Browsing by Author "Mbuyane, Lethiwe Lynett"

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    Investigating polyol and acetic acid metabolism in wine related non-Saccharomyces yeasts
    (Stellenbosch : Stellenbosch University, 2017-12) Mbuyane, Lethiwe Lynett; Divol, Benoit; Bauer, Florian; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.
    ENGLISH SUMMARY: Glycerol is the main polyol produced in Saccharomyces cerevisiae not only to counterbalance osmotic pressure but also to adjust redox balance. Incidentally, it may also contribute to the smooth mouthfeel of wine. Whereas glycerol is closely linked to acetic acid production in S. cerevisiae, this correlation is not as clear in non-Saccharomyces yeasts (particularly Torulaspora delbrueckii). Additional polyols - which function as stress protectants and could potentially influence wine mouthfeel - have been reported in wine but the producing yeasts were never isolated. Lachancea thermotolerans, Starmerella bacillaris and T. debrueckii have been recently described as producing other polyols in addition to glycerol with the latter producing the highest amounts. However, the enzyme assays used were limited to polyol detection in combination. Thus, the aim of this study was to optimize chromatography-based methods for the separation of polyols and to investigate the production of these compounds in non-Saccharomyces yeasts under a variety of environmental conditions. Gas Chromatography-Mass Spectrophotometry was successful for the separation of polyols but only in fermentation samples with no residual sugars. Since non-Saccharomyces yeasts do not ferment to completion, other methods are required for the individual detection of polyols in order to follow production throughout fermentation. Our data show that in addition to glycerol, three T. delbrueckii strains increasingly produced similar amounts of D-sorbitol, D-arabitol and D-mannitol throughout fermentation. Furthermore with the exception of glycerol, T. delbrueckii produced higher amounts of polyols in grape must when compared to synthetic must. Whereas glycerol is limited to NADH recycling, these additional polyols may increase the co-factor recycling pool in T. delbrueckii. Our data also show that D-sorbitol, D-mannitol and D-arabitol production was influenced by initial sugar concentration with the highest amounts detected for D-arabitol in T. delbrueckii. In contrast to D-arabitol which was produced at the highest amounts, D-mannitol and D-sorbitol were not induced by NaCl. It is possible that these compounds may have accumulated within the cell as a consequence of the osmotic gradients or mechanisms related to the prevention of ion toxicity as observed in literature. Polyol production was repressed in acetic acid media in this study and induced in ethanol supplemented media. The intake of acetic acid could have resulted in a change in redox balance and a reduced need for polyols as reported in literature. The presence of ethanol could have resulted in readjustment of polyol retention within the cell and release of polyols. Overall this study shows that non-Saccharomyces yeasts (particularly T. delbrueckii) are capable of polyol production. The amounts of polyols produced in some non-Saccharomyces yeasts may have a direct impact on wine but further investigations are required on this.
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    Investigating the lipid requirements of wine-related non-Saccharomyces yeast
    (Stellenbosch : Stellenbosch University, 2022-03) Mbuyane, Lethiwe Lynett; Divol, Benoit; Bauer, Florian; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: Lipids are structural components of the yeast plasma membrane which is a barrier between the intra- and extracellular environment. The lipid bilayer also contains proteins responsible for transport, signalling and cell function. Changes in the lipid composition are necessary for yeast to adapt to unfavourable conditions that occur during alcoholic fermentation. Numerous studies focused on the lipid metabolism of Saccharomyces cerevisiae exist and previous reports have shown that ergosterol, oleic and palmitoleic acids enhance fermentation performance, ethanol stress resistance and impact aroma production. On the other hand, little is known about non- Saccharomyces yeasts. Instead, previous studies showed that selected non-Saccharomyces yeasts produce lipids different from S. cerevisiae but the impact of these differences on lipid metabolism and fermentation performance was not evaluated. The aim of this study was to evaluate the impact of exogenous lipids on fermentation kinetics, metabolite production and ethanol stress resistance in wine relevant non-Saccharomyces yeasts. Protocols focused on fatty acid and sterol extraction are tedious, requiring large sample and solvent volumes. Furthermore, the analytical methods focused on detecting sterols and fatty acids on one instrument are limited. Lipid extraction and analysis via Gas Chromatography – Mass Spectrometry was therefore optimized in yeast cells and synthetic media. The data showed that while only monounsaturated fatty acids (oleic and palmitoleic acids) were detected in S. cerevisiae cells, the non-Saccharomyces yeasts in this study accumulated linoleic and linolenic acids in addition to monounsaturated fatty acids. The data in this study showed that Metschnikowia pulcherrima, Kluyveromyces marxianus and Torulaspora delbrueckii are sensitive to the presence of lipids in the extracellular environment. Indeed, the impact of fatty acid and sterol containing mixtures on fermentation performance was species-specific. This species-specific response to the different lipid mixtures could be linked to acetyl-CoA availability and redox balance regulation based on the differences in alpha- ketoglutarate, acetate, malate and succinate biosynthesis during fermentation. K. marxianus and M. pulcherrima were found sensitive to ethanol shock with the latter being the most sensitive. The addition of unsaturated fatty acids did not drastically increase ethanol tolerance. Instead, K. marxianus and M. pulcherrima were sensitive to ethanol in media supplemented with polyunsaturated fatty acids indicating that when these compounds (which impact membrane fluidity) are taken up into the cell, they do not counteract the inhibitory effects of ethanol in these strains. Furthermore, the presence of unsaturated fatty acids increased lipid droplet accumulation following ethanol shock, possibly in an effort to reduce the toxic effects of these compounds on the cell during ethanol stress. Overall, this study showed that M. pulcherrima, K. marxianus as well as T. delbrueckii require specific lipid combinations for an increase in fermentation rate. Furthermore, the species-specific response to lipid mixtures may be due to differences in the lipid composition, acetyl-CoA metabolism and redox balance. Moreover, this study showed that the presence of polyunsaturated fatty acids during ethanol shock may be detrimental for selected non- Saccharomyces yeasts. Specific lipid mixtures and oxygen sparging could be suggested to winemakers interested in increasing non-Saccharomyces yeast persistence during alcoholic fermentation and ultimately the metabolic footprint as well as quality of wine.