deeply explore the potential of residual stalks in the wine industry from a circular economy perspective. Polyphenols were extracted from stalks with new sustainable technologies in the frame of “green chemistry” without the use of hazardous solvents. Extracts were analyzed for total phenolic compound content (TPC) and their antioxidant activity was determined. As polyphenols can have antimicrobial activity, the effect of the extracted polyphenols against wine-spoiling yeast Brettanomyces bruxellensis was determined. The percent reduction of the culture optical density, in the presence or absence of polyphenols, was compared to assess the antimicrobial activity of the samples. The results obtained underline the importance of winemaking by-products (stalks) and their eco-friendly valorization to obtain molecules for food, nutraceutical and cosmetic industries.

Effect of Brown Algae and Lichen Extracts on the SCOBY Microbiome and Kombucha Properties

Kombucha tea was made by the fermentation of SCOBY culture of green tea broth with the addition of Fucus vesiculosus algae extract, Cetraria islandica lichen extract and their mixture. Kombucha was also made without the herbal supplements as a control. After 11 days of fermentation, in addition to the yeast Brettanomyces bruxellensis and the bacteria Komagataeibacter rhaeticus and Komagataeibacter hansenii contained in all of the samples, the yeast Zygosaccharomyces bailii and bacteria Komagataeibacter cocois were detected in the samples with the herbal extracts. In all of the kombucha with herbal additives, the total fraction of yeast was decreased as compared to the control. The total content of polyphenols and the antioxidant activity of the beverages with and without the addition of herbal extracts were comparable. The kombucha made with the algae extract showed an increased content of sucrose and organic acids, while the fructose and glucose content in the samples with algae and the mixture of extracts were lower than in the other samples. The samples with the algae extract had the highest organoleptic indicators "aroma", "clarity" and "acidity", while the control samples had slightly higher indicators of "taste" and "aftertaste". The results of this study indicate the potential of algae and lichens as functional supplements for obtaining non-alcoholic fermented beverages with additional nutraceutical value.

Microbial Diversity and Interaction Specificity in Kombucha Tea Fermentations.

ABSTRACTDespite the popularity of kombucha tea, the distribution of different microbes across kombucha ferments and how those microbes interact within communities are not well characterized. Using metagenomics, comparative genomics, synthetic community experiments, and metabolomics, we determined the taxonomic, ecological, and functional diversity of 23 distinct kombuchas from across the United States. Shotgun metagenomic sequencing demonstrated that the bacterium Komagataeibacter rhaeticus and the yeast Brettanomyces bruxellensis were the most common microbes in the sampled kombucha communities. To determine the specificity of bacterium-yeast interactions, we experimentally quantified microbial interactions within kombucha biofilms by measuring densities of interacting species and biofilm production. In pairwise combinations of bacteria and yeast, B. bruxellensis and individual strains of Komagataeibacter spp. were sufficient to form kombucha fermentations with robust biofilms, but Zygosaccharomyces bisporus, another yeast found in kombucha, did not stimulate bacteria to produce biofilms. Profiling the spent media of both yeast species using nuclear magnetic resonance spectroscopy suggested that the enhanced ability of B. bruxellensis to ferment and produce key metabolites in sucrose-sweetened tea may explain why it stimulates biofilm formation. Comparative genomics demonstrated that Komagataeibacter spp. with >99% genomic similarity can still have dramatic differences in biofilm production, with strong producers yielding five times more biofilm than the weakest producers.IMPORTANCE Through an integration of metagenomic and experimental approaches, our work reveals the diversity and nature of interactions among key taxa in kombucha microbiomes through the construction of synthetic microbial pairs. Manipulation of these microbes in kombucha has the potential to shape both the fermentation qualities of kombucha and the production of biofilms and is valuable for kombucha beverage producers, biofilm engineers, and synthetic ecologists.

Wine Spoilage Control: Impact of Saccharomycin on Brettanomyces bruxellensis and Its Conjugated Effect with Sulfur Dioxide.

The yeast Brettanomyces bruxellensis is one of the most dangerous wine contaminants due to the production of phenolic off-flavors such as 4-ethylphenol. This microbial hazard is regularly tackled by addition of sulfur dioxide (SO2). Nevertheless, B. bruxellensis is frequently found at low levels (ca 103 cells/mL) in finished wines. Besides, consumers health concerns regarding the use of sulfur dioxide encouraged the search for alternative biocontrol measures. Recently, we found that Saccharomyces cerevisiae secretes a natural biocide (saccharomycin) that inhibits the growth of different B. bruxellensis strains during alcoholic fermentation. Here we investigated the ability of S. cerevisiae CCMI 885 to prevent B. bruxellensis ISA 2211 growth and 4-ethylphenol production in synthetic and true grape must fermentations. Results showed that B. bruxellensis growth and 4-ethylphenol production was significantly inhibited in both media, although the effect was more pronounced in synthetic grape must. The natural biocide was added to a simulated wine inoculated with 5 × 102 cells/mL of B. bruxellensis, which led to loss of culturability and viability (100% dead cells at day-12). The conjugated effect of saccharomycin with SO2 was evaluated in simulated wines at 10, 12, 13 and 14% (v/v) ethanol. Results showed that B. bruxellensis proliferation in wines at 13 and 14% (v/v) ethanol was completely prevented by addition of 1.0 mg/mL of saccharomycin with 25 mg/L of SO2, thus allowing to significantly reduce the SO2 levels commonly used in wines (150–200 mg/L).

Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic activities have been described to date, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of S. cerevisiae. In this study, we identified seven Moco biosynthesis genes in the non-conventional yeast Ogataea parapolymorpha by SpyCas9-mediated mutational analysis and expressed them in S. cerevisiae. Functionality of the heterologously expressed Moco biosynthesis pathway in S. cerevisiae was assessed by co-expressing O. parapolymorpha nitrate-assimilation enzymes, including the Moco-dependent nitrate reductase. Following two-weeks of incubation, growth of the engineered S. cerevisiae strain was observed on nitrate as sole nitrogen source. Relative to the rationally engineered strain, the evolved derivatives showed increased copy numbers of the heterologous genes, increased levels of the encoded proteins and a 5-fold higher nitrate-reductase activity in cell extracts. Growth at nM molybdate concentrations was enabled by co-expression of a Chlamydomonas reinhardtii high-affinity molybdate transporter. In serial batch cultures on nitrate-containing medium, a non-engineered S. cerevisiae strain was rapidly outcompeted by the spoilage yeast Brettanomyces bruxellensis. In contrast, an engineered and evolved nitrate-assimilating S. cerevisiae strain persisted during 35 generations of co-cultivation. This result indicates that the ability of engineered strains to use nitrate may be applicable to improve competitiveness of baker's yeast in industrial processes upon contamination with spoilage yeasts.

<em>Brettanomyces bruxellensis</em> biofilms: a mode of life to withstand environmental stresses?

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.

Fermentation profiles of the yeast Brettanomyces bruxellensis in d‐xylose and l‐arabinose aiming its application as a second‐generation ethanol producer

The yeast Brettanomyces bruxellensis is able to ferment the main sugars used in first-generation ethanol production. However, its employment in this industry is prohibitive because the ethanol productivity reached is significantly lower than the observed for Saccharomyces cerevisiae. On the other hand, a possible application of B. bruxellensis in the second-generation ethanol production has been suggested because this yeast is also able to use d-xylose and l-arabinose, the major pentoses released from lignocellulosic material. Although the latter application seems to be reasonable, it has been poorly explored. Therefore, we aimed to evaluate whether or not different industrial strains of B. bruxellensis are able to ferment d-xylose and l-arabinose, both in aerobiosis and oxygen-limited conditions. Three out of nine tested strains were able to assimilate those sugars. When in aerobiosis, B. bruxellensis cells exclusively used them to support biomass formation, and no ethanol was produced. Moreover, whereas l-arabinose was not consumed under oxygen limitation, d-xylose was only slightly used, which resulted in low ethanol yield and productivity. In conclusion, our results showed that d-xylose and l-arabinose are not efficiently converted to ethanol by B. bruxellensis, most likely due to a redox imbalance in the assimilatory pathways of these sugars. Therefore, despite presenting other industrially relevant traits, the employment of B. bruxellensis in second-generation ethanol production depends on the development of genetic engineering strategies to overcome this metabolic bottleneck.

Carbohydrate composition of red wines during early aging and incidence on spoilage by Brettanomyces bruxellensis

Wine is generally considered as hostile medium in which spoilage microbes have to manage with many abiotic factors among which low nutrient content. Wines elaborated in 8 wineries were sampled during the first summer of aging over two consecutive vintages, and analysed for carbohydrate composition. This revealed the systematic presence of many carbohydrates including those useful for the spoilage yeast Brettanomyces bruxellensis. However, during the first summer of aging, the changes in wine carbohydrate composition were low and it was difficult to assess how much carbohydrate composition contributed to wine spoilage by B. bruxellensis. Subsequent laboratory experiments in inoculated wines showed that the sugars preferentially consumed in wine by the spoilage yeast are d-glucose, d-fructose, and trehalose, whatever the yeast strain considered. The addition of these sugars to red wines accelerates the yeast growth and the volatile phenols formation. Although probably not the only promoting factor, the presence of high amounts of metabolisable sugars thus really increases the risk of “brett” spoilage.

New advances on the Brettanomyces bruxellensis biofilm mode of life

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.

Real-time PCR assays for the quantification of native yeast DNA in grape berry and fermentation extracts

Native yeasts comprise part of the microbial community in grape vineyards and play roles in alcoholic fermentation and wine quality. Monitoring populations of native yeast in vineyards, during fermentation and after bottling will provide viticulturalists and oenologists with information needed to help control spoilage and to enhance desirable wine properties. This is especially crucial for low-intervention winemaking, in which fermentation is driven by native rather than starter microbes. In this study, we report real-time polymerase chain reaction (qPCR) assays for rapid quantification of seven grape yeast species or species combinations that occur in vineyards of Washington State and throughout the world. The assays targeted Candida californica, Curvibasidium pallidicorallinum, Metschnikowia spp., Meyerozyma caribbica/Me. guilliermondii, and Saccharomyces cerevisiae/S. bayanus. We also developed assays for the spoilage yeast Brettanomyces bruxellensis, and the yeast-like fungus Aureobasidium pullulans. Primers were designed for sequences in the internal transcribed spacer (ITS) and large ribosome subunit (LSU) gene. Known populations of yeast cells, added to fermentation extract, were significantly correlated to amounts of purified DNA in picograms (pg) for most of the yeasts; exceptions were A. pullulans and Cu. pallidicorallinum. The utility of the Metschnikowia, Meyerozyma and Saccharomyces assays was further validated by good correlations (R2 = 0.75–0.83) between the number of target sequences and pg of DNA from qPCR for selected vineyard and fermentation samples. Overall, the assays will aid in species identification and monitoring of specific yeasts from cultures, vineyards and fermentation samples.Topics: Food Microbiology, Microbiological Method.

Identification and characterisation of two high-affinity glucose transporters from the spoilage yeast Brettanomyces bruxellensis.

ABSTRACTThe yeast Brettanomyces bruxellensis (syn. Dekkera bruxellensis) is an emerging and undesirable contaminant in industrial low-sugar ethanol fermentations that employ the yeast Saccharomyces cerevisiae. High-affinity glucose import in B. bruxellensis has been proposed to be the mechanism by which this yeast can outcompete S. cerevisiae. The present study describes the characterization of two B. bruxellensis genes (BHT1 and BHT3) believed to encode putative high-affinity glucose transporters. In vitro-generated transcripts of both genes as well as the S. cerevisiae HXT7 high-affinity glucose transporter were injected into Xenopus laevis oocytes and subsequent glucose uptake rates were assayed using 14C-labelled glucose. At 0.1 mM glucose, Bht1p was shown to transport glucose five times faster than Hxt7p. pH affected the rate of glucose transport by Bht1p and Bht3p, indicating an active glucose transport mechanism that involves proton symport. These results suggest a possible role for BHT1 and BHT3 in the competitive ability of B. bruxellensis.

Novel antimicrobial peptides produced by Candida intermedia LAMAP1790 active against the wine-spoilage yeast Brettanomyces bruxellensis.

Brettanomyces bruxellensis negatively impacts on the sensorial quality of wine by producing phenolic compounds associated with unpleasant odors. Thus, the control of this spoilage yeast is a critical factor during the winemaking process. A recent approach used to biocontrol undesired microorganisms is the use of yeast released antimicrobial peptides (AMPs), but this strategy has been poorly applied to wine-related microorganisms. The aim of this study was to evaluate the antifungal capacity of Candida intermedia LAMAP1790 against wine-spoilage strains of B. bruxellensis and fermentative strains of Saccharomyces cerevisiae, and also to determine the chemical nature of the compound. The exposure of strains to the supernatant of C. intermedia saturated cultures showed antifungal activity against B. bruxellensis, without affecting the growth of S. cerevisiae. By fractionation and concentration of C. intermedia supernatants, it was determined that the antifungal activity was related to the presence of heat-labile peptides with molecular masses under 5kDa. To our knowledge, this is the first report of AMPs secreted by C. intermedia that control B. bruxellensis. This could lead to the development of new biocontrol strategies against this wine-spoilage yeast.

Influence of Oenococcus oeni and Brettanomyces bruxellensis on Wine Microbial Taxonomic and Functional Potential Profiles

The interactions between lactic acid bacteria, yeast, and other wine microbiota impact on wine quality. Some strains of lactic acid bacteria Oenococcus oeni possess cinnamoyl esterase activity that releases hydroxycinnamic acids (HCAs) that can subsequently be processed by some strains of the spoilage yeast Brettanomyces bruxellensis and lead to off-flavor compounds. Using metagenomic analyses, this study investigated the influence of O. oeni and B. bruxellensis on the taxonomic and functional potential profiles of the microbial community of Cabernet Sauvignon wine, particularly regarding flavor formation. Metagenomic datasets were generated from inoculations of three strains of B. bruxellensis, in combination with two O. oeni strains, with and without cinnamoyl esterase activity. Effect on the microbial profiles was found to depend on the O. oeni and B. bruxellensis strains being combined and on the abundance that the inoculants reach in the final wine, which depends on unidentified conditions (e.g., derived from microbial interactions). This study confirmed that the potential of B. bruxellensis to produce off-flavor compounds from HCAs is strain dependent. Interestingly, the samples without microbial inoculants also had this potential, suggesting that microbiota from the native grape could also influence the levels of HCAs. The metagenomic analyses complemented by experimental validation also found that the presence of B. bruxellensis did not interfere with the microbial functional potential to transform l-malic acid into l-lactic acid, which typically leads to a less acidic flavor. We show that metagenomic approaches can help uncover the complex wine microbial community traits, such as flavor, impacted by the simultaneous presence of O. oeni and B. bruxellensis.

The carbon consumption pattern of the spoilage yeast Brettanomyces bruxellensis in synthetic wine-like medium

The wine matrix contains limited carbon compounds to sustain microbial life. Brettanomyces bruxellensis is one of very few yeast species that has adapted to this environment. Indeed, the presence of growth-inhibiting compounds and conditions do not prevent its proliferation. Literature regarding the nutritional requirements of this yeast is surprisingly poor, given the observation that B. bruxellensis produces biomass with apparently less nutrients than other yeasts. In this study, various carbon sources were screened in a synthetic wine medium, under anaerobic and semi-aerobic growth conditions, in order to determine which compounds B. bruxellensis assimilates. Slight differences were observed between strains but overall, B. bruxellensis produced biomass from limited nutrients consumed in a specific order regardless of the oxygen conditions. Upon initial consumption of the simple sugars, B. bruxellensis was able to remain viable, by concurrently utilising ethanol (only in the presence of oxygen) and malic acid. Although initially beneficial, oxygen was found detrimental in the long term. Formation of volatile phenols occurred during the consumption of the sugars but not as a mechanism to help correct the redox imbalance. The study confirms that B. bruxellensis is able to survive using limited amount of nutrients, making this yeast a challenge for winemakers.

Candida pyralidae killer toxin disrupts the cell wall of Brettanomyces bruxellensis in red grape juice.

The control of the wine spoilage yeast Brettanomyces bruxellensis using biological methods such as killer toxins (instead of the traditional chemical methods, e.g. SO2 ) has been the focus of several studies within the last decade. Our previous research demonstrated that the killer toxins CpKT1 and CpKT2 isolated from the wine yeast Candida pyralidae were active and stable under winemaking conditions. In this study, we report the possible mode of action of CpKT1 on B.bruxellensis cells in red grape juice. Brettanomyces bruxellensis cells were exposed to CpKT1 either directly or through co-inoculation with C. pyralidae. This exposure yielded a temporary or permanent decline of the spoilage yeast population depending on the initial cell concentration. Scanning electron microscopy revealed cell surface abrasion while propidium iodide viability staining showed that CpKT1 caused plasma membrane damage on B.bruxellensis cells. Our data show that the exposure to CpKT1 resulted in increased levels of β-glucan, suggesting a compensatory response of the sensitive cells. The toxin CpKT1 causes cell membrane and cell wall damage in B. bruxellensis. Candida pyralidae shows potential to be used as a biocontrol agent against B. bruxellensis in grape juice/wine.

Comparative morphological characteristics of three Brettanomyces bruxellensis wine strains in the presence/absence of sulfur dioxide

The red wine spoilage yeast Brettanomyces bruxellensis has been the subject of numerous investigations. Some of these studies focused on spoilage mechanisms, sulfur dioxide tolerance and nutrient requirements. Pseudomycelium formation, although a striking feature of this species, has however been poorly investigated. Furthermore, literature regarding the induction mechanism of pseudomycelium formation in this yeast is limited and lacks clarity, as results published are contradictory. This study elucidates this phenomenon among strains from geographically different areas. Potential environmental cues were investigated, to attain a better understanding of this mechanism and its role as a survival strategy. SO2 was previously reported to induce this morphological change however results obtained in this study did not support this. Nevertheless, the results obtained using scanning and transmission electron microscopy illustrate, for the first time in this yeast, deformity to the cell membrane and alterations to the fibrillar layers in SO2 treated cells. In addition, the SO2 exposed cultures displayed cell size variations, with cells displaying a decrease in length as well as delayed growth, with a prolonged lag phase. Fluorescence microscopy demonstrated a decrease in metabolic activity and the appearance of inclusion body-like structures in the cells following exposure to SO2.