Browsing Masters Degrees (Institute for Wine Biotechnology) by Subject "Brettanomyces bruxellensis"
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- ItemInvestigating the impact of sulphur dioxide on Brettanomyces bruxellensis at a molecular and cellular level(Stellenbosch : Stellenbosch University, 2012-03) Duckitt, Edward; Divol, Benoit; Du Toit, Maret; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: The yeast Brettanomyces was isolated from beer in 1904 and associated with wine thereafter. A sporulating form, Dekkera, was discovered later. Brettanomyces bruxellensis produces high levels of volatile phenol off-flavours in wine. Sulphur dioxide (SO2) is the most widely used chemical preservative in wine. Yeasts have several mechanisms to cope with the SO2, namely Ssu1p, a membrane bound SO2 transporter; sulphite reduction, sulphite oxidation and acetaldehyde production. In unfavourable environmental conditions, certain yeasts can enter a viable-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 by chemical stress such as high SO2 levels. The objectives of this study were to examine the SO2 tolerance of B. bruxellensis and Saccharomyces cerevisiae, to quantify their rates of SO2 accumulation and efflux, determine the effect of SO2 on their energy metabolism and investigate if B. bruxellensis possesses an orthologue to S. cerevisiae SSU1. In this study, the identity of a number of Brettanomyces/Dekkera strains was confirmed using 5.8S rDNA-ITS RFLP analysis and DNA sequencing. Sporulation assays were used to confirm whether these strains belonged to the Dekkera or Brettanomyces genus. A method to accurately quantify SO2 in laboratory conditions was optimised. Molecular SO2 tolerance was tested by spotting fresh yeast cultures on media with SO2 and/or ethanol. Tolerance to SO2 and/or ethanol showed highly strain dependent results with S. cerevisiae showing the highest tolerance levels while B. bruxellensis tolerated SO2 and ethanol poorly but certain strains grew well 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 the same trends as previously found in literature whereas B. bruxellensis strains showed similar trends but displayed highly variable strain-dependent results. B. bruxellensis CB63 and S. cerevisiae VIN13 were investigated for their response to SO2 in two different 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 regularly monitored over the time course of each experiment. SO2 had the greatest impact on B. bruxellensis with decreased rates of glucose consumption and ethanol production as well as increased acetic acid. Acetaldehyde peaked shortly after SO2 addition with the subsequent restarting of sugar consumption for certain samples. This suggests that sufficient acetaldehyde was produced to bind free SO2 to reduce SO2 stress. Volatile phenols were quantified for day 32 of the SWM experiment. An increase of 4-ethyl guaiacol was correlated to higher molecular SO2 levels. SO2 negatively affected both yeasts energy metabolism, forcing the yeasts metabolism to adapt to ensure survival. In general, SO2 was shown to have a negative impact on all aspects of a yeasts growth and metabolism and that SO2 tolerance is highly strain dependent and a far more complicated characteristic than currently understood.