Ensuring the continued efficacy of Brettanomyces control strategies for avoidance of spoilage

Abstract

Spoilage of wine by Brettanomyces yeasts remains one of the foremost microbiological problems faced by winemakers. Current strategies to minimise risk of spoilage do not eliminate Brettanomyces yeasts from wineries and are heavily reliant upon use of the preservative sulfite to stabilise wine against Brettanomyces growth. This project demonstrated that existing Brettanomyces wine strains have potential to evolve greater tolerance to sulfite and found evidence for this occurring in industry. The importance of developing new research tools for these yeasts was underscored by observed variation in efficacy of an emerging sulfite alternative, chitosan, which was ineffective against some strains.

Summary

Spoilage of wine by Brettanomyces yeasts remains one of the foremost microbiological problems faced by winemakers. The risk of ‘Brett’ spoilage is currently managed through application of a multi-faceted strategy previously developed by the AWRI in collaboration with Australian winemakers, which facilitated industry-wide decreases in levels of spoilage compounds in finished wines. This strategy does not, however, eliminate Brettanomyces yeasts from wineries, and is heavily reliant upon use of the preservative sulfite to stabilise wine against Brettanomyces growth. To ensure Australian winemakers’ continued ability to manage ‘Brett’ in a cost-effective manner, this project examined the interactions of sulfite with Brettanomyces biology at the population, strain and molecular levels.

Contrary to work done elsewhere, this project did not find evidence that Brettanomyces cells enter a viable but non-culturable (VBNC) state in response to sulfite. Exhaustive, long-term experiments instead suggest that resumption of population growth is dependent upon low numbers of viable, culturable cells, that may fall below limits of detection for standard microbiological and rapid tests.

For the first time, laboratory experiments revealed potential for common Australian winery strains of Brettanomyces bruxellensis to evolve greater tolerance to sulfite. Strikingly, when new industry isolates were obtained and compared to strains isolated over the past two decades it was evident that selection for sulfite-tolerance is already occurring. Attempts to characterise the mechanisms by which B. bruxellensis gains tolerance to sulfite did not reveal a common pattern, though parallel work showed that variations in amino acid sequence of the sulfite efflux pump Ssu1p can partly explain strain variation.

Efforts to identify genetic determinants of sulfite tolerance were hampered by the lack of molecular genetics tools for Brettanomyces yeasts. In response to this limitation, new tools were developed for the genetic transformation of B. bruxellensis, which were applied to study competitiveness of different strains under sulfite stress. The importance of developing tools for further study of Brettanomyces biology was emphasised by observed strain-level variation in efficacy of an increasingly popular alternative control agent, chitosan, which was shown to be ineffective against a key Australian wine strain.