Masters Degrees (Microbiology)

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Browsing Masters Degrees (Microbiology) by Subject "Alcoholic beverages -- Flavor and odor"

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    The use of enzymes for increased aroma formation in wine
    (Stellenbosch : Stellenbosch University, 2001-12) Stidwell, Tanya Gwendryth; Pretorius, I. S.; Van Rensburg, P.; Lambrechts, M. G.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Monoterpene alcohols (monoterpenols) play an important role in the flavour and aroma of grapes and wine. This is especially applicable to wines of a muscat variety, but these flavour compounds are also present in other non-muscat grape varieties, where they supplement other varietal flavours and aromas. These monoterpenols can be found in grapes and wine as free, volatile and odorous molecules, as well as in flavourless, nonvolatile glycosidic complexes. These complexes most often occur as 6-0-a-L-arabinofuranosyl-p-D-glucopyranosides (vicianosides), 6-0-P-D-xylopyranosyl- P-D-gluco-pyranosides (primverosides), 6-0-P-D-glucopyranosyl-p-D-glucopyranosides (gentio-biosides ), 6-0-a-L -rhamnopyra nosyl-p-D-g lucopyra nos ides (rutinos ides), or 6-0-p-D-apiofuranosyl-p-D-glucopyranosides of mainly linalool, geraniol, nerol, a-terpineol and hotrienol. These precursors are, however, hydrolyzed only to a limited extent by endogenous glycosidases during the fermentation process, as they exhibit very low activity in wine conditions. The monoterpenols can be released from their sugar moieties by one of two methods: either an acid or an enzymatic hydrolysis. The enzymatic hydrolysis mechanism is fully understood, and the process functions in two successive steps: firstly, depending on the precursor, the glycosidic linkage is cleaved by an a-L-arabinofuranosidase, an a-L-rhamnosidase, a p-D-xylosidase, or a p-D-apiosidase. The second step involves the liberation of the monoterpene alcohol by a p-glucosidase. This enzymatic hydrolysis does not influence the intrinsic aromatic characteristics of the wine, as opposed to acid hydrolysis. As the endogenous grape glycosides of Vitis vinifera and the yeast Saccharomyces cerevisiae show very low activity towards these aromatic precursors during the handling of the juice and winemaking processes, the focus has increasingly fallen on introducing exogenous p-glucosidases to wines and juices. Genes encoding p-glucosidases and a-L-arabinofuranosidases have been cloned from various organisms, including bacteria, fungi and yeasts. However, the activities and properties of these enzymes are not always suitable for exploitation under winemaking conditions, where a low pH, low temperatures, and high ethanol and glucose concentrations prevail. A genetically engineered wine yeast strain of S. cerevisiae that expresses glycosidases that are active in these conditions would be useful in improving the flavour and aroma of wines, thereby adding to the complexity and value of the wine. Two p-glucosidase genes, BGL 1 and BGL2 from Saccharomycopsis fibufigera, were subcloned into two Escherichia coli-yeast shuttle vectors. A dominant selectable marker gene (SMR1) was also inserted onto these plasmids. These plasmids were designated pBGL 1 (containing the BGL 1 gene) and pBGL2 (containing the BGL2 gene) respectively. Introduction of the two plasmids into two strains of S. cerevisiae then followed. A laboratory strain, L1278, was transformed to confirm the effective secretion of the expressed protein. An industrial yeast strain, VIN13, was subsequently transformed by making use of the selectable marker (resistance against sulfometuron). Enzyme assays with the synthetic substrate p-nitrophenol-j3-D-glucopyranoside (pNPG) were performed to determine the activity of the j3-glucosidases over a period of days, as well as at certain temperatures and pH values. The stability of the enzymes was also investigated. These recombinant yeasts were able to degrade the pNPG efficiently. They showed promising results concerning pH optima, with a substantial amount of activity found at the pH levels as found in the wine environment. There was also a slight increase in specific activity at lower temperatures. The recombinant yeast strains were also tested in smallscale fermentations. Three wines were made, of which two were from white cultivars (Chenin blanc and GewOrtztraminer) and one from red (Pinotage). Results obtained from micro-extraction from the finished wines showed that the terpenol content did increase, although this was not the only wine component influenced. Other flavour compounds also showed increases, especially the esters. This also played a role in the flavour increase in the wine. Future work would include optimizing the available results. This would entail the addition of another glycosidic enzyme, such as a-L-arabinofuranosidase, to the genome of the wine yeast to aid the further breakdown of glycosidic bonds. The cloning or engineering of a j3-glucosidase enzyme that is more active at low temperatures would also yield better results and release even more of the aroma of the wine.