INVESTIGATION ON CYSTEINYL THIOL COMPOUNDS FROM YEAST AFFECTING WINE PROPERTIES

Thiol compounds carry out several activities in must and wine. Among them, our attention was focused on the cysteinyl thiols (glutathione and free and protein bound cysteine). The glutathione (GSH) properties in oenological matrices are well known: it reduces the o-quinones arising from the hydroxycinnamic acids esters, limiting the formation of brown polymers. GSH can decrease the loss of thiol-related aromas and it prevents the sotolon (3-hydroxy-4,5-dimethyl-2(5H)furanone) formation, the main responsible of the atypical white wine ageing. The GSH constitutes the main thiol compound on grape, in must and wine and its content is affected by the winemaking practices. The cysteinyl residues on the yeast cell wall can limit the reduced/oxidized defects in wine ageing as they occur when the ageing sur lies or the addition of commercial yeast cell-wall fractions (YCWF) are carried out. The addition of such adjuvants is allowed by the European law (EU Regulation 2165/2005) and they exert several activities improving the sensorial properties of wine. The evaluation of their cysteinyl thiols level was the object of this research. The analytical approaches proposed in the literature are hard-to-apply for the routine analysis since the thiols quantification of YCWF was not reliable applying the methods described for the biological system proteins. The aims of this research were to set up and validate a sensitive and reliable analytical method for the thiols content determination in the YCWF. The cysteinyl thiols content of some commercial preparations and active dry yeast was assessed and it was correlated to the heat damage occurring during the industrial preparation. The same analytical approach was applied and validated to the quantification of GSH in grape juice, must and wine. The winemaking practices affecting the GSH level were evaluated in real processes both in vintage 2009 (8) and vintage 2010 (10). Finally, also the oxidation rate in white wine was estimated through the evaluation of the interactions between GSH, sulfur dioxide and phenols. The analytical approach proposed was based on the reaction between the thiol and p-benzoquinone (pBQ); the thio-substituted hydroquinones were separated by liquid chromatography and detected by spectrophotometry. The pBQ was added in excess and the unreacted amount was bound to an excess of 3-mercaptopropanoic acid. The derivatization was fast, accurate and stoichiometric at room temperature. The YCWF showed an heterogeneous thiols content; the lysate samples had the highest thiols concentration, as well as the GSH which could have been added by the producer to increase the antioxidant properties. The lowest amount of cysteinyl thiols was detected in the mannoprotein samples which were also able to deplete the free cysteine and GSH. Such a behavior can decrease the content of the low molecular weight thiols (e.g. flavour related thiols) naturally occurring in wine. Moreover, the heat damage was higher in the mannoprotein samples and it could be correlated to the low thiols level. The GSH content was assessed in some grape and wine samples. The sample preparation is reliable, fast and easy-to-apply. The levels detected were in accordance to the literature. The GSH level during the winemaking was low after pressing according to the must exposure to air. The GSH increased during the fermentation dependent to the yeast strain, the fermentation course and must aeration, as well as to the readily assimilable nitrogen and the copper content. At the end of the alcoholic fermentation, the decrease of GSH concentration was faster if the must preparation was performed in oxidative condition. The oxidation rate in white wine was evaluated through 2 experiments: in the first one the attention was focused on the interactions between GSH, sulfur dioxide and caffeic acid in depleted phenols wine and synthetic wine solution (12% ethanol, 2.5 g L-1 tartaric acid and pH adjusted at 3.5). In the second experiment, 13 South Africans Sauvignon blanc wines were added with sulfur dioxide and the phenols oxidation was evaluated. The quantification of GSH, caffeic acid and catechin was carried out by the validated UPLC method which was previously described. The statistical analysis showed the oxidation rate was mainly affected by the sulfur dioxide content; when the level of the latter was high the oxygen consumption decreased faster in presence of GSH, too. When GSH and sulfur dioxide were added at higher concentration, the comparison between wine and synthetic wine solution was allowed. The oxygen consumption was at least 2 times higher when the sulfur dioxide was added for 12 of the 13 Sauvignon blanc analyzed. Among the 21 parameters considered, it was mainly affected by the copper, free sulfur dioxide and ferulic acid level which constituted the 68% of the variation of the oxidation rate. Further investigations are needed to better understand the role of copper either during the grape pressing or the wine ageing as well as the interactions between GSH, sulfur dioxide and phenols occurring in white wine.