Investigating the impact of sulphur dioxide on Brettanomyces bruxellensis at a molecular and cellular level

[摘要] ENGLISH ABSTRACT: The yeast Brettanomyces was isolated from beer in 1904 and associated with wine thereafter. Asporulating form, Dekkera, was discovered later. Brettanomyces bruxellensis produces highlevels of volatile phenol off-flavours in wine. Sulphur dioxide (SO2) is the most widely usedchemical preservative in wine. Yeasts have several mechanisms to cope with the SO2, namelySsu1p, a membrane bound SO2 transporter; sulphite reduction, sulphite oxidation andacetaldehyde production. In unfavourable environmental conditions, certain yeasts can enter aviable-but-non-culturable (VBNC) state which is characterised by reduced metabolic rate,inability to reproduce on solid media and a reduction of cell size. VBNC can be triggered bychemical stress such as high SO2 levels. The objectives of this study were to examine the SO2tolerance of B. bruxellensis and Saccharomyces cerevisiae, to quantify their rates of SO2accumulation and efflux, determine the effect of SO2 on their energy metabolism and investigateif B. bruxellensis possesses an orthologue to S. cerevisiae SSU1.In this study, the identity of a number of Brettanomyces/Dekkera strains was confirmed using5.8S rDNA-ITS RFLP analysis and DNA sequencing. Sporulation assays were used to confirmwhether these strains belonged to the Dekkera or Brettanomyces genus. A method toaccurately quantify SO2 in laboratory conditions was optimised. Molecular SO2 tolerance wastested by spotting fresh yeast cultures on media with SO2 and/or ethanol. Tolerance to SO2and/or ethanol showed highly strain dependent results with S. cerevisiae showing the highesttolerance levels while B. bruxellensis tolerated SO2 and ethanol poorly but certain strains grewwell with only SO2. The SO2 accumulation and efflux rates of 3 S. cerevisiae strains and 3 B.bruxellensis strains were determined. It was shown that the S. cerevisiae strains followed thesame trends as previously found in literature whereas B. bruxellensis strains showed similartrends but displayed highly variable strain-dependent results.B. bruxellensis CB63 and S. cerevisiae VIN13 were investigated for their response to SO2 in twodifferent media, TA and SWM, over a 48-hour and 32-day period respectively. Acetic acid,acetaldehyde, D-glucose, D-fructose (only in SWM) and ethanol (only in TA) were regularlymonitored over the time course of each experiment. SO2 had the greatest impact onB. bruxellensis with decreased rates of glucose consumption and ethanol production as well asincreased acetic acid. Acetaldehyde peaked shortly after SO2 addition with the subsequentrestarting of sugar consumption for certain samples. This suggests that sufficient acetaldehydewas produced to bind free SO2 to reduce SO2 stress. Volatile phenols were quantified for day 32of the SWM experiment. An increase of 4-ethyl guaiacol was correlated to higher molecular SO2levels. SO2 negatively affected both yeasts energy metabolism, forcing the yeasts metabolismto adapt to ensure survival.In general, SO2 was shown to have a negative impact on all aspects of a yeasts growth andmetabolism and that SO2 tolerance is highly strain dependent and a far more complicatedcharacteristic than currently understood.

[发布机构] Stellenbosch University