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<i>Brettanomyces bruxellensis</i> is found in several fermented matrices and produces relevant alterations to the wine quality. The methods usually used to identify <i>B. bruxellensis</i> contamination are based on conventional microbiological techniques that require long procedures (15 days), causing the yeast to spread in the meantime. Recently, a flow cytometry kit for the rapid detection (1–2 h) of <i>B. bruxellensis</i> in wine has been developed. The feasibility of the method was assessed in a synthetic medium as well as in wine samples by detecting <i>B. bruxellensis</i> in the presence of other yeast species (<i>Saccharomyces cerevisiae</i> and <i>Pichia</i> spp.) and at the concentrations that produce natural contaminations (up to 10⁵ cells/mL), as well as at lower concentrations (10³–10² cells/mL). Wine samples naturally contaminated by <i>B. bruxellensis</i> or inoculated with four different strains of <i>B. bruxellensis</i> species together with <i>Saccharomyces cerevisiae</i> and <i>Pichia</i> spp., were analyzed by flow cytometry. Plate counts were carried out in parallel to flow cytometry. We provide evidence that flow cytometry allows the rapid detection of <i>B. bruxellensis</i> in simple and complex mixtures. Therefore, this technique has great potential for the detection of <i>B. bruxellensis</i> and could allow preventive actions to reduce wine spoilage.