When yeast-bacterial interactions depend on the quality of wines

International audienceno abstrac

Oligopeptide assimilation and transport by Oenococcus oeni

International audienceAims: Oenococcus oeni is a slow-growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino-acid medium. We aimed to characterize the oligopeptide use of this micro-organism. Methods and Results: Several peptides of two to eight amino-acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium. Conclusions: Oenococcus oeni was able to transport the oligopeptides with two to five amino-acid residues tested and to hydrolyse them further. Significance and Impact of the Study: This study has clear implications for the relationship between wine nitrogen composition and the ability of O.oeni to cope with its environment

Evaluation of three Brettanomyces qPCR commercial kits: results from an interlaboratory study

Aim: Brettanomyces bruxellensis is well adapted to high ethanol concentrations and low pH which allows it to develop in difficult environments, such as wine. B. bruxellensis is mainly found in red wine and is regarded as a spoilage yeast due to its production of ethylphenols and other compounds responsible for organoleptic defects. The detection and quantification of this yeast is essential to preventing wine spoilage. Several specific detection and quantification kits based on real time quantitative PCR (qPCR) are commercially available. Although these kits are frequently used by private enological and research laboratories, no scientific reports on the reliability and performance of these kits, including interlaboratory and interassay comparisons, have been published. The aim of this work was to compare commercially available kits for the quantification of B. bruxellensis in red wine to classical method (plate counting on selective medium) in an interlaboratory study. Methods and results: Three different commercial kits were tested on three different wines from Bordeaux, Cotes du Rhone, and Burgundy inoculated with B. bruxellensis at four different concentrations. Five naturally contaminated wines from different French wine regions were also tested. Our results suggest that all the kits tested probably over or underestimate the quantity of B. bruxellensis in red wine and, under specific conditions, give false positives. Conclusion: Quantification may be very heterogeneous depending on the wine, laboratory, or population level. Underestimations or false negative results may have serious consequences for winemakers. Overestimation may be partly due to the quantification of dead cells by qPCR. Significance and impact of the study: This study highlights that quantification of B. bruxellensis in red wine using commercial kits requires a high level of expertise in molecular biology. We recommend that all users use a microbiological internal control to validate DNA extraction yield

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis

International audienceBrettanomyces 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