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.     

Ustilago maydis killer toxin as a new tool for the biocontrol of the wine spoilage yeast Brettanomyces bruxellensis

Brettanomyces bruxellensis is one of the most damaging species for wine quality, and tools for controlling its growth are limited. In this study, thirty-nine strains belonging to Saccharomyces cerevisiae and B. bruxellensis have been isolated from wineries, identified and then tested against a panel of thirty-nine killer yeasts. Here, for the first time, the killer activity of Ustilago maydis is proven to be effective against B. bruxellensis. Mixed cultures in winemaking conditions show that U. maydis CYC 1410 has the ability to inhibit B. bruxellensis, while S. cerevisiae is fully resistant to its killer activity, indicating that it could be used in wine fermentation to avoid the development of B. bruxellensis without undesirable effects on the fermentative yeast. The characterization of the dsRNAs isolated and purified from U. maydis CYC 1410 indicated that this strain produces a KP6-related toxin. Killer toxin extracts were active against B. bruxellensis at pH values between 3.0 and 4.5 and temperatures comprised between 15 °C and 25 °C, confirming their biocontrol activity in winemaking and wine aging conditions. Furthermore, small amounts (100 AU/ml) of killer toxin extracts from U. maydis significantly reduced the amount of 4-ethylphenol produced by B. bruxellensis, indicating that in addition to the growth inhibition observed for high killer toxin concentrations (ranging from 400 to 2000 AU/ml), small amounts of the toxin are able to reduce the production of volatile phenols responsible for the aroma defects in wines caused by B. bruxellensis.     

Glycerol formation during wine fermentation is mainly linked to Gpd1p and is only partially controlled by the HOG pathway

Glycerol 3-phosphate dehydrogenase, a key enzyme in the production of glycerol, is encoded by GPD1 and GPD2. The isoforms encoded by these genes have different functions, in osmoregulation and redox balance, respectively. We investigated the roles of GPD1, GPD2 and HOG1-the kinase involved in the response to osmotic stress-in glycerol production during wine fermentation. We found that the deletion of GPD2 in a wine yeast-derived strain did not affect growth or fermentation performance and reduced glycerol production by only 20%. In contrast, a gpd1delta mutant displayed a prolonged lag phase, and produced 40% less glycerol than the wild-type strain. The deletion of HOG1 resulted in a slight decrease in growth rate and a 20% decrease in glycerol production, indicating that the HOG pathway operates under wine fermentation conditions. However, the hog1delta mutant was not as severely affected as the gpd1delta mutant during the first few hours of fermentation, and continued to express GPD1 strongly. The hog1delta mutant was able to increase glycerol production in response to high sugar concentration (15-28% glucose), to almost the same extent as the wild-type, whereas this response was totally abolished in the gpd1delta mutant. These data show that Gpd1p plays a major role in glycerol formation, particularly during the first few hours of exposure to high sugar concentration, and that GPD2 is only of little significance in anaerobic fermentation by wine yeast. The results also demonstrate that the HOG pathway exerts only limited control over GPD1 expression and glycerol production during wine fermentation.     

Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production

The spoilage yeast Brettanomyces/Dekkera can persist throughout the winemaking process and has the potential to produce off-flavours that affect the sensory quality of wine. The main objective of this study was to select different strains of Brettanomyces bruxellensis isolated from red wines and to compare their volatile phenol production. From a collection of 63 strains, eight strains of B. bruxellensis were selected for volatile phenol production after the application of molecular techniques such as ISS–PCR, PCR–DGGE and REA–PFGE. All strains showed three large chromosomes of similar size with PFGE. However, unique restriction profiles of the chromosomes were visible after NotI digestion that clearly distinguished the strains. All strains were capable of producing large quantities of 4-ethylphenol and 4-ethylguaiacol from p-coumaric acid and ferulic acid, respectively in synthetic media. However, the diversity among strains for volatile phenol production differed between synthetic media and wine with regard to the maximum production levels of 4-ethylphenol and 4-ethylguaiacol. This study illustrated the diversity of B. bruxellensis strains that occur during winemaking.     

Preliminary evaluation of infrared spectroscopy for the differentiation of Brettanomyces bruxellensis strains isolated from red wines

The objective of this study was to evaluate different infrared spectroscopy methods in combination with chemometrics for the differentiation between Brettanomyces bruxellensis strains. These methods of discrimination were applied to intact yeast cells of B. bruxellensis strains and on wines spoiled by the same strains. Eleven wine isolates of B. bruxellensis were evaluated for volatile phenol production in red wine and their genetic diversity was determined by Restriction Endonuclease Analysis-Pulsed Field Gel Electrophoresis (REA-PFGE). Fourier transform mid-infrared (FTMIR) spectroscopy was used to obtain spectral fingerprints of the spoiled wines. Attenuated total reflectance (ATR) was used to obtain spectral fingerprints from the intact cells of the 11 B. bruxellensis strains. The groupings from the genetic fingerprints obtained with REA-PFGE were used as reference firstly for comparison with the groupings observed with the FTMIR spectral fingerprint of the wines and secondly for the FTIR-ATR spectral fingerprints from the whole cells. Results indicated that ATR-IR spectra obtained by scanning whole cells of B. bruxellensis could be useful for rapid strain typing in comparison or complementary to molecular techniques and FTMIR spectra from wines provide a useful resource for the discrimination between B. bruxellensis contaminated wines.     

Positioning of cell growth and division after osmotic stress requires a map kinase pathway

The yeast Saccharomyces cerevisiae has a genetic program for selecting and assembling a bud site on the cell cortex. Yeast cells confine their growth to the emerging bud, a process directed by cortical patches of actin filaments within the bud. We have investigated how cells regulate budding in response to osmotic stress, focusing on the role of the high osmolarity glycerol response (HOG) pathway in mediating this regulation. An increase in external osmolarity induces a growth arrest in which actin filaments are lost from the bud. This is followed by a recovery phase in which actin filaments return to their original locations and growth of the original bud resumes. After recovery from osmotic stress, haploid cells retain an axial pattern of bud site selection while diploids change their bipolar budding pattern to an increased bias for forming a bud on the opposite side of the cell from the previous bud site. Mutants lacking the mitogen-activated protein (MAP) kinase encoded by HOG1 or the MAP kinase kinase encoded by PBS2 (previously HOG4) show a similar growth arrest after osmotic stress. However, in the recovery phase, the mutant cells (a) do not restart growth of the original bud but rather start a new bud, (b) fail to restore actin filaments to the original bud but move them to the new one, and (c) show a more random budding pattern. These defects are elicited by an increase in osmolarity and not by other environmental stresses (e.g., heat shock or change in carbon source) that also cause a temporary growth arrest and shift in actin distribution. Thus, the HOG pathway is required for repositioning of the actin cytoskeleton and the normal spatial patterns of cell growth after recovery from osmotic stress.     

Influence of Dekkera bruxellensis on the contents of anthocyanins,organic acids and volatile phenols of Dão red wine

The anthocyanin, organic acid and volatile phenol compositions of red wine obtained from Touriga Nacional grapes growing in the Dão region (Portugal) were determined by HPLC/DAD, HPLC/UV and GC/FID, respectively. By these means, nine anthocyanic compounds (malvidin-3,5-O-diglucoside, cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside, delphinidin, cyanidin, pelargonidin and malvidin), six organic acids (ketoglutaric, tartaric, malic, quinic, lactic and shikimic acids) and two volatile phenols (4-ethylguaiacol and 4-ethylphenol) were identified and quantified. Malvidin-3-O-glucoside, the pair lactic plus shikimic acids and 4-ethylguaiacol were the main anthocyanin, organic acids and volatile phenol, respectively. The effects of nine different Dekkera bruxellensis strains on these chemical parameters were also evaluated. The results obtained indicate that some strains of D. bruxellensis yeast are able to cause deterioration of red wine from the Dão region during its maturation by the production of volatile phenols, namely 4-ethylphenol.     

Pulsed electric field treatment of red wine: Inactivation of Brettanomyces and potential hazard caused by metal ion dissolution

Pulsed electric field (PEF) technology is an attractive alternative method of wine preservation by inactivating Brettanomyces bruxellensis, a major spoilage concern affecting wines worldwide. Currently, wine preservation using SO₂ can have negative effects on consumers including headaches and allergic reactions. Therefore, the objectives of this study were to investigate the effect of PEF processing conditions, B. bruxellensis yeast strain and alcohol concentration on B. bruxellensis inactivation in red wine, as well as whether PEF treatment could have a harmful effect on wine through the release of metal ions. Electric field intensity was found to have a greater impact on inactivation than specific energy, with 31, 40 and 50 kV/cm treatments resulting in B. bruxellensis D values of 181.8, 36.1 and 13.0 μs, respectively. At 50 kV/cm, a temperature rise of almost 10 °C, doubled inactivation to 3.0 log reductions (cfu/mL). Yeast strain and alcohol concentration were also shown to influence inactivation, even though cell size comparisons of the three yeasts tested proved inconclusive. Overall, PEF treatment of wine was shown to be a possible preservation alternative for the wine industry. After PEF treatment, the wine produced remained safe for human consumption, with Fe, Cr and Ni ions contents well below dangerous levels.     

Oleic acid and ergosterol supplementation mitigates oxidative stress in wine strains of Saccharomyces cerevisiae

During fermentation of high-sugar-containing medium lacking lipid nutrients, wine yeasts undergo oxidative stress and oxidative damage to cell membranes and proteins. Considering that cell membranes are important stress sensors, and that under hypoxic conditions wine yeasts modulate cell membranes composition by incorporating lipids available in the growth medium, in the present work, the effects of lipid nutrition on wine yeast oxidative stress response were evaluated on two strains of Saccharomyces cerevisiae. Biomarkers of oxidative stress, oxidative damage and antioxidant response were evaluated together with viability and acetic acid production during fermentation of a synthetic must lacking lipid nutrients as compared to added oleic acid and ergosterol. The results show that the availability of lipid nutrients causes a significant reduction in the intracellular content of reactive oxygen species and in the oxidative damage to membranes and proteins, as indicated by flow cytometry of cells stained with dihydroethidum (DHE) and propidium iodide (PI) and by Western blot of protein carbonyls. Accordingly, lipid nutrients feeding results in the increase in cell viability and superoxide activity, and the reduction in trehalose accumulation, proteinase A activity and production of acetic acid. In summary, these results are compatible with the hypothesis that the supplementation of lipid nutrients mitigates oxidative stress and oxidative damage in wine strains of S. cerevisiae during growth under unfavourable conditions.     

Identification of yeasts isolated from wine-related environments and capable of producing 4-ethylphenol

The ability to produce 4-ethylphenol from the substrate p-coumaric acid in synthetic media was evaluated for several yeast species associated with wine production. Molar conversion rates as high as 90% were found by only Dekkera bruxellensis, D. anomala and by some unidentified strains isolated from wine-related environments. Other unidentified strains produced traces of 4-ethylphenol. All unidentified strains showed the same cultural characteristics as D. bruxellensis when grown on DBDM (Dekkera/Brettanomyces differential medium) agar. The determination of long-chain fatty acid compositions and the utilization of peptide nucleic acid (PNA) probes specific for D. bruxellensis showed that the unidentified strains did not belong to this species. Further identification, by restriction pattern generated from PCR-amplification of the 5.8S rRNA gene and the two internal transcribed spacers (ITS), assigned the unidentified strains to Candida cantarelli, C. wickerhamii, Debaryomyces hansenii, Kluyveromyces lactis and Pichia guilliermondii. However, only some strains of P. guilliermondii were capable of converting p-coumaric acid into 4-ethylphenol with efficiencies close to those observed in D. bruxellensis and D. anomala.     

Effect of lysozyme on "flor" velum yeasts in the biological aging of sherry wines

Biological aging is a key step in the production of Sherry wine classified as “fine”. During this stage, a film of yeast referred to as “flor velum” covers the surface of the wine and substantially alters its characteristics. Other microorganisms may coexist with flor yeasts, such as lactic acid bacteria and non-Saccharomyces yeasts, whose growth may be favored under certain conditions, causing organoleptic deviations and deterioration of the wine. To prevent the development of lactic bacteria, lysozyme usage has been introduced. Lysozyme is a hydrolytic enzyme with muramidase activity that can lyse gram-positive bacteria; its use in winemaking was approved by the OIV in 1997 (resolution OENO 10/97). Thus far, the use of lysozyme during the production of Sherry wines is not widespread despite its effectiveness in controlling lactic acid bacteria. However, there have been no studies on the effect of lysozyme on flor velum. The aim of this study was to determine the influence of lysozyme on yeast growth and the formation, development and metabolism of flor velum during the biological aging process of Sherry wine. The results indicate that lysozyme does not affect the flor yeast during the fermentative stage or biofilm stage. However, if yeast inoculation is carried out under submerged culture conditions during biological aging, low doses of lysozyme (≥12.5 g/hL) affect cell multiplication and the membrane hydrophobicity of the yeast, inhibiting their aggregation and flotation and the subsequent development of flor velum. Thus, the yeast inoculation protocol and the methodology used for the addition of lysozyme influence velum development, its metabolism and the wine characteristics.     

Brettanomyces susceptibility to antimicrobial agents used in winemaking: in vitro and practical approaches

Brettanomyces bruxellensis is a spoiling yeast responsible for developing off-odors in wine described as “Brett-character.” The objective of this study was to evaluate the antimicrobial activity of four enological compounds against Brettanomyces: potassium metabisulfite (PMB), chitosan, enological tannins and dimethyl dicarbonate. Minimal inhibitory concentrations and minimal biocidal concentrations of the antimicrobial agents were determined, and a comparative study between B. bruxellensis and Saccharomyces cerevisiae was performed under in vitro controlled conditions. All tested compounds showed inhibitory effect on the growth of Brettanomyces. Chitosan and the enological tannins showed selectivity against Brettanomyces, and PMB showed the highest efficacy in concentrations under the currently permitted limits for enological use; consequently, PMB was further evaluated in red wines naturally contaminated by Brettanomyces. Volatile phenol concentrations were determined after long-term storage of the wines treated with PMB. A direct correlation was demonstrated between the concentrations of 4-ethylphenol, 4-ethylguaiacol, 4-propylguaiacol and Brettanomyces populations in the studied wines, and these parameters correlated inversely with the concentrations of PMB employed. This is the first time that 4-propylguaiacol is shown to correlate with Brettanomyces population in wine. It is of enological significance that a concentration of 100 mg/L of total PMB efficiently prevented Brettanomyces growth in the aging red wines of our study and that volatile phenol concentrations were significantly (p < 0.05) higher in those poorly protected wines.     

Diversity in Spoilage Yeast Dekkera/Brettanomyces bruxellensis Isolated from French Red Wine. Assessment During Fermentation of Synthetic Wine Medium

Yeast Brettanomyces bruxellensis is a contaminant found worldwide and is responsible for red wine spoilage due to the development of animal and phenolic off‐odours. During this study, 24 Brettanomyces bruxellenis isolates were obtained from red wine samples from two French wineries and these were discriminated as 23 strains. Nine strains coming from 2 wineries and 4 vintages were cultivated in synthetic wine medium for 1500 hours and they gave nine different behaviours. Four main growth patterns (with different growth steps and durations) and three main different sugar consumption profiles were obtained. Glucose and fructose were not limiting substrates for all strains. The production level of 4‐ethylphenol was found to vary from strain to strain (from 0.350 to 2.773 mg L^?1) and was independent of the biomass concentration. Some strains presented a coupled‐to‐growth production of volatile phenols, others did not. This study showed that different strains of Brettanomyces bruxellensis behaved differently, one from another, under the given conditions taking into consideration several aspects. The results thus demonstrate a large intraspecific diversity.     

Production of sensory compounds by means of the yeast Dekkera bruxellensis in different nitrogen sources with the prospect of producing cachaça

The distilled spirit made from sugar cane juice, also known as cachaça, is a traditional Brazilian beverage that in recent years has increased its market share among international distilled beverages. Several volatile compounds produced by yeast cells during the fermentation process are responsible for the unique taste and aroma of this drink. The yeast Dekkera bruxellensis has acquired increasing importance in the fermented beverage production, as the different metabolites produced by this yeast may be either beneficial or harmful to the end‐product. Since D. bruxellensis is often found in the fermentation processes carried out in ethanol fuel distillation in Brazil, we employed this yeast to analyse the physiological profile and production of aromatic compounds and to examine whether it is feasible to regard it as a cachaça‐producing microorganism. The assays were performed on a small scale and simulated the conditions for the production of handmade cachaça. The results showed that the presence of aromatic and branched‐chain amino acids in the medium has a strong influence on the metabolism and production of flavours by D. bruxellensis. The assimilation of these alternative nitrogen sources led to different fermentation yields and the production of flavouring compounds. The influence of the nitrogen source on the metabolism of fusel alcohols and esters in D. bruxellensis highlights the need for further studies of the nitrogen requirements to obtain the desired level of sensory compounds in the fermentation. Our results suggest that D. bruxellensis has the potential to play a role in the production of cachaça. Copyright © 2014 John Wiley & Sons, Ltd.     

Highlight on the problems generated by p-coumaric acid analysis in wine fermentations

p-Coumaric acid is a natural hydroxycinnamic acid existing in grapes and wine. It is the precursor of the 4-ethylphenol molecule through the bioconversion reaction by Brettanomyces yeast. Chromatographic methods are the most common techniques to detect p-coumaric acid. It is known that this acid is highly unstable in analysis and fermentation experiments. This paper highlights the problems occurring in p-coumaric acid analysis in wine fermentation conditions when studying its bioconversion. First, it was shown that p-coumaric acid was unstable at elevated temperature. On the other hand, it was found that in our experimental conditions p-coumaric acid reacted with ethanol. This work revealed also that the p-coumaric acid is partially adsorbed on Brettanomyces yeast, certainly on cell walls. Because of these phenomena the quantity of p-coumaric acid which can participate to the bioconversion into ethylphenol decreases.     

Modulation of the glycerol and ethanol syntheses in the yeast Saccharomyces kudriavzevii differs from that exhibited by Saccharomyces cerevisiae and their hybrid

In the last years there is an increasing demand to produce wines with higher glycerol levels and lower ethanol contents. The production of these compounds by yeasts is influenced by many environmental variables, and could be controlled by the choice of optimized cultivation conditions. The present work studies, in a wine model system, the effects of temperature, pH and sugar concentration on the glycerol and ethanol syntheses by yeasts Saccharomyces cerevisiae T73, the type strain of Saccharomyces kudriavzevii IFO 1802^T, and an interspecific hybrid between both species (W27), which was accomplished by the application of response surface methodology based in a central composite circumscribed design. Results show that carbon flux could be especially directed towards glycerol synthesis instead of ethanol at low pH, high sugar concentrations and low temperatures. In general, the non-wine yeast S. kudriavzevii produced higher glycerol levels and lower ethanol content than wine strains S. cerevisiae T73 and the hybrid W27, with specific and different glycerol production profiles as a function of temperature and pH. These results were congruent with the higher glycerol-3-phosphate dehydrogenase activities estimated for this species, chiefly at low temperatures (14 °C), which could explain why S. kudriavzevii is a cryotolerant yeast compared to S. cerevisiae.     

Determination of viable wine yeast using DNA binding dyes and quantitative PCR

The detection and quantification of wine yeast can be misleading due to under or overestimation of these microorganisms. Underestimation may be caused by variable growing rates of different microorganisms in culture media or the presence of viable but non-cultivable microorganisms. Overestimation may be caused by the lack of discrimination between live and dead microorganisms if quantitative PCR is used to quantify with DNA as the template. However, culture-independent methods that use dyes have been described to remove the DNA from dead cells and then quantify the live microorganisms. Two dyes have been studied in this paper: ethidium monoazide bromide (EMA) and propidium monoazide bromide (PMA). The technique was applied to grape must fermentation and ageing wines. Both dyes presented similar results on yeast monitoring. Membrane cell recovery was necessary when yeasts were originated from ethanol-containing media. When applied to grape must fermentation, differences of up to 1 log unit were seen between the QPCR estimation with or without the dye during the stationary phase. In ageing wines, good agreement was found between plating techniques and QPCR. Most of the viable cells were also culturable and no differences were observed with the methods, except for Zygosaccharomyces bailii and Dekkera bruxellensis where much higher counts were occasionally detected by QPCR. The presence of excess dead cells did not interfere with the quantification of live cells with either of the dyes.     

Comparative study of physiological adaptation to salt stress in the genome shuffled Candida versatilis and a wild-type salt-tolerant yeast strain

Candida versatilis is a yeast with a complex salt-tolerant system. It can maintain normal physiological activities and metabolic fermentation under a high-salt environment. The cellular mechanisms of adaptation to salt stress in strains of a wild type of C. versatilis (WT) and S3–5, genome shuffling strains of C. versatilis with improved tolerance to salt, were investigated. The content of intra- and extra-cellular glycerol, intra-cellular Na⁺, as well as membrane fluidity and permeability, were determined under salt-stressed yeast growth conditions. The results showed that Na⁺/H⁺-antiporter played a primary role in Na⁺ extrusion and H⁺-ATPase has been associated with yeast survival under salt stress. Considerable amounts of glycerol were produced and secreted by the yeast to outside the cell under this salt stress. Changes in the portion of membrane saturated and unsaturated fatty acid composition of C. versatilis in response to osmotic stress lead to membrane permeability and fluidity decreases. They could restrict the influx of Na⁺, enhance H⁺-ATPase activity, and prevent leakage of glycerol across the cell membrane under osmotic stress. The salt tolerance of genome shuffled strain S3–5 was higher than WT. It could be correlated with a higher level of intra-cellular accumulation of glycerol and sodium ions in cells of S3–5 than WT as well as a higher portion of oleic fatty acid (C18: 1) and a lower level of linoleic acid (C18: 2) in cell membranes of the studied yeast mutant. It can be concluded that S3–5 improved physiological regulatory mechanisms of response to salt stress, such as decreased membrane fluidity and a permeability that rapidly adjusted to osmotic stress.