The development of polysaccharide degrading wine yeast strains

[摘要] ENGLISH ABSTRACT: The polysaccharides that are present in wine originate from the grapes, the fungi thatgrow on the grapes and from other microorganisms that come into contact with themust during winemaking. The grape-derived polysaccharides of most concern inwinemaking are pectin, glucan and xylan that can be enzymatically degraded bypectinases, glucanases and xylanases, respectively. These are the main structuralpolysaccharides of the cell wall of the grape cell. Degradation of the cell walls willresult in the separation and rupture of the grape cells, and cell wall-boundcompounds will be released into the must. Treating the must with pectinase andmacerating enzyme preparations can result in an increase in free-flow juice, animprovement in must clarification and filtration, and an increased extraction ofphenols and tannins. The tannins that are extracted polymerise with anthocyanins inred wine during ageing, resulting in increased colour intensity and stability. Winearoma is also influenced by enzyme treatment. The degradation of the cell wallcontributes to the release of glycosidically-bound terpene or alcohol precursors fromthe berries. The hydrolysis of these precursors during fermentation can result in animprovement in aroma. It can thus be seen that it is possible to improve wine qualityand processing by supplementing the endogenous enzymes that are present in thefermentation with commercial enzyme preparations. Commercial enzymes aretypically crude fungal preparations.The majority of commercial pectinase and glucanase preparations are derivedfrom Aspergillus and Trichoderma, respectively. Since the endogenouspolysaccharase activity of Saccharomyces cerevisiae is very limited, theheterologous expression of specific polysaccharase genes in an industrial yeaststrain can improve the winemaking process, resulting in a higher quality wine withoutthe addition of expensive commercial enzyme preparations. Since only the desiredenzymes are secreted by the recombinant strain, there will be no undesired sideactivities,which can be detrimental to wine quality. Several pectinase-, glucanaseandxylanase-encoding genes, cloned from a variety of organisms, have beenexpressed successfully in laboratory strains of S. cerevisiae. Attempts have alsobeen made to construct industrial wine yeast strains that express thesepolysaccharase genes and secrete the encoded enzymes. Fermentation with someof these strains resulted in a decrease in total phenolics and turbidity, an increase injuice extraction, and alterations in the colour and aromatic profile of the resultingwines.In this study, four polysaccharide-degrading, recombinant wine yeast strains wereconstructed. The endo-β-1,4-xylanase gene, XYN2, and the endo-β-1,4-glucanasegene, end1, were previously cloned from the soft rot fungus Trichoderma reesei andthe rumen bacterium Butyrivibrio fibrisolvens, respectively. These genes weresubcloned into different expression cassettes which were used to construct the fourintegration plasmids. The recombinant plasmids contained the following genecassettes: TEF1P-XYN2-ADH2T (plasmid pDLG29) ADH1P- MFα1S -end1-TRP5T (plasmid pDLG30) ADH1P-MFα1S-end1-TRP5T andADH2P-XYN2-ADH2T (plasmid pDLG33), ADH1P-MFα1S-end1-TRP5T and YG100PXYN2-ADH2T (plasmid pDLG39). These four plasmids were then separatelyintegrated into the ILV2 locus of the commercial wine yeast strain S. cerevisiaeVIN13. Wine was made with the four strains constructed in this study, a pectolyticstrain, VIN13[pPPK], a glucanase- and xylanase-secreting strain, VIN13[pEX], anuntransformed VIN13 strain, and an untransformed strain with the addition of thecommercial enzyme preparation Rapidase EX Colour. Microvinification experimentswere carried out on Pinot noir, Ruby Cabernet and Muscat d'Alexandria wines.Fermentation with the polysaccharide-degrading strains resulted in significantimprovements in juice extraction, colour intensity and stability, and in alterations inthe aromatic profiles of the wines produced.Subject to the approval by the regulatory authorities and eventual consumeracceptance of the use of genetically modified organisms (GMOs) in fermented foodsand beverages, it might be required that the GM status of the yeast that is usedappears on the label. Currently, there is no robust technique available with which theuse of GM yeast can be revealed in a finished wine because the yeast cells and theirDNA are removed from or denatured in the wine during filtration and processing. Oneway with which the undeclared use of a GM yeast in winemaking could be exposedwould be to compare the chemical profile of a suspect wine with that of non-GM wine.In order to explore this concept further, a secondary aim of this study was toinvestigate whether Fourier Transformation Infra Red (FT-IR) spectroscopy coupledwith multivariate data analysis could distinguish between wines fermented withtransgenic and non-transgenic yeast strains, or between wines fermented withdifferent transgenic strains. The results showed that this method could be used toclassify wines fermented with different yeast strains if fermentation with the strainresulted in a unique chemical profile in the resulting wine. This was a preliminarystudy and these findings were summarised as an addendum to the thesis.

[发布机构] Stellenbosch University