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 mg L 1 day 1 of 4-ethylphenol was only observed in the presence of high numbers (106 CFU mL 1) of culturable cells, being stimulated by increasing glucose concentrations.info:eu-repo/semantics/publishedVersio

Saccharomyces cerevisiae and Dekkera bruxellensis interactions in alcoholic fermentations: growth and 4-ethylphenol production

The yeast Dekkera/Brettanomyces bruxellensis can cause enormous economic losses both in wine industry and fuel-ethanol processes due to production of phenolic off-flavour compounds and low ethanol productivities. In winemaking this microbial hazard is usually tackled by the use of chemical preservatives such as sulphur dioxide. In spite of this, D. bruxellensis strains are frequently found in wines at low levels (ca 103 cells/ml) where they can metabolise residual sugars producing phenolic off-flavours compounds, such as 4-ethyl phenol. In the present work we investigated S. cerevisiae and D. bruxellensis interactions during alcoholic fermentations and evaluated the effectiveness of antimicrobial peptides secreted by S. cerevisiae to prevent growth of the main wine spoilage yeast and the production of 4-ethylphenol. Several fermentations were performed with single cultures of D. bruxellensis and mixed cultures of S. cerevisiae and D. bruxellensis, both in synthetic grape juice (SGJ) and grape must. Yeast growth (culturability and viability) and fermentation performance (i.e. sugars consumption, ethanol and 4-ethylphenol production) of those fermentations was accessed by different methods, namely by florescence in situ hybridization and flow cytometry. Results showed that S. cerevisiae significantly reduced the growth of D. bruxellensis and the production of 4-ethylphenol both in SGJ and grape must fermentations performed with mixed cultures. Moreover, our work also showed that antimicrobial peptides secreted by S. cerevisiae are effective to prevent growth of D. bruxellensis and production of phenolic off-flavor compounds in wine

Factors affecting the production of 4-ethylphenol by the yeast Dekkera bruxellensis in enological conditions

"Food Microbiology" article in PressThe conversion of p-coumaric acid into 4-ethylphenol was studied in Dekkera bruxellensis ISA 1791 under defined conditions in synthetic media. The production of 4-ethylphenol occurred roughly between mid-exponential growth phase and the beginning of the stationary phase. This behaviour was observed when glucose was the only energy and carbon source, the conversion rate being close to 90%. Ethanol, as the single energy source, yielded conversion rates close to 80% while in the presence of trehalose and acetic acid conversion rates lower than 10% were obtained. The production of 4-ethylphenol was not observed when the cells were maintained in buffer solution without carbon and energy sources. The precursor of 4-ethylphenol, p-coumaric acid, was not utilized as energy and carbon source. Furthermore, it was shown that 4-vinylphenol may be used as a precursor of 4-ethylphenol in the absence of pcoumaric acid. Growth and 4-ethylphenol production were inhibited by increasing concentrations of ethanol, being fully prevented at 13% (v/v) ethanol. The cultivation of strain ISA 1791 in mixed culture with Saccharomyces cerevisiae, in synthetic medium, showed that the cell numbers of D. bruxellensis increased from 104 cfu/ml to 5 109 cfu/ml. Laboratory microvinifications of white and red juices inoculated with as low as 10 cfu/ml of D. bruxellensis and 107 cells/ml of S. cerevisiae showed growth of D. bruxellensis to levels of about 5 108 cfu/ml. In addition, 4-ethylphenol production by D. bruxellensis was observed only after complete fermentation of the grape juice

Yeasts and wine off-flavours: a technological perspective

Review article. Part of the special issue "Wine microbiology and safety: from the vineyard to the bottle (Microsafety Wine)", 19-20 Nov. 2009, ItalyIn wine production, yeasts have both beneficial and detrimental activities. Saccharomyces cerevisiae is the yeast mainly responsible for turning grape juice into wine but this species and several others may also show undesirable effects in wines. Among such effects, technologists are particularly concerned with the production of offflavours that may occur during all stages of winemaking. Typical spoiling activities include the production of ethyl acetate by apiculate yeasts before fermentation, hydrogen sulphide by S. cerevisiae during fermentation phases, acetaldehyde by film-forming yeasts during bulk storage, and volatile phenols by Dekkera bruxellensis during storage or after bottling. The occurrence of these hazards depends on the technological operations designed to obtain a given type of wine and most can be avoided by current preventive or curative measures. On the contrary, good manufacturing practices must be strengthened to deal with the problem of volatile phenol production in red wines. Appropriate monitoring of D. bruxellensis populations and quantification of 4-ethylphenol is advised during storage, particularly when oak barrels are used, and absence of viable cells must be guaranteed in bottled wines. This work, which is based on our experience at winery level, aims to provide information on appropriate technological strategies to deal with the problem of off-flavours produced by yeasts

Molecular typing of the yeast species Dekkera bruxellensis and Pichia guilliermondii recovered from wine related sources

A total of 63 strains of Dekkera bruxellensis and 32 strains of Pichia guilliermondii isolated from wine related environments were identified by restriction analysis of the 5.8S-ITS region of the rDNA. These strains were subjected to intraspecific discrimination using mtDNA restriction and RAPD-PCR analysis. The isolates identified as D. bruxellensis yielded 3 different molecular patterns of mtDNA restriction using the endonuclease HinfI. The pattern A was the most frequent (58 strains) among strains from different sources, regions and countries. Pattern B (4 strains) and C (one strain) were determined in isolates from Portuguese wines. The discrimination among the pattern A strains was achieved by a RAPD-PCR assay with 3 primers (OPA-2, OPA-3 and OPA-9). A total of 12 haplotypes were obtained with the combination of the patterns provided by the 3 OPAs. The pattern 2 was the most frequent and extensively distributed being found in strains from different countries and from different sources like wine, barrique wood and insects. The strains of P. guilliermondii were characterized with restriction of mtDNA using the endonuclease HinfI yielding 7 different restriction patterns. These patterns were associated with different efficiencies of 4-ethylphenol production. Patterns A to D corresponded to 19 strains producing low levels of 4-ethylphenol (50 mg/l), when grown in synthetic media supplemented with 100 mg/l of p-coumaric acid. The high degree of polymorphism observed shows that intraspecific typing is essential for accurate yeast dissemination studies in wine related environments.www.elsevier.com/locate/ijfoodmicro DOI: 10.1016/j.ijfoodmicro.2005.05.01

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

Impact of volatile phenols and their precursors on wine quality and control measures of Brettanomyces/Dekkera yeasts

Volatile phenols are aromatic compounds and one of the key molecules responsible for olfactory defects in wine. The yeast genus Brettanomyces is the only major microorganism that has the ability to covert hydroxycinnamic acids into important levels of these compounds, especially 4-ethylphenol and 4-ethylguaiacol, in red wine. When 4-ethylphenols reach concentrations greater than the sensory threshold, all wine’s organoleptic characteristics might be influenced or damaged. The aim of this literature review is to provide a better understanding of the physicochemical, biochemical, and metabolic factors that are related to the levels of p-coumaric acid and volatile phenols in wine. Then, this work summarizes the different methods used for controlling the presence of Brettanomyces in wine and the production of ethylphenols