The use of enzymes for increased aroma formation in wine

[摘要] ENGLISH ABSTRACT:Monoterpene alcohols (monoterpenols) play an important role in the flavour and aroma ofgrapes and wine. This is especially applicable to wines of a muscat variety, but theseflavour compounds are also present in other non-muscat grape varieties, where theysupplement 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, nonvolatileglycosidic complexes. These complexes most often occur as6-0-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), or6-0-p-D-apiofuranosyl-p-D-glucopyranosides of mainly linalool, geraniol, nerol, a-terpineoland hotrienol. These precursors are, however, hydrolyzed only to a limited extent byendogenous glycosidases during the fermentation process, as they exhibit very low activityin wine conditions.The monoterpenols can be released from their sugar moieties by one of twomethods: either an acid or an enzymatic hydrolysis. The enzymatic hydrolysis mechanismis fully understood, and the process functions in two successive steps: firstly, depending onthe precursor, the glycosidic linkage is cleaved by an a-L-arabinofuranosidase, ana-L-rhamnosidase, a p-D-xylosidase, or a p-D-apiosidase. The second step involves theliberation of the monoterpene alcohol by a p-glucosidase. This enzymatic hydrolysis doesnot influence the intrinsic aromatic characteristics of the wine, as opposed to acidhydrolysis.As the endogenous grape glycosides of Vitis vinifera and the yeast Saccharomycescerevisiae show very low activity towards these aromatic precursors during the handling ofthe juice and winemaking processes, the focus has increasingly fallen on introducingexogenous p-glucosidases to wines and juices. Genes encoding p-glucosidases anda-L-arabinofuranosidases have been cloned from various organisms, including bacteria,fungi and yeasts. However, the activities and properties of these enzymes are not alwayssuitable for exploitation under winemaking conditions, where a low pH, low temperatures,and high ethanol and glucose concentrations prevail. A genetically engineered wine yeaststrain of S. cerevisiae that expresses glycosidases that are active in these conditions wouldbe useful in improving the flavour and aroma of wines, thereby adding to the complexityand value of the wine.Two p-glucosidase genes, BGL 1 and BGL2 from Saccharomycopsis fibufigera, weresubcloned into two Escherichia coli-yeast shuttle vectors. A dominant selectable markergene (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 laboratorystrain, L1278, was transformed to confirm the effective secretion of the expressed protein.An industrial yeast strain, VIN13, was subsequently transformed by making use of theselectable marker (resistance against sulfometuron). Enzyme assays with the syntheticsubstrate p-nitrophenol-j3-D-glucopyranoside (pNPG) were performed to determine theactivity of the j3-glucosidases over a period of days, as well as at certain temperatures andpH values. The stability of the enzymes was also investigated.These recombinant yeasts were able to degrade the pNPG efficiently. They showedpromising results concerning pH optima, with a substantial amount of activity found at thepH levels as found in the wine environment. There was also a slight increase in specificactivity at lower temperatures. The recombinant yeast strains were also tested in smallscalefermentations. Three wines were made, of which two were from white cultivars(Chenin blanc and GewOrtztraminer) and one from red (Pinotage). Results obtained frommicro-extraction from the finished wines showed that the terpenol content did increase,although this was not the only wine component influenced. Other flavour compounds alsoshowed increases, especially the esters. This also played a role in the flavour increase inthe wine.Future work would include optimizing the available results. This would entail theaddition of another glycosidic enzyme, such as a-L-arabinofuranosidase, to the genome ofthe wine yeast to aid the further breakdown of glycosidic bonds. The cloning or engineeringof a j3-glucosidase enzyme that is more active at low temperatures would also yield betterresults and release even more of the aroma of the wine.

[发布机构] Stellenbosch University