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In this study, the antimicrobial activity of a commercial beta-glucanase preparation against wine spoilage yeasts such as Cryptococcus albidus, Dekkera bruxellensis, Pichia membranifaciens, Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Zygosaccharomyces bisporus has been evaluated. Yeast species tested showed different sensitivities to the enzyme preparation. In vitro assays in laboratory medium (GPY) showed inhibition by the beta-glucanase preparation of D. bruxellensis and Z. bailii growth with IC(50) and MIC values approximately 3 to 4-fold greater than the recommended dose for improving wine filtration. Wine spoilage experiments showed antimicrobial action against D. bruxellensis and Z. bailii although with reduced effectiveness to that showed in laboratory medium. Under the conditions tested, the addition of beta-glucanase did not affect wine enological parameters. Our data suggest the potential use of beta-glucanases as antimicrobial agents in wine and indicate that the application of antimicrobial enzymes for yeast spoilage control deserves further investigation.