Flocculation character in Brettanomyces bruxellensis strains: a potential innovative approach to counteract the spoilage

Flocculation is a reversible and asexual adaptive mechanism involving Ca2+-dependent surface proteins, called flocculins, responsible for the phenomenon allowing the interaction with ɑ-mannan residues receptors of the other cells. Moreover, calcium bivalent ions (Ca2+) allows the aggregation and the subsequent precipitation of the so-called floc. In particular, the bivalent Ca2+-ion acts as a cofactor for the correct maintenance of the active conformation of the surface proteins, favoring their binding with the receptors of the other cells [1]. These peculiar structures are under the control of the FLO gene family [2]. Flocculation has been deeply investigated in Saccharomyces cerevisiae for its dependence on the growth conditions [3,4], improving the clarification of the sparkling wine during the remuage step of the champenoise method. Indeed, flocculation facilitates yeast aggregation and sedimentation and if in vinification processes it is not favorable, it could be an efficient method to remove the sedimented yeasts. In this context, this work aimed at evaluating the flocculation character in the spoilage yeast Brettanomyces bruxellensis to verify its occurrence at strain level and environmental condition. The results could be exploited to setup innovative approaches for yeast removal. One hundred and thirteen strains were characterized at phenotypic level for their capability to flocculate under laboratory conditions, by a Ca2+-dependent method [5]. Bearing in mind the preliminary phenotypic analysis, CBS2499 and UMY321 strains identified as medium and low-flocculant strains, were selected for the next experiments. A genetic analysis was performed on the three main genes of the FLO family, FLO1, FLO8 and FLO11, to check for possible mutations. Then, a Box–Behnken experimental design was performed in a model wine (SMW) to assess the impact on the flocculation occurrence and the FLO genes’ expression of three oenological parameters: sulfur dioxide and ethanol concentrations and pH. While no significant differences in flocculation capability were observed in UMY321, CBS2499 strain showed to be affected by the oenological conditions under study at genetic and phenotypic level, indeed, already the basal condition in SMW (permissive condition) showed an increase in the flocculation character. In particular, in two conditions in which the extreme levels of the three oenological parameters were combined, CBS2499 revealed a higher flocculation percentage and expression of the FLO1 gene, in comparison with the permissive condition. These results suggest that flocculation can be considered a strain-dependent character that could have a potential application in vinification processes.