Improvement of Wine Quality Through the Release of Terpenes

Project Number
MIC 01

Project title
Improvement of wine quality through the release of terpenes

Project leader
Killian, S G

University of Orange Free State

Team members
Makhele, N L
Butler, M

Project description
Terpenols, which are important flavour compounds in many wines, mostly occur in grape juice in the form of flavourless terpenyl glucosides. Wine yeasts have a limited ability to hydrolyse these compounds with the release of free terpenols. An investigation into the nature of the wine yeast enzyme or enzymes involved in the hydrolysis of terpenyl glucosides was, therefore, undertaken. The long-term aim is to improve the terpenyl glucoside splitting ability of wine yeasts by increasing the production and secretion of the relevant enzymes.

We attempted to synthesise and isolate terpenyl glucosides to use as natural substrates for the evaluation of protein fractions with ß-glucosidic activity isolated from Saccharomyces cerevisiae. A method using a transglycosylating enzyme was used to transfer glucose from cellobiose to terpenols that are important to wine flavour. The resulting glucosides were extracted from the reaction mixture using an adaptation of a microwave extraction method with ethyl acetate. The results of thin layer chromatography (TLC) analysis and analysis of products of hydrolysis by a commercial ß-glucosidase were compatible with the products being terpenyl glucosides. However, subsequent gas chromatographic (GC) analysis and proton nuclear magnetic resonance (NMR) spectroscopic analysis showed that the products were ethyl glucosides and not terpenyl glucosides. It was concluded that isolation of terpenyl glucosides from wine or grape juice is a better alternative to obtain the necessary substrates for enzyme assays.

Eleven commercial wine yeasts, five laboratory strains of Saccharomyces cerevisiae and yeasts that are known producers of ß-glucosidase were tested against the synthetic ß-glucosides PNPG and b-MUG as substrates. All the yeasts tested produced both cell associated and extracellular ß-glucosidic activity, although greater levels of activity were produced by the known b-glucosidase producers. The occurrence of ß-glucosidic activity in different cell fractions of Saccharomyces cerevisiae CBS2444 was further investigated. Partially purified fractions with activity against b-MUG were isolated from the culture supernatant, cell extract and insoluble cellular material using column chromatographic techniques. These fractions can be tested against isolated terpenyl glucosides to identify the fraction or fractions responsible for the hydrolysis of terpenyl glucosides.

Reports in literature indicate that the ability to hydrolyse ß-glucosides is rare among strains of Saccharomyces cerevisiae. Our results show that, in fact, this activity is present in all strains we tested. Our results also show that ß-glucosidic activity is present in all cellular fractions of Saccharomyces cerevisiae and in different protein fractions. This knowledge can be applied to devise strategies for the improvement of terpenyl glucoside hydrolysis during wine fermentation through enhancement of the production and secretion of these enzymes by wine yeasts.

Nieuwoudt, H H, Prior, B A, Pretorius, I S, Bauer, F F. 2002. Glycerol in South African table grapes: an assessment of its contribution to wine quality, South African Journal of Enology and Viticulture, v. 23 (p. 22-30)


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