Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Proteins from various sources, including grape berry cells, yeast, bacteria and fining agents
e.g. albumin and casein, have previously been identified in wine. These proteins play various
critical roles in the functioning and survival of the organisms that produced them but also
exhibit oenological properties, once secreted in the juice/wine. Some of them can indeed be
beneficial to winemaking, by releasing aroma compounds from grape-derived precursors, or
detrimental to wine quality, by causing protein haze. Yeasts contribute significantly to the
protein pool during and after alcoholic fermentation. However, while the extracellular proteins
of Saccharomyces cerevisiae, the main wine yeast species, have been characterised, those
of non-Saccharomyces yeasts remain largely unknown, especially under winemaking
conditions. Although specific extracellular enzymes released by non-Saccharomyces yeasts
have been the focus of many studies in recent years, the targeted approaches used have
restricted our knowledge to these specific enzymes and excluded the other secreted
proteins. A more comprehensive insight into entire secretomes could improve our
understanding of how yeasts survive in wine and interact with other species in mixed culture
fermentations.
This study aims to characterise the exo-proteome of Saccharomyces and selected
non-Saccharomyces yeasts in pure and mixed cultures in a wine-like medium.
Fermentation kinetics were monitored and the extracellular proteins isolated at the end of
fermentation. M. pulcherrima hardly fermented whereas L. thermotolerans fermented slowly
but steadily. As expected S. cerevisiae completed the fermentation rapidly. In sequential
fermentations, the kinetics resembled those of the non-Saccharomyces yeasts for a period
before switching to that of S. cerevisiae. This period varied from 4 to 15 days for M. pulcherrima and L. thermotolerans respectively.
Visual observations of the protein content of the medium at the end of fermentation using 1D
and 2D SDS-PAGE gels as well as identification of these proteins using mass fingerprinting
revealed the large variety of proteins secreted and the influence of yeast interactions on
each other’s secretome. The fermentation kinetics observed could partially be explained by
the extent of the contribution of the different yeast to the protein content.
Proteins secreted by non-Saccharomyces yeasts lowered the potential of wine to form
protein haze, with both M. pulcherrima and L. thermotolerans in pure and mixed culture
fermentations showing lower haze formation than S. cerevisiae.
As far as we know, this is the first report on the secretome of non-Saccharomyces under
winemaking condition and the influence non-Saccharomyces proteins have on the protein
haze potential of wine, providing the basis for future investigations. / AFRIKAANSE OPSOMMING: Proteïene vanaf verskeie bronne (insluitend druiwe korrels, gis, bakterieë en verhelderings
agente bv. albumien en kaseïen) is reeds in wyn identifiseer. Hierdie proteïene speel
verskeie rolle in die funksionering en oorlewing van die organismes wat dit produseer, maar
beskik ook oor wynkundige eienskappe sodra dit in die sap of wyn uitgeskei word. Hoewel
sommige proteïene in wyn wel voordelig mag wees as gevolg van die vrystelling van aroma
komponente vanuit druif‐voorlopers, kan dit ook nadelig wees vir wyn kwaliteit deur die troebelheid
wat dit kan veroorsaak Gis dra aansienlik by tot die totale proteïen inhoud van wyn, beide
gedurende asook na alkoholiese fermentasie. Alhoewel die ekstrasellulêre proteïene van
Saccharomyces cerevisiae (die mees algemeen gebruikte gis vir wynmaak) reeds goed
gekarakteriseer is, is die proteïene van nie-Saccharomyces giste grootliks onbekend, veral
die wat tydens wynmaak vrygestel word. Gedurende die laaste paar jaar het verskeie
studies gefokus op spesifieke ekstrasellulêre ensieme wat deur nie-Saccharomyces giste
produseer word, maar geteikende benaderings het ons kennis beperk tot net hierdie
spesifieke ensieme, en enige ander afgeskeide proteïene uitgesluit. ʼn Meer omvattende
insig oor die algehele afgeskeide proteoom kan ons begrip van hoe gis in wyn oorleef en
interaksies tussen gis spesies in gemengde kultuur fermentasies verbeter
Hierdie studie streef om die sekretoom van Saccharomyces en geselekteerde
nie-Saccharomyces giste in suiwer en gemengde kultuur fermentasies van sintetiese wyn
medium te karakteriseer. Fermentasie kinetika is gemonitor en die ekstrasellulêre proteïene is teen die einde van
fermentasie geïsoleer. Metschnikowia pulcherrima het swak fermenteer terwyl Lachancea
thermotolerans stadig tog reëlmatig fermenteer het. Soos verwag, het S. cerevisiae vinnig tot
droog fermenteer. In agtereenvolgend geïnokuleerde fermentasies is die kinetika vir ʼn
tydperk soortgelyk aan die van die nie-Saccharomyces giste voordat dit oorskakel na die van
S. cerevisiae. Hierdie tydperk wissel respektiewelik vanaf 4 tot 15 dae vir M. pulcherrima en
L. thermotolerans.
Visuele waarnemings van die proteïen-inhoud van die medium aan die einde van die gisting
met behulp van 1D en 2D SDS-PAGE gels asook identifisering van hierdie proteïene met
behulp van massa vingerafdrukke onthul die groot verskeidenheid proteïene wat afgeskei
word, asook die invloed van die giste se interaksies op mekaar se sekretoom. Die
fermentasie kinetika waargeneem kan gedeeltelik verklaar word deur die omvang van die
bydrae van die verskillende gis tot die proteïen-inhoud. Proteïene wat afgeskei word deur
nie-Saccharomyces giste verlaag die potensiaal van wyn om proteïen troebelheid te vorm, met beide M. pulcherrima en L. thermotolerans (in suiwer en gemengde kultuur
fermentasies) wat minder troebelheid vorm as fermentasies met S. cerevisiae.
Sover ons kennis strek, is hierdie die eerste verslag oor die sekretoom van nie-
Saccharomyces onder wynmaak toestande en ook oor die invloed wat nie-Saccharomyces
proteïene op die proteïen troebelheid van wyn het, en vorm die basis vir toekomstige
navorsing. / Winetech and THRIP
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/79989 |
| Date | 03 1900 |
| Creators | Mostert, Talitha Tanya |
| Contributors | Divol, B. T., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 88 p. : ill. |
| Rights | Stellenbosch University |
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