Winemaking practices affecting glutathione concentrations in white wine

Kritzinger, Engela Cornelia (2012-03)

Thesis (MScAgric)--Stellenbosch University, 2012.

Thesis

ENGLISH ABSTRACT: Glutathione (GSH), a tripeptide consisting of glutamate, cysteine and glycine, is the most ubiquitous non‐protein intracellular thiol in a large variety of organisms, including plants, animals and fungi. The thiol moiety of the cysteine residue confers unique redox and nucleophilic properties. In plant cells, GSH fulfils an indispensible role in the antioxidant system, sulphur metabolism and detoxification of xenobiotics. Upon grape crushing, GSH is extracted into the juice where it exerts several protective effects during the vinification process. In must, it reacts with oxidized phenolic compounds to form the colourless grape reaction product (GRP) which limits must browning to a certain extent. During wine ageing, GSH impedes the decrease of important aroma compounds, including certain esters, terpenes and volatile thiols, while at the same time preventing the development of atypical ageing off‐flavours. GSH may also inhibit the yellowing of wine during ageing. It is thus evident that elevated GSH levels in wine, in particular white wine which is more sensitive to oxidation, may be highly valuable for wine quality. The reductive crushing and pressing of white grape varieties, which limits oxidation and the consequent incorporation of GSH into GRP, promotes higher GSH levels in the juice. The reductive handling of juice also limits the formation of oxidized glutathione (GSSG). However, during alcoholic fermentation and maturation, levels generally decrease as a result of assimilation by the yeast Saccharomyces cerevisiae, and inevitable oxidation that takes place during the vinification process. The principal focus of this study was to gain a better understanding of the fate of glutathione during alcoholic fermentation and to establish whether certain oenological applications could result in elevated wine GSH levels. The application studied, included choice of yeast strain, extended lees contact, nitrogen supplementation and supplementation with glutathione enriched inactive dry yeast preparations (GHS‐IDYs). In addition, the need for a rapid analytical method for the simultaneous quantification of both GSH and GSSG in must and wine which does not involve derivatization or require extensive sample preparation, led to the development of a novel UPLC‐MS/MS method. The method was also employed to determine intracellular GSH and GSSG contents of the yeast S. cerevisiae and was studied for the first time in winemaking conditions. It was shown that the GSH levels fluctuated during alcoholic fermentation, suggesting the uptake and release by yeast. At the end of alcoholic fermentation, levels were generally lower than those initially present in grape juice, but in some cases, concentration increases were also observed. This finding indicates that, in some cases, endogenously‐produced GSH may be secreted into must during alcoholic fermentation, contributing to higher GSH levels in wine. Albeit small, significant differences in GSH content could be seen in wines fermented with different yeast strains, implying that yeast strain may to a certain extent influence wine GSH levels. While the effects of lees ageing and nitrogen supplementation seem to be insignificant in contributing to higher GSH levels in wine, the supplementation of must with GSH‐IDYs could result in increased wine GSH levels, provided the supplementation is done early during fermentation. This study has broadened our knowledge of several oenological factors, influencing GSH levels in wine and provided a new baseline for future research studies.

AFRIKAANSE OPSOMMING: Glutatioon (GSH), ’n tripeptied bestaande uit glutamaat, sisteïen en glisien, is die mees algemene nieproteïenagtige intrasellulêre tiool in ‘n wye verskeidenheid organismes, insluitende plante, diere en fungi. Die tioolfunksiegedeelte van die sisteïenresidu verleen unieke redoks‐ en nukleofiliese eienskappe. GSH vervul ‘n onmisbare rol in die antioksidantsisteem, swaelmetabolisme en die ontgiftiging van xenobiotika in plantselle. Tydens die maal van druiwe word glutatioon in die sap geëkstraeer waar dit verskeie beskermende effekte tydens die wynbereidingsproses uitoefen. GSH reageer met geöksideerde fenoliese verbindings om die kleurlose druifreaksieproduk (DRP) te vorm wat die verbruining van mos in ‘n sekere mate beperk. GSH verminder ook die afname van belangrike aromaverbindings tydens wynveroudering, insluitende sekere esters, terpene en vlugtige tiole, terwyl dit terselfdertyd die vorming van atipiese verouderingswangeure belemmer. So ook kan GSH die vergeling van wyn tydens veroudering inhibeer. Dit is dus voor die hand liggend that verhoogde GSH‐vlakke in wyn, in die besonder witwyn, wat meer oksidasie‐sensitief is, van waarde kan wees vir wynkwaliteit. Die reduktiewe maal en pers van witdruifvariëteite wat oksidasie en gevolglike inkorporasie van GSH in DRP beperk, bevorder hoër GSH‐vlakke in sap. So ook beperk die reduktiewe behandeling van sap die vorming van geoksideerde glutatioon (GSSG). Gedurende alkoholiese gisting en veroudering neem GSH‐vlakke egter af as gevolg van assimilasie deur die gis, Saccaromyces cerevisiae, asook onvermydelike oksidasie wat gedurende die wynbereidingsproses plaasvind. Die hooffokus van die studie was om ‘n beter begrip van die lot van glutatioon tydens alkoholiese gisting te verkry en om vas te stel of sekere wynkundige praktyke verhoogde GSH‐vlakke in wyn tot gevolg kan hê. Die studie het gisraskeuse, verlengde gismoerkontak, stikstofaanvulling en aanvulling met glutatioon‐verrykte, onaktiewe droëgis ingesluit. Daarbenewens het die behoefte aan ‘n vinnige analitiese metode vir die gelyktydige kwantifisering van sowel GSH as GSSG in mos en wyn wat nie derivatisering of uitgebreide monstervoorbereiding vereis nie, gelei tot die ontwikkeling van ‘n nuwe UPLC‐MS/MS metode. Hierdie metode is ook gebruik om die intrasellulêre GSH‐ en GSSG‐inhoud van die gis S. cerevisiae te bepaal wat vir die eerste keer in wynbereiding bestudeer is. Daar is bewys dat GSH‐vlakke tydens alkolholiese gisting fluktueer, wat dui op die opname en vrystelling daarvan deur die gis. Die vlakke aan die einde van alkoholiese gisting was oor die algemeen laer as vlakke aanvanklik teenwoordig in die sap. In sommige gevalle is konsentrasietoenames egter ook waargeneem. Hierdie bevinding dui daarop dat intrasellulêr‐vervaardige GSH, in sommige gevalle, in die mos uitgeskei kan word, wat tot hoër GSH‐vlakke in wyn lei. Klein, dog beduidende verskille in GSHinhoud is waargeneem in wyne wat met verskillende gisrasse berei is, wat daarop dui dat gisras in ‘n sekere mate die GSH‐vlakke in wyn kan beïnvloed. Alhoewel die effek van gismoerveroudering en stikstofaanvulling onbeduidend is, kan die aanvulling van mos met glutatioon‐verrykte, onaktiewe droëgis tot verhoogde GSH‐vlakke in wyn lei, mits die aanvulling vroeg tydens alkoholiese gisting gedoen word. Hierdie studie verbreed ons kennis van verskeie wynkundige praktyke wat GSH‐vlakke in wyn beïnvloed en vorm ‘n nuwe basis vir toekomstige navorsingstudies.

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