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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.

THE yeast Brettanomyces is the major microbial cause for wine spoilage worldwide and causes significant economic loss. This yeast produces from grape acid phenols, 4-ethylphenol and 4-ethylguaiacol, which confer characteristic aroma described as mousy, smoky, burnt plastic, barnyard, horse sweat, leather, and wet wool.Moreover, these yeasts are high acetic acid producers and are capable of producing a mousy taint caused by 2-acetyl-1,4,5,6-tetrahydropyridine and 2-acetyl-3,4,5,6-tetrahydropyridine. Thus, this peculiar yeast metabolism is deleterious to wine quality.The monitoring of Brettanomyces remains a problem for most wine cellars. Recently, flow cytometry (FCM) has been proposed to quantify Brettanomyces in wine (1). However, cells are dyed with fluorescein diacetate, which stained all viable wine microorganisms and thus is not specific to Brettanomyces. By contrast, methods with some specificity, which are available to identify Brettanomyces, present other disadvantages. Most of them take 1 or 2 weeks and spoilage may occur before it is noticed by the winemaker.Detection of Brettanomyces by FCM using antibodies coupled to fluorochrome could be another strategy as it has been used successfully for other microorganisms (2). However, Brettanomyces antibodies are not available.Among the many molecular methods for analyzing microorganisms, fluorescence in situ hybridization (FISH) is a widely used method for monitoring microorganisms. Indeed, the FISH method presents advantages comparing with other molecular methods. It can target DNA or RNA regions and combines a counting technique with an identification technique