Isolation, biofilm formation and control of the wine spoilage yeast Brettanomyces bruxellensis

Abstract

Brettanomyces bruxellensis, commonly referred to as “Brett,” is one of the most notorious microorganisms implicated in wine spoilage. This yeast species has developed a noteworthy resistance to sulfur dioxide, a widely used preservative in winemaking, prompting the wine industry to seek new antimicrobial agents. The development of B. bruxellensis in wine can change the sensory properties of wine due to the production of undesirable aromas. The present work focuses on i) the biofilm-forming ability of B. bruxellensis, derived from Greek wines, on stainless steel surfaces ii) the ability of the adhered cell to cause wine spoilage iii) new treatment to handle the contamination. Three wines from different regions of Greece were collected and subjected in molecular analyses and identification at species level. RAPD (Random Amplified Polymorphic DNA) genomic fingerprinting with the oligo-nucleotide primer M₁₃ was used, combined with Matrix Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. Strain differentiation of B. bruxellensis different strains was achieved by rep-PCR fingerprinting method with the oligo-nucleotide primer GTG₅. For the biofilm formation assay, stainless steel coupons were placed in test tubes containing sterilized Ringer solution and pure cultures of the different B. bruxellensis strains was inoculated at an initial population of approximately 10⁷ CFU/mL. The tubes were incubated at 28 °C for 3 hours to allow attachment of the yeast cells onto the coupons surface. Biofilm growth was evaluated with the bead vortexing method. Finally, different treatments were applied in order to prevent the adherence of B. bruxellensis strains to the coupons. Overall, we showed that the attachment and biofilm formation capacity of the spoilage yeast is influenced by the strain effect and tree different types of adherences were noticed. Additionally, all tested treatments achieved to decrease the attached yeast cells proposing a new way of handling B. bruxellensis contamination.

References

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