Biocontrol of Brettanomyces bruxellensis by Metschnikowia pulcherrima: from pulcherriminic acid production to preventive barrel treatment.

Brettanomyces bruxellensis is a major spoilage yeast in winemaking due to its persistence in winery environments and its ability to produce volatile phenols responsible for sensory defects. This study evaluated the biocontrol potential of selected Metschnikowia pulcherrima strains, focusing on the role of pulcherriminic acid production and its application in winery-relevant conditions. Twelve M. pulcherrima strains were screened for pulcherriminic acid production under different iron conditions, revealing strong strain-dependent variability. Three strains (1521, 1558, and DSM 70336) were identified as the most efficient producers and exhibited significant iron depletion capacity. The produced metabolite remained stable over an 18-day period. Cell-free supernatants from selected strains inhibited the growth of B. bruxellensis in a strain- and concentration-dependent manner, with the strongest effect observed for strain M. pulcherrima DSM 70742. Biofilm assays on oak wood demonstrated that the supernatants reduced viable cell counts by up to 80-86% when applied at the initial stage of colonization, whereas significantly lower reductions (10-55%) were observed for pre-established biofilms. Application of a mixed supernatant under winery conditions successfully prevented Brettanomyces contamination in treated barrels without affecting wine sensory properties. These findings indicate that M. pulcherrima-derived metabolites primarily act by limiting iron availability and interfering with early biofilm formation. The results highlight the potential of M. pulcherrima as a practical and sustainable biocontrol agent for reducing spoilage yeast contamination and limiting the need for chemical preservatives in winemaking.