Survival and metabolism of hydroxycinnamic acids by Dekkera bruxellensis in monovarietal wines.

Volatile phenols in wines are responsible for unpleasant aromas, which negatively affect the quality of the wine. These compounds are produced from the metabolism of hydroxycinnamic acids, mainly by the yeasts Brettanomyces/Dekkera. Relevant data, potentially useful to support decisions on how to manage the risk of contamination of wines by Brettanomyces/Dekkera, according to the grape varieties used in the vinification, is important to the wine industry. Therefore, the aim of this work was to evaluate the survival and the metabolism of hydroxycinnamic acids by Dekkera bruxellensis in monovarietal wines. Yeast growth and survival were monitored in fifteen wines, five from each of the grape varieties Touriga Nacional, Cabernet Sauvignon and Syrah, inoculated with a strain of D. bruxellensis. Yeast culturable populations of 107 CFU mL-1 were reduced to undetectable numbers in 24 h in all wines. Plate counts of 104-106 CFU mL-1 were, however, detected after 48 h in most of Touriga Nacional and Cabernet Sauvignon wines and later in Syrah. Viability measurement by flow cytometry showed that a significant part of the populations was in a viable but non-culturable state (VBNC). The time required for the recovery of the culturable state was dependent on the wine, being longer on Syrah wines. Besides the production of ethylphenols, the metabolism of hydroxycinnamic acids by VBNC cells led to the accumulation of vinylphenols at relatively high levels, independently of the grape variety. The flow cytometry methodology showed a higher survival capacity of D. bruxellensis in Touriga Nacional wines, which corroborates with the higher amounts of volatile phenols found on this variety.

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.

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.

Brettanomyces bruxellensis phenotypic diversity, tolerance to wine stress and wine spoilage ability.

Brettanomyces bruxellensis is a yeast species found in many fermented matrices. A high level of genetic diversity prevails in this species and was recently connected with tolerance to sulfur dioxide, the main preservative used in wine. We therefore examine other phenotypes that may modulate the ability of the species to spoil wine, in a selection of representative strains. The species shows a fairly high homogeneity with respect to the carbohydrates that can support growth, but more diverse behaviors regarding tolerance to low pH or ethanol. Thought no clear link can be drawn with genotype, some strains appear more tolerant than the others, mainly in the AWRI1499 like genetic group. Volatile phenol production is ubiquitous within the species, independent from yeast growth profile and not affected by the nature of the growth substrate. The specific production. n rate of volatile phenol production raises in case of increased aeration. It is little affected by pH decrease until 3.0 or by ethanol concentration increase up to 12% vol, but it decreased in case of increased constraint (pH < 3.0, Ethanol ≥14% vol) or combination of constraints. All the strain studied have thus the ability to spoil wine but some outstanding dangerous strains can even spoil the wine with high level of constrainst.

Brettanomyces bruxellensis wine isolates show high geographical dispersal and long persistence in cellars.

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.

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 5 kDa. 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.

The raise of Brettanomyces yeast species for beer production.

The adequate application of Brettanomyces species could raise a potential opportunity for the beer industry, generating new products and optimizing production processes. Several valuable properties like high ethanol yield, tolerance to low pH and production of unique flavors have brought this yeast species into the spotlight. Aroma and flavor production of Brettanomyces in beer is currently under discussion, and it can be adjusted if the mechanism insights are understood. This review summarizes the recent findings in physiological, genetic and biochemical traits related to the application of Brettanomyces species for brewing.

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.

Spoilage potential of brettanomyces bruxellensis strains isolated from Italian wines.

Brettanomyces bruxellensis is an important wine spoilage agent. In this study a population of Brettanomyces strains isolated from Italian wines was thoroughly investigated to evaluate adaptability to wine conditions and spoilage potential. The presumptive isolates of Brettanomyces were identified at species level with 26S rRNA gene sequencing and species-specific PCR, and subsequently subjected to analysis of intra-species variability through the study of intron splice sites (ISS-PCR). Although, some strains were tracked in wines from different regions, extensive genetic biodiversity was observed within the B. bruxellensis population investigated. All strains were evaluated for their growth ability in the presence of ethanol, high sugar content, low pH, different temperatures and sulphur dioxide, using optical density and flow cytometry measurement. The ability of yeasts to produce ethyl phenols in red wines with different chemical compositions was evaluated by means of high performance liquid chromatography with electrochemical detection (HPLC-ECD). The results highlighted wide variability in B. bruxellensis in response to wine limiting factors and in terms of the accumulation of ethyl phenols. As regards this last aspect, the differences found among strains were closely related to chemical composition of wine and strain resistance to environmental stress factors, making a priori evaluation of risk of wine alteration quite difficult. These results suggest that strategies for the control of Brettanomyces should be tailored on the basis of strain distribution and wine characteristics.

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.

Interactive effects between total SO2 , ethanol and storage temperature against Brettanomyces bruxellensis.

Although Brettanomyces bruxellensis continues to be a problem during red winemaking due to formation of off-odours and flavours, few interactions between intrinsic and extrinsic conditions that would limit spoilage have been identified. Using a commercially prepared Merlot wine, a 3 × 2 × 2 complete factorial design was implemented with total SO2 (0, 60 or 100 mg l-1 ), ethanol (13% or 14·5% v v-1 ) and storage temperature (15° or 18°C) as variables. Populations of two strains of B. bruxellensis isolated from Washington wines (I1a and F3) were monitored for 100 days before concentrations of 4-ethylphenol, 4-ethylguaiacol and volatile acidity were measured. In wines with 13% v v-1 ethanol and stored at 15°C, addition of 100 mg l-1 total SO2 resulted in much longer lag phases (>40 days) compared with wines without sulphites. At 14·5% v v-1 ethanol, culturability did not recover from wines with 100 mg l-1 total SO2 regardless of the storage temperature (15° or 18°C). A few significant interactions were noted between these parameters which also affected synthesis of metabolites. Thus, SO2 , ethanol concentration and storage temperature should be together used as means to reduce infections by B. bruxellensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for utilizing SO2 along with the ethanol and storage temperature was studied to inhibit the spoilage yeast, Brettanomyces bruxellensis, during cellar ageing of red wines. This report is the first to identify the existence of interactions between these parameters that affect growth and/or metabolism of the yeast (i.e., synthesis of 4-ethylphenol, 4-ethylguaiacol and volatile acidity). Based on current and past findings, recommendations are presented related to the use of potential antimicrobial synergies between SO2 , ethanol concentration and storage temperatures.

Brettanomyces bruxellensis yeasts: impact on wine and winemaking.

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.

High pressure inactivation of Brettanomyces bruxellensis in red wine.

Brettanomyces bruxellensis ("Brett") is a major spoilage concern for the wine industry worldwide, leading to undesirable sensory properties. Sulphur dioxide, is currently the preferred method for wine preservation. However, due to its negative effects on consumers, the use of new alternative non-thermal technologies are increasingly being investigated. The aim of this study was to determine and model the effect of high pressure processing (HPP) conditions and yeast strain on the inactivation of "Brett" in Cabernet Sauvignon wine. Processing at 200 MPa for 3 min resulted in 5.8 log reductions. However higher pressure is recommended to achieve high throughput in the wine industry, for example >6.0 log reductions were achieved after 400 MPa for 5 s. The inactivation of B. bruxellensis is pressure and time dependent, with increased treatment time and pressure leading to increased yeast inactivation. It was also found that yeast strain had a significant effect on HPP inactivation, with AWRI 1499 being the most resistant strain. The Weibull model successfully described the HPP "Brett" inactivation. HPP is a viable alternative for the inactivation of B. bruxellensis in wine, with the potential to reduce the industry's reliance on sulphur dioxide.

Impedance technology reduces the enumeration time of Brettanomyces yeast during beer fermentation.

Brettanomyces yeasts are increasingly being used to produce lambic style beers and craft beers with unique flavors. Currently, the industry monitors Brettanomyces bruxellensis using time consuming plate counting. B. bruxellensis is a fastidious slow growing organism, requiring five days of incubation at 30°C for visible growth on agar plates. Thus, a need exists to develop a quicker, feasible method to enumerate this yeast. The aim of this study was therefore to determine the feasibility of using the 'direct' and 'indirect' impedance methods for the enumeration of B. bruxellensis in beer and to monitor the growth of the yeast during fermentation. The impedance methods were able to decrease the incubation time of beer samples containing Brettanomyces from 120 h down to 2 and 84 h for samples containing 107 and 103 cfu/mL, respectively. The 'indirect' method was more successful than the 'direct' method, presenting a smaller error and wider detection range. Overall, the 'indirect' impedance method is a viable alternative to plate counting for the enumeration of yeasts in the brewing industry because it decreases preparation and incubation times, thereby increasing throughput and decreasing the chance of contamination.

Enhancing the Feasibility of Microcystis aeruginosa as a Feedstock for Bioethanol Production under the Influence of Various Factors.

Microcystis aeruginosa, a freshwater microalga, is capable of producing and accumulating different types of sugars in its biomass which make it a good feedstock for bioethanol production. Present study aims to investigate the effect of different factors increasing growth rate and carbohydrates productivity of M. aeruginosa. MF media (modified BG11 media) and additional ingredients such as aminolevulinic acid (2 mM), lysine (2.28 mM), alanine (1 mM), and Naphthalene acetic acid (1 mM) as cytokine promoted M. aeruginosa growth and sugar contents. Salmonella showed growth-assisting effect on M. aeruginosa. Enhanced growth rate and carbohydrates contents were observed in M. aeruginosa culture grown at 25°C under red LED light of 90 µmolm(-2)s(-1) intensity. More greenish and carbohydrates rich M. aeruginosa biomass was prepared (final OD660 nm = 2.21 and sugar contents 10.39 mM/mL) as compared to control (maximum OD660 nm = 1.4 and sugar contents 3 mM/mL). The final algae biomass was converted to algae juice through a specific pretreatment method. The resulted algae Juice was used as a substrate in fermentation process. Highest yield of bioethanol (50 mM/mL) was detected when Brettanomyces custersainus, Saccharomyces cerevisiae, and Pichia stipitis were used in combinations for fermentation process as compared to their individual fermentation. The results indicated the influence of different factors on the growth rate and carbohydrates productivity of M. aeruginosa and its feasibility as a feedstock for fermentative ethanol production.

Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of VBNC behaviour using a transcriptomic approach.

The spoilage potential of Brettanomyces bruxellensis in wine is strongly connected with the aptitude of this yeast to enter in a Viable But Non Culturable (VBNC) state when exposed to the harsh wine conditions. In this work, we characterized the VBNC behaviour of seven strains of B. bruxellensis representing a regional intraspecific biodiversity, reporting conclusive evidence for the assessment of VBNC as a strain-dependent character. The VBNC behaviour was monitored by fluorescein diacetate staining/flow cytometry for eleven days after addition of 0.4, 0.6, 0.8, 1 and 1.2 mg/L of molecular SO2 (entrance in the VBNC state) and after SO2 removal (exit from the VBNC state). Furthermore, one representative strain was selected and RNA-seq analysis performed after exposure to 1.2 mg/L SO2 and during the recovery phase. 30 and 1634 genes were identified as differentially expressed following VBNC entrance and 'resuscitation', respectively. The results reported strongly suggested that the entrance in the SO2-induced VBNC state in B. bruxellensis is associated with both, sulfite toxicity and oxidative stress response, confirming the crucial role of genes/proteins involved in redox cell homeostasis. Among the genes induced during recovery, the expression of genes involved in carbohydrate metabolism and encoding heat shock proteins, as well as enriched categories including amino acid transport and transporter activity was observed. The evidences of a general repression of genes involved in DNA replication suggest the occurrence of a true resuscitation of cell rather than a simple regrowth.

Comparison of the behaviour of Brettanomyces bruxellensis strain LAMAP L2480 growing in authentic and synthetic wines.

Brettanomyces bruxellensis is the main microorganism responsible for the production of off-flavours in wine. Studies have been carried out in synthetic cultures using p-coumaric acid for the production of vinyl and ethylphenols. The results obtained have been extrapolated to authentic wine, but there is no evidence that this correlation will be correct. We studied the behaviour of B. bruxellensis native strain LAMAP L2480 in authentic wine and in a synthetic medium with a chemical composition similar to the authentic wine used in this study (basal synthetic wine + pH, ethanol and hydroxycinnamic acid concentrations of commercial wine). In some assays, B. bruxellensis has been studied using media containing 100 mg L(-1) p-coumaric acid, so we also used the same concentration added to the authentic and synthetic wines. The microorganism showed better growth in authentic wine, regardless of the presence of p-coumaric acid. In the case of synthetic wine, the addition of p-coumaric acid caused a delay in yeast growth and an increase in the production of volatile phenols. The coumarate decarboxylase activity did not show any difference regardless of the media and the presence of p-coumaric acid. Vinylphenol reductase showed higher activity when a higher concentration of p-coumaric acid was added in synthetic wine, but no change was observed in authentic wine.

Impact of available nitrogen and sugar concentration in musts on alcoholic fermentation and subsequent wine spoilage by Brettanomyces bruxellensis.

The level of yeast assimilable nitrogen (YAN) supplementation required for Saccharomyces cerevisiae to complete fermentation of high sugar musts in addition to the impact of non-metabolized nitrogen on post-alcoholic spoilage by Brettanomyces bruxellensis was studied. A 2 × 3 factorial design was employed using a synthetic grape juice medium with YAN (150 or 250 mg N/L) and equal proportions of glucose/fructose (230, 250, or 270 g/L) as variables. S. cerevisiae ECA5 (low nitrogen requirement) or Uvaferm 228 (high nitrogen requirement) were inoculated at 10(5) cfu/mL while B. bruxellensis E1 or B2 were added once alcoholic fermentation ceased. Regardless of YAN concentration, musts that contained 230 or 250 g/L glucose/fructose at either nitrogen level attained dryness (mean = 0.32 g/L fructose) while those containing 270 g/L generally did not (mean = 2.5 g/L fructose). Higher concentrations of YAN present in musts yielded wines with higher amounts of α-amino acids and ammonium but very little (≤ 6 mg N/L) was needed by B. bruxellensis to attain populations ≥ 10(7) cfu/mL. While adding nitrogen to high sugar musts does not necessarily ensure completion of alcoholic fermentation, residual YAN did not affect B. bruxellensis growth as much as ethanol concentration.

A Response Surface Methodology study on the role of factors affecting growth and volatile phenol production by Brettanomyces bruxellensis ISA 2211 in wine.

The present study was aimed at determining the effect of glucose, ethanol and sulphur dioxide on the growth and volatile phenol production by Brettanomyces bruxellensis in red wines using a response surface methodology approach. Sulphur dioxide proved to have a significant (p < 0.05) negative linear and quadratic effect on growth and 4-ethylphenol production. Concentrations of sulphur dioxide higher than 20 mg L(-1), at pH 3.50, induced immediate loss of cell culturability under growth permissive levels of ethanol. Under high ethanol concentrations (14% v/v), the lag phase increased from 3 to 10 days, growth being fully arrested at 15% (v/v). Glucose up to 10 g L(-1) was found to be a significant factor (quadratic level) in biomass increase under low ethanol (<12.5% v/v) and low sulphite concentrations. However, when cells were inactivated by sulphur dioxide and ethanol, glucose (up to 10 g L(-1)) did not prevent cell death. Production of more than 50 µg L(-1) day(-1) of 4-ethylphenol was only observed in the presence of high numbers (10(6) CFU mL(-1)) of culturable cells, being stimulated by increasing glucose concentrations.

Use of propidium monoazide for the enumeration of viable Brettanomyces bruxellensis in wine and beer by quantitative PCR.

Brettanomyces bruxellensis is a current problem in winemaking all over the world, and the question if B. bruxellensis has a positive or negative impact on wine is one of the most controversial discussions in the world. The presence of live B. bruxellensis cells represents the risk of growth and an increase in cell numbers, which is related to the potential production of volatile phenols. In this work, the optimisation of a PMA-quantitative PCR (qPCR) method to enumerate only viable cells was carried out using the standard strain B. bruxellensis DSMZ 70726. The obtained detection limits were 0.83 log CFU/mL in red wine, 0.63 log CFU/mL in white wine and 0.23 log CFU/mL in beer. Moreover, the quantification was also performed by Reverse Transcription quantitative PCR (RT-qPCR), and the results showed a higher detection limit for all of the trials.