Thesis (MScAgric)--Stellenbosch University, 2012. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/20295 |
| Date | 03 1900 |
| Creators | Kritzinger, Engela Cornelia |
| Contributors | Du Toit, W. J., Bauer, F. F., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. |
| Publisher | Stellenbosch: Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 95 p. : ill. |
| Rights | Stellenbosch University |
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