Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: In the present-day competitive global market, wine industries are constantly
aiming to improve the wine-making process,including the role of yeast. The
most commonly used wine yeast is Saccharomyces cerevisiae, which is able to
produce high quality wines, but problem fermentations do sometimes arise.
The occurrence of stuck and sluggish fermentations pose a serious problem
leading to loss of productivity and quality. Although the precise mechanism
leading to stuck fermentations is unknown, they are often correlated with
high fructose to glucose ratios in the wine-must. S. cerevisiae is a glucophylic
yeast, indicating its preference for consuming glucose over fructose. Both these
hexose sugars are present in unfermented wine must, mostly in equal concentrations.
As fermentation progresses, glucose is consumed at a faster rate than
fructose, leading to an increase in the fructose to glucose ratio. Yeast are left
with the undesirable fructose at the later stages of fermentation, when the
environmental stresses on the yeast can lead to stuck or sluggish fermentation.
This residual fructose can lead to undesirable sweetness, as fructose is
about twice as sweet as glucose. Even with the extensive research into yeast
metabolism, there is as yet no definitive explanation as to why yeasts ferment
glucose faster than fructose.
This study aimed to investigate the mechanism responsible for the faster consumption
of glucose over fructose of a commercially used wine yeast strain
S. cerevisiae VIN 13. The first two steps of sugar metabolism, uptake and
phosphorylation, were investigated as the possible sites of discrepancy in fermentation
rates. Enzyme rates and affinities for both glucose and fructose as substrates for the relevant enzymes were experimentally determined. These
kinetic parameter values were used to improve an existing model of yeast glycolytic
pathway to model wine fermentations. The feasibility of constructing
and validating a kinetic model of wine fermentations were investigated, by
comparing model predicted fluxes with experimentally determined fluxes.
Another aspect of this study was an investigation into the effect of hexose
sugar type on fermentation profiles. Wine fermentations were done with only
one hexose sugar as carbon source to determine if it has an effect on the flux
through metabolism.
This work succeeded in the construction of a kinetic model that distinguished
between glucose and fructose as carbon source. The glucose was consumed
faster than fructose, with control lying in the hexose transport step. It was
also established that fermentation prfiles of fermentations with only one sugar
was the same for both one sugar type fermentations. Fermentation with either
glucose or fructose as the sole carbohydrate source had the same specfic
production and consumption rates as normal fermentations with both sugars.
Construction of detailed kinetic models can aid in the metabolic and cellular
engineering of novel yeast strains. By identifying the importance of hexose
transport, and thus the glucophilic character of the yeast, in flux control, yeast
transporters can be targeted for strain improvement. This may in turn lead to
more effective fermentation practices for controlling problem fermentations, or
to the development of novel strains that utilizes fructose in the same manner as
glucose, and in so doing lower the risk of stuck or sluggish wine fermentation. / AFRIKAANSE OPSOMMING: In die hedendaagse kompeterende wynmark is wynmakers aanhoudend besig
om die wynmaak proses te verbeter en dit sluit die verbetering van wyngis
in. Die mees algemeenste gebruikte wyngis is Saccharomyces cerevisiae, omdat
dit wyn van gehalte produseer, maar probleem fermentasies kom wel voor.
Die verskynsel van vasval of stadige fermentasies kan lei tot die verlies van
produksie en kwaliteit. Die oorsaak van probleem fermentasies is gewoontlik
veelvoudig, maar die verhouding van glukose tot fruktose in die wyn-mos kan
ongunstig raak om fermentasies te onderhou. S. cerevisiae is 'n glukofiliese
gis, wat sy voorkeur om glukose bo fruktose te gebruik beskryf. Albei hierdie
heksose suikers is teenwoordig in ongefermenteerde wyn-mos, meestal in gelyke
hoeveelhede. Soos fermentasies vorder word glukose vinniger verbruik as
fruktose wat lei tot 'n toename in die fruktose tot glukose verhouding. Die gis
moet dus die fruktose in die later stadium van fermentasie gebruik wanneer
die omgewings druk op die gis kan lei tot probleem fermentasies. Die oorblywende
fruktose kan lei tot ongewenste soetheid aangesien fruktose twee keer
soeter is as glukose. Selfs met die ekstensiewe navorsing met betrekking tot
gis metabolisme is daar nog nie 'n verduideliking hoekom gis glukose vinniger
as fruktose gebruik nie.
Hierdie studie het beoog om die meganisme wat lei tot die vinniger verbruik
van glukose oor fruktose te ondersoek vir 'n kommersieël gebruikte gis S. cerevisiae VIN 13. Die eerste twee stappe van suiker metabolisme, suiker opname
en fosforilasie, was ondersoek as die moontlike punt van die verskil in fermentasie
tempo. Ensiem snelhede en affiniteite vir beide glukose en fruktose as substrate vir die ensieme van belang was eksperimenteel bepaal. Hierdie
waardes is gebruik om 'n bestaande model van gis glikolise aan te pas vir wyn
fermentasies. Die uitvoerbaarheid van saamstel en valideer van 'n kinetiese
model van wyn fermentasies was ondersoek, deur model voorspelde fluksie
waardes met eksperimentele fluksie waardes te vergelyk.
'n Ander aspek van die studie was die ondersoek van die effek van heksose
suiker tipe op fermentasie profiel. Wyn fermentasies is gedoen met slegs een
heksose suiker as koolstof bron om te bepaal of dit 'n invloed het op die fluksie
deur metabolisme.
Hierdie werk het daarin geslaag om 'n kinetiese model saamtestel wat onderskei
tussen glukose en fruktose as koolstof bron. Die glukose is vinniger verbruik as
fruktose, met beheer gesetel in die heksose opname stap. Dit was ook vasgestel
dat fermentasie profiele van fermentasies met slegs een suiker nie verskil het
vir fermentasies met slegs fruktose of glukose. Fermentasies met slegs een
suiker het dieselfde spesifieke produksie en konsumpsie tempo gehad as die
normale fermentasie met albei suikers. Die konstruksie van 'n gedetailleerde
kinetiese model kan gebruik word in die metaboliese en sellulêre ontwikkeling
van nuwe gisstamme. Deur die ontdekking van die belangrikheid van heksose
opname in fluksie beheer, wat lei tot die glukofiliese karakter van gis, kan gis
opname geteiken word vir gis ontwikkeling. Dit mag om die beurt lei tot meer
effektiewe fermentasie praktyk in die beheer van probleem fermentasies, of die
ontwikkeling van nuwe stamme wat fruktose in dieselfde manier as glukose
benut, en sodoende die risiko van vasval of stadige wyn fermentasies verlaag. / National Research Foundation / Post-graduate Merit Bursary
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/80316 |
| Date | 03 1900 |
| Creators | Mocke, Leanie |
| Contributors | Snoep, J. L., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xiii, 64 p. : ill. |
| Rights | Stellenbosch University |
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