Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine

Aims:  Brettanomyces / Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine.
Methods and Results:  Sterile red wines were inoculated with 5 × 10³ viable cells ml⁻¹ of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10⁶–10⁷ colony forming units (CFU) ml⁻¹ and volatile phenol concentrations ranged from 500 to 4000 μg l⁻¹. Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C₂ to C₁₀), short chain acid ethyl-esters and the 'mousy off-flavour' component 2-acetyl-tetrahydropyridine.
Conclusions:  Different kinds of 'Brett character' characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs.
Significance and Impact of the Study:  We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.     

Modelling the growth and ethanol production of Brettanomyces bruxellensis at different glucose concentrations

Aim: To study the effect of glucose concentrations on the growth by Brettanomyces bruxellensis yeast strain in batch experiments and develop a mathematical model for kinetic behaviour analysis of yeast growing in batch culture. Methods and Results: A Matlab algorithm was developed for the estimation of model parameters. Glucose fermentation by B. bruxellensis was studied by varying its concentration (5, 9·3, 13·8, 16·5, 17·6 and 21·4%). The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol and biomass production; at a substrate concentration of 50–138 g l^?1, the ethanol and biomass production were 24, 59 and 6·3, 11·4 g l^?1, respectively. However, an increase in glucose concentration to 165 g l^?1 led to a drastic decrease in product formation and substrate utilization. Conclusions: The model successfully simulated the batch kinetic observed in all cases. The confidence intervals were also estimated at each phase at a 0·95 probability level in a t‐Student distribution for f degrees of freedom. The maximum ethanol and biomass yields were obtained with an initial glucose concentration of 138 g l^?1. Significance and Impact of the Study: These experiments illustrate the importance of using a mathematical model applied to kinetic behaviour on glucose concentration by B. bruxellensis.     

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis

Brettanomyces is the major microbial cause for wine spoilage worldwide and causes significant economic losses. The reasons are the production of ethylphenols that lead to an unpleasant taint described as 'phenolic odour'. Despite its economic importance, Brettanomyces has remained poorly studied at the metabolic level. The origin of the ethylphenol results from the conversion of vinylphenols in ethylphenol by Brettanomyces hydroxycinnamate decarboxylase. However, no information is available on the vinylphenol reductase responsible for the conversion of vinylphenols in ethylphenols. In this study, a vinylphenol reductase was partially purified from Brettanomyces bruxellensis that was active towards 4-vinylguaiacol and 4-vinylphenol only among the substrates tested. First, a vinylphenol reductase activity assay was designed that allowed us to show that the enzyme was NADH dependent. The vinylphenol reductase was purified 152-fold with a recovery yield of 1.77%. The apparent K(m) and V(max) values for the hydrolysis of 4-vinylguaiacol were, respectively, 0.14 mM and 1900 U mg(-1). The optimal pH and temperature for vinylphenol reductase were pH 5-6 and 30 degrees C, respectively. The molecular weight of the enzyme was 26 kDa. Trypsic digest of the protein was performed and the peptides were sequenced, which allowed us to identify in Brettanomyces genome an ORF coding for a 210 amino acid protein.     

A simple cultural method for the presumptive detection of the yeasts Brettanomyces/Dekkera in wines

AIMS: The development of a simple and reliable procedure, compatible with routine use in wineries, for the presumptive detection of Brettanomyces/Dekkera from wine and wine-environment samples. METHODS AND RESULTS: The method of detection of these yeasts employs a selective enrichment medium. The medium contains glucose (10 g l(-1)) as carbon and energy source, cycloheximide (20 mg l(-1)) to prevent growth of Saccharomyces, chloramphenicol (200 mg l(-1)) to prevent growth of bacteria and p-coumaric acid (20 mg l(-1)) as the precursor for the production of 4-ethyl-phenol. After the inoculation with wine, the medium is monitored by visual inspection of turbidity and by periodic olfactive analysis. Contaminated wines will develop visible turbidity in the medium and will produce the 4-ethyl-phenol off-odour, which can be easily detected by smelling. CONCLUSIONS: A selective enrichment liquid medium was developed to differentially promote the growth and activity of Brettanomyces/Dekkera. The method is simple to execute, employing a simple-to-prepare medium and a periodic olfactive detection. SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of the procedure make it particularly applicable in a wine-making environment thus presenting important advantages to the wine industry.     

Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking

AIMS: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. METHODS AND RESULTS: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. CONCLUSIONS: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.     

Development of a molecular method for the typing of Brettanomyces bruxellensis (Dekkera bruxellensis) at the strain level

AIMS: In recent years, Brettanomyces/Dekkera bruxellensis has caused increasingly severe quality problems in the wine industry. A typing method at the strain level is needed for a better knowledge of the dispersion and the dynamics of these yeasts from grape to wine. METHODS AND RESULTS: Three molecular tools, namely random-amplified polymorphic DNA, PCR fingerprinting with microsatellite oligonucleotide primers and SAU-PCR, were explored for their relevance to typing strains of Brettanomyces bruxellensis. The results indicated that discrimination of each individual strain was not possible with a single PCR typing technique. We described a typing method for B. bruxellensis based on restriction enzyme analysis and pulse field gel electrophoresis (REA-PFGE). Results showed that electrophoretic profiles were reproducible and specific for each strain under study. CONCLUSIONS: Consequently, REA-PFGE should be considered for the discrimination of B. bruxellensis strains. This technique allowed a fine discrimination of B. bruxellensis, as strains were identified by a particular profile. SIGNIFICANCE AND IMPACT OF THE STUDY: This study constitutes a prerequisite for accurate and appropriate investigations on the diversity of strains throughout the winemaking and ageing process. Such studies will probably give clearer and more up-to-date information on the origin of the presence of Brettanomyces in wine after vinification when they are latent spoilage agents.     

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

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

Detection and quantification of Brettanomyces bruxellensis and 'ropy' Pediococcus damnosus strains in wine by real-time polymerase chain reaction

AIMS: Brettanomyces bruxellensis is a well-known wine spoilage yeast that causes undesirable off-flavours. Likewise, glucan-producing strains of ropy Pediococcus damnosus are considered as spoilage micro-organisms because the synthesis of glucan leads to an unacceptable viscosity of wine. METHODS AND RESULTS: We developed a real-time PCR method to detect and quantify these two spoilage micro-organisms in wine. It is based on specific primer pairs for amplification of target DNA, and includes a melting-curve analysis of PCR products as a confirmatory test. CONCLUSIONS: The detection limit in wine was 10(4) CFU ml(-1) for B. bruxellensis and 40 CFU ml(-1) for ropy Pediococcus damnosus. The real-time PCR proved to be reliable for the early, sensitive detection and quantification of B. bruxellensis and ropy P. damnosus in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR-based method described in this study provides a new tool for monitoring spoilage micro-organisms in wine. Time-consuming culture and colony isolation steps are no longer needed, so winemakers can intervene before spoilage occurs.     

Wine phenolic compounds influence the production of volatile phenols by wine-related lactic acid bacteria

Aims: To evaluate the effect of wine phenolic compounds on the production of volatile phenols (4‐vinylphenol [4VP] and 4‐ethylphenol [4EP]) from the metabolism of p‐coumaric acid by lactic acid bacteria (LAB). Methods and Results: Lactobacillus plantarum, Lactobacillus collinoides and Pediococcus pentosaceus were grown in MRS medium supplemented with p‐coumaric acid, in the presence of different phenolic compounds: nonflavonoids (hydroxycinnamic and benzoic acids) and flavonoids (flavonols and flavanols). The inducibility of the enzymes involved in the p‐coumaric acid metabolism was studied in resting cells. The hydroxycinnamic acids tested stimulated the capacity of LAB to synthesize volatile phenols. Growth in the presence of hydroxycinnamic acids, especially caffeic acid, induced the production of 4VP by resting cells. The hydroxybenzoic acids did not significantly affect the behaviour of the studied strains. Some of the flavonoids showed an effect on the production of volatile phenols, although strongly dependent on the bacterial species. Relatively high concentrations (1 g l^?1) of tannins inhibited the synthesis of 4VP by Lact. plantarum. Conclusions: Hydroxycinnamic acids were the main compounds stimulating the production of volatile phenols by LAB. The results suggest that caffeic and ferulic acids induce the synthesis of the cinnamate decarboxylase involved in the metabolism of p‐coumaric acid. On the other hand, tannins exert an inhibitory effect. Significance and Impact of the Study: This study highlights the capacity of LAB to produce volatile phenols and that this activity is markedly influenced by the phenolic composition of the medium.     

Growth and fermentation behaviour of Brettanomyces/Dekkera yeasts under different conditions of aerobiosis

Brettanomyces/Dekkera yeasts grow in wine and their presence is often associated with spoiling activity. In this report, we investigated on the influence of different conditions of aerobiosis on growth and fermentation behaviour of these spoilage yeasts in wine. Results showed that in all conditions tested the Brettanomyces strain consumed all sugars, taking wine fermentation to completion. Strict-anaerobic conditions limited the growth of Brettanomyces. Both anaerobiosis (using a fermentation trap) and strict anaerobiosis did not negatively affect the principal by-products of fermentation whereas semi-anaerobiosis caused an increase of acetic acid, acetaldehyde and ethyl acetate that negatively affected the fermentation profile of resulting products.     

The effect of sulphur dioxide and oxygen on the viability and culturability of a strain of Acetobacter pasteurianus and a strain of Brettanomyces bruxellensis isolated from wine

AIMS: The objective of this study was to investigate the effects of free molecular and bound forms of sulphur dioxide and oxygen on the viability and culturability of a selected strain of Acetobacter pasteurianus and a selected strain of Brettanomyces bruxellensis in wine. METHODS AND RESULTS: Acetic acid bacteria and Brettanomyces/Dekkera yeasts associated with wine spoilage were isolated from bottled commercial red wines. One bacterium, A. pasteurianus strain A8, and one yeast, B. bruxellensis strain B3a, were selected for further study. The resistance to sulphur dioxide and the effect of oxygen addition on these two selected strains were determined by using plating and epifluorescence techniques for monitoring cell viability in wine. Acetobacter pasteurianus A8 was more resistant to sulphur dioxide than B. bruxellensis B3a, with the latter being rapidly affected by a short exposure time to free molecular form of sulphur dioxide. As expected, neither of these microbial strains was affected by the bound form of sulphur dioxide. The addition of oxygen negated the difference observed between plate and epifluorescence counts for A. pasteurianus A8 during storage, while it stimulated growth of B. bruxellensis B3a. CONCLUSIONS: Acetobacter pasteurianus A8 can survive under anaerobic conditions in wine in the presence of sulphur dioxide. Brettanomyces bruxellensis B3a is more sensitive to sulphur dioxide than A. pasteurianus A8, but can grow in the presence of oxygen. Care should be taken to exclude oxygen from contact with wine when it is being transferred or moved. SIGNIFICANCE AND IMPACT OF THE STUDY: Wine spoilage can be avoided by preventing growth of undesirable acetic acid bacteria and Brettanomyces/Dekkera yeasts through the effective use of sulphur dioxide and the management of oxygen throughout the winemaking process.     

Methionine catabolism and production of volatile sulphur compounds by OEnococcus oeni

AIMS: During malolactic fermentation (MLF), the secondary metabolisms of lactic acid bacteria (LAB) contribute to the organoleptic modification of wine. To understand the contribution of MLF, we evaluated the capacity of various wine LAB to metabolize methionine. METHODS AND RESULTS: Using gas chromatography (GC) coupled either with mass spectrometry (MS) or a flame photometry detector in sulphur mode (FPD), we studied this metabolism in laboratory media and wine. In laboratory media, several LAB isolated from wine were able to metabolize methionine. They formed methanethiol, dimethyl disulphide, 3-(methylsulphanyl)propan-1-ol and 3-(methylsulphanyl)propionic acid. These are known to have powerful characteristic odours and play a role in the aromatic complexity of wine. In various red wines, after MLF only the 3-(methylsulphanyl)propionic acid concentration increased significantly, as verified with several commercial starter cultures. This compound, which is characterized by chocolate and roasted odours, could contribute to the aromatic complexity produced by MLF. CONCLUSIONS: This study shows that LAB isolated from wine, especially OEnococcus oeni strains, the major species in MLF, are able to metabolize methionine to form volatile sulphur compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate the capacity of wine LAB to metabolize methionine.     

Quantification of glycosidase activities in selected yeasts and lactic acid bacteria

Using a model system, the activities of α-L-arabinofuranosidase, β-glucosidase, and α-L-rhamonopyranosidase were determined in 32 strains of yeasts belonging to the genera Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Hansenula, Kloeckera, Metschnikowia, Pichia, Saccharomyces, Torulaspora and Brettanomyces (10 strains); and seven strains of the bacterium Leuconostoc oenos. Only one Saccharomyces strain exhibited β-glucosidase activity, but several non-Saccharomyces yeast species showed activity of this enzyme. Aureobasidium pullulans hydrolyzed α-L-arabinofuranoside, β-glucoside, and α-L-rhamnopyranoside. Eight Brettanomyces strains had β-glucosidase activity. Location of enzyme activity was determined for those species with enzymatic activity. The majority of β-glucosidase activity was located in the whole cell fraction, with smaller amounts found in permeabilized cells and released into the growth medium. Aureobasidium pullulans hydrolyzed glycosides found in grapes. Received 02 February 1999/ Accepted in revised form 26 June 1999     

葡萄酒中重要挥发性硫化物的代谢及基因调控

葡萄酒中的挥发性硫化物是由酿酒微生物在葡萄酒发酵过程中代谢所产生的,主要包括硫化氢、硫醇、硫醚、硫醇酯、含硫杂醇油及杂环化合物等,它们对葡萄酒的风味会产生重要影响。本综述介绍了葡萄酒中重要的挥发性硫化物的主要代谢途径及相关基因的调控机制,并提出酿酒微生物的相关研究是提高优良风味物质含量,同时抑制不良风味产生的有效途径。     

Effect of fermentation, aging and thermal storage on total glycosides, phenol-free glycosides and volatile compounds of White Riesling (Vitis vinifera L.) wines

There is growing recognition of the significance of the products of glycoside hydrolysis to varietal wine aroma. White Riesling wines were produced from four strains of Saccharomyces cerevisiae. Wines underwent conventional aging or anaerobic thermal storage (20 days at 45°C) either 2 or 40 months post-fermentation to quantify influences on total glycosides, phenol-free glycosides and selected volatiles. Glycoside and free volatile concentrations were estimated by analysis of glycosyl-glucose and gas chromatography/mass spectrometry, respectively. Thermal storage of wines 2 months post-fermentation reduced the total glycosides by an average of 33% for all yeasts and increased the concentration of free benzyl alcohol while decreasing the concentration of free linalool and geraniol. Conventional aging for 40 months reduced the total and phenol-free glycosides equally among yeasts by an average of 60%, with phenol-free glycosides averaging 80% of the total. Thermal storage of aged wines reduced the total glycoside concentration by an additional 29%. The effect of thermal storage on selected volatile phenols, higher alcohols, esters, acids, terpenes, carbonyl compounds, C-13 norisoprenoids and six-carbon alcohols was variable depending upon the component. Received 29 September 1998/ Accepted in revised form 16 January 1999     

Changes in volatile compounds and aromatic series in sherry wine with high gluconic acid levels subjected to aging by submerged flor yeast cultures

Volatile compounds of sherry wine containing gluconic acid under aging by submerged flor yeast cultures were analyzed. The aroma profile was obtained by grouping the compounds in nine aromatic series. The balsamic, fatty, herbaceous and empyreumatic series increased significantly as consequence of the increase of pantolactone, acids (butanoic, 2-methylbutanoic and 3-methylbutanoic), methionol and gamma-butyrolactone compounds, respectively. The decrease of higher alcohols promoted solvent series diminished. These changes are consistent with those observed in the production of commercial sherry wine using traditional biological aging.     

Increased resveratrol production in wines using engineered wine strains Saccharomyces cerevisiae EC1118 and relaxed antibiotic or auxotrophic selection

Resveratrol is a polyphenolic compound with diverse beneficial effects on human health. Red wine is the major dietary source of resveratrol but the amount that people can obtain from wines is limited. To increase the resveratrol production in wines, two expression vectors carrying 4‐coumarate: coenzyme A ligase gene (4CL) from Arabidopsis thaliana and resveratrol synthase gene (RS) from Vitis vinifera were transformed into industrial wine strain Saccharomyces cerevisiae EC1118. When cultured with 1 mM p‐coumaric acid, the engineered strains grown with and without the addition of antibiotics produced 8.249 and 3.317 mg/L of trans‐resveratrol in the culture broth, respectively. Resveratrol content of the wine fermented with engineered strains was twice higher than that of the control, indicating that our engineered strains could increase the production of resveratrol during wine fermentation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:650–655, 2015