Unveiling the Flocculation Potential of Brettanomyces bruxellensis: Innovating Spoilage Mitigation Strategies

Flocculation is a reversible and asexual adaptive mechanism involving Ca2+-dependent surface proteins called flocculins. These proteins interact with ɑ-mannan residues on other cells, with calcium ions facilitating aggregation and precipitation of the floc. Calcium acts as a cofactor, maintaining the active conformation of surface proteins and enhancing binding with cell receptors. This process is controlled by the FLO gene family [1,2]. Flocculation has been extensively studied in Saccharomyces cerevisiae due to its dependence on growth conditions [3,4]. In sparkling wine production, particularly during the remuage step of the champenoise method, flocculation helps in yeast aggregation and sedimentation. Although not always favorable in vinification, it can efficiently remove sedimented yeasts. This study aimed to evaluate flocculation in the spoilage yeast Brettanomyces bruxellensis, examining its occurrence at the strain level and under different environmental conditions. The findings could lead to innovative yeast removal methods. One hundred and thirteen strains were phenotypically characterized for flocculation ability under laboratory conditions using a Ca2+-dependent method [5]. Based on preliminary analyses, strains CBS2499 and UMY321, identified as medium and low-flocculant respectively, were selected for further experiments. Genetic analysis of the FLO1 and FLO11 genes, comparing 11 other B. bruxellensis strains, was performed to check for mutations. A Box–Behnken experimental design in a model wine assessed the impact of sulfur dioxide, ethanol concentrations, and pH on flocculation and FLO gene expression. While UMY321 showed no significant changes, CBS2499 was affected by the oenological conditions, with increased flocculation under certain conditions. Specifically, CBS2499 showed higher flocculation and FLO1 expression when extreme levels of the three parameters were combined, compared to the permissive condition. Additionally, the analysis of FLO1 and FLO11 sequences showed multiple SNPs, revealing potential polymorphisms in the FLO genes. These results suggest that flocculation is a strain-dependent character and could be utilized in vinification processes.