Thesis (PhD)--Stellenbosch University, 2010. / ENGLISH ABSTRACT: Popular wine styles prepared from fully-ripened, more mature grapes are characterized
by intense fruitiness and varietal flavors. However, lengthy maturation of
grapes in the vineyard does not only translate into higher flavor intensity but also
into higher sugar levels, which, in turn, leads to wines with higher concentrations of
alcohol. Excessive alcohol levels can compromise wine flavor and render wine unbalanced.
This, along with health issues and anti-social behavior linked to high-risk
alcohol consumption patterns, stricter legislation and increased tax rates associated
with high-alcohol wines, have increased demand for wines with reduced alcohol
concentrations, without loss of the intense fruity aromas. Although low-alcohol
wines can be made using physical post-fermentation processes, such approaches are
often expensive and can impact adversely on wine flavor. As an alternative strategy,
yeast strains are being developed by several research groups to convert some of the
grape sugars into metabolites other than ethanol.
Based on promising results from previous preliminary work, this study focused
on the development of an industrial Saccharomyces cerevisiae wine strain producing
glucose oxidase (GOX; b-D-glucose:oxygen oxidoreductase, EC 1.1.3.4).
GOX oxidizes b-D-glucose to D-glucono-d-lactone and gluconic acid (GA) extracellularly,
thus preventing its entry into glycolysis, thereby diverting a portion of the sugar carbon away from ethanol. The GOX-encoding gene from the foodgrade
fungus, Aspergillus niger was used to construct three cassettes (GOX1, GOX2
and GOX2LOX). In these gene cassettes, the A. niger GOX gene was placed under
the regulation of the S. cerevisiae phosphoglycerate-kinase-1 gene promoter
(PGK1P) and terminator (PGK1T ). To facilitate secretion, in GOX1 the yeast mating
pheromone-factor a secretion signal (MFa1S) was fused to the GOX gene, and
in GOX2 the native A. niger secretion signal of GOX was used. These gene cassettes
were each integrated into the genome of two laboratory yeast strains (BY4742 and
S1278b) and one industrial wine yeast strain (VIN13). An additional integration
cassette, designated GOX2LOX, was constructed to knock out the IME1 gene in S.
cerevisiae. In GOX2LOX, GOX2 was fused to a loxP cassette. VIN13-D1 was obtained
by integrating a single copy of GOX2LOX into the IME1 locus. To generate
an asporogenic, GOX-producing wine yeast, VIN13-D2 was created by sporulation,
micromanipulation and re-diploidisation of VIN13-D1. Comparative analysis indicated
that (i) GOX2 resulted in higher levels of extracellular glucose oxidase activity
than GOX1; and that (ii) the levels of secreted glucose oxidase activity in the wine
yeast transformants were sufficiently high to conduct follow-up small-scale wine
fermentation trials.
The wine yeast transformant, VIN13-D1 was evaluated under red and white experimental
winemaking conditions. Results from this work indicated that glucose
oxidase was produced and secreted by VIN13-D1 that dominated the fermentation
to the end, but also that the enzyme was not highly active under the evaluated winemaking
conditions. Consequently, no significant decrease in ethanol concentrations
was observed in the wine made from VIN13-D1 when compared to that from
VIN13. Wine samples were analyzed by Fourier transform-middle infrared spectrometry
(FT-MIR) to determine the chemical composition and Gas chromatography
with a flame ionization detector (GC-FID) to evaluate the concentrations of
aroma compounds. The levels of gluconic acid were determined by enzymatic assays.
Multivariate data analysis (PCA and PLS1-discrim) was applied to highlight
significant differences between the wines made by VIN13 (wild-type) and VIN13-
D1. Chemometric projections of the score plots for all results allowed insight into
all significant variation up to three principal components (PCA) or PLS components,
which showed very clearly that GA is a key factor in evaluating the effect of
GOX in VIN13-D1 fermentation with regard to VIN13 fermentations. The VIN13-
D1 effect manifestations were best shown on PLS1-discrim score plots that revealed that, of the restricted variable subsets the FT-MIR-compounds and GC-compounds
yielded better results, with the GC-compounds displaying greater discriminability
between cultivars and VIN13 / VIN13-D1. It can be concluded from these results
that the greatest influence of VIN13-D1 produced wines could be observed in the
aroma components, but, because there were also discriminability effects discernable
in the FT-MIR-compounds, thus the flavor components were also affected.
The activity of GOX in grape juice was further investigated in controlled small
scale fermentations performed in a bio-reactor. It was confirmed that GOX is active
under aerobic conditions, inactive under anaerobic conditions, and can be activated
instantly when an anaerobic culture is switched to aerobic conditions (simulated
micro-oxygenation). These fermentations showed that glucose oxidase is active in
grape juice, and that oxygen play a key-role in the enzyme’s activation. Finally, it
was shown with the help of a simplified model, that under ideal conditions, GOX
secreted from VIN13-D1, can be employed to reduce the ethanol by a predefined
concentration for the production of low alcohol wines.
This work gives more insight into how to employ a GOX-producing wine yeast
during winemaking and strongly suggests the use of micro-oxygenation to activate
the enzyme in order to reduce available glucose, thereby diverting a portion of the
sugar carbon away from ethanol production. / AFRIKAANSE OPSOMMING: Gewilde wynstyle word dikwels gemaak van volryp, goed ontwikkelde druiwe,
gekarakteriseer deur intense aromas en smaakkomponente wat direk met spesifike
kultivars geassosieer word. ’n Nadelige gevolg om druiwe te lank aan die wingerdstok
te laat bly hang sodat meer intense geurkomponente kan ontwikkel, is die
toename in suikerinhoud. Hierdie addisionele suiker lei tot wyne met hoër alkoholvlakke.
Te hoë alkoholvlakke kan wyn ongebalanseerd laat voorkom en die
smaak nadelig beïnvloed. Dit, tesame met gesondheidsredes en anti-sosiale gedrag
wat gekoppel kan word aan die inname van te veel alkohol, strenger wetgewing
aangaande dronkbestuur en die toename in belasting op wyne met ’n hoër alkoholinhoud,
het aanleiding gegee tot ’n aanvraag vir wyn met ’n verlaagte alkoholinhoud,
sonder dat aroma- en geurkomponente ingeboet word. Alhoewel daar sekere
fisiese/gemeganiseerde prosesse beskikbaar is om die alkohol in wyn te verwyder of
te verminder, is ’n nadeel dat hierdie prosesse baie duur en arbeidsintensief is, en dat
dit deur sommige wynpuriste as te ingrypend in die ‘natuurlike’ proses van wynmaak
beskou word. Sommige van hierdie alkoholverwyderingsprosesse kan ook die wyn se geur- en aromakomponente nadelig beïnvloed. As alternatief tot hierdie
fisies-chemiese prosesse word wyngiste reg oor die wêreld deur verskillende
navorsingsgroepe ontwikkel sodat van die druifsuikers nie na alkohol omgeskakel
word nie, maar eerder ander metaboliete.
Belowende navorsingsresultate in ’n voorafgaande studie het aanleiding gegee
tot hierdie navorsingsprojek. In hierdie studie word daar klem gelê op die ontwikkeling,
deur middel van genetiese manipulering, van ’n industriële wynras van
Saccharomyces cerevisiae sodat dit in staat sal wees om glukose-oksidase (GOX;
b-D-glukose:suurstof oksidoreduktase, EC 1.1.3.4) te produseer. GOX kan reeds
b-D-glukose in die medium oksideer na glukoonsuur (GA), wat sodoende verhoed
dat dit verder gemetaboliseer word via glukolise, en dit het tot gevolg dat
’n gedeelte van die beskikbare suiker nie omgeskakel word na alkohol nie. Die
strukturele glukose-oksidase-geen (GOX) van die voedsel-gegradiëerde fungus, Aspergillus
niger is gebruik tydens die konstruksie van drie kassette (GOX1, GOX2 en
GOX2LOX). Binne hiedie geenkassette is A. niger se GOX-geen se transkripsieinisiëring
en -terminering onafhanklik deur die fosfogliseraat-kinase-1-promotor
(PGK1P) en termineerder (PGK1T ) bewerkstellig. Om uitskeiding van GOX uit die
gis te bewerkstellig, is daar van die a-spesifieke gisferomoon-a-faktor (MFa1S)
in GOX1 gebruik gemaak, en in GOX2, van A. niger se eie natuurlike sekresiesein.
Hierdie geenkassette is binne-in die genoom van twee labaratoriumgisrasse
van S. cerevisiae (BY4742 en S1278b) asook een industriële wyngisras (VIN13)
geintegreer. ’n Addisionele integreringskasset (die sogenaamde GOX2LOX-kasset)
is gemaak om die IME1-geen van S. cerevisiae te elimineer. Binne die GOX2LOXkasset
is GOX2 aan ’n loxP-kasset gekoppel. Die nuwe wyngis VIN13-D1 is verkry
deur ’n genomiese integrasie van GOX2LOX binne-in die IME1-lokus. Om die niesporulerende
GOX-produserende wyngis VIN13-D2 te verkry, is VIN13-D1 gesporuleer,
onderwerp aan mikromanipulasie en toegelaat om te herdiploidiseer. Ontledings
het aangedui dat (i) GOX2 aanleiding gegee het tot hoër vlakke van ekstrasellulêre
glukose-oksidase aktiwiteit in vergelyking met GOX1; en (ii) dat die
vlakke van uitgeskeide biologies-aktiewe glukose-oksidase vir die wyngisrasse aansienlik
hoër was. Dit het verdere kleinskaalse wynfermentasies geregverdig.
Die getransformeerde wyngis VIN13-D1 is op eksperimentele skaal in die maak
van rooi- en witwyn geëvalueer. Ontledings van hierdie eksperimentele wyne het
daarop gedui dat glukose-oksidase deur die VIN13-D1-gisselle geproduseer en suksesvol
uitgeskei tydens die wynmaakproses is, en dat VIN13-D1 die fermentasie gedomineer het en die alkoholiese gisting voltooi het. Resultate het egter ook aangedui
dat die geproduseerde glukose-oksidase nie baie aktief was onder die wynmaaktoestande
wat in hierdie eksperimentele wynmaakproses gegeld het nie, en gevolglik
was daar nie ’n drastiese verlaging in die alkoholvlakke sigbaar toe VIN13-D1
se wyne met VIN13 se wyne vergelyk is nie. Wynmonsters is deur middel van
Fourier-transformasie-mid-infrarooispektroskopie (FT-MIR) ontleed ten einde die
chemiese samestelling te bepaal, en gaschromatografie-massaspektrometrie (GCMS)
is aangewend om die wynaromakomponente te bepaal. Die vlakke van glukoonsuur
is deur middel van ensiematiese reaksies bepaal. Multiveranderlike data-analise
[hoofkomponentanalise (PCA) en parsiële kleinte kwadrate (PLS1) diskriminantanalise]
is op die data van die verskeie analitiese tegnieke toegepas om onderliggende
veskille tussen die wyne van VIN13 (wilde-tipe) en VIN13-D1 uit te wys. Chemometriese
projeksies het aangetoon dat daar wel beduidende variasies sigbaar was tot en
met drie hoofkomponente en/of PLS-komponente wat duidelik aandui dat glukoonsuur
’n sleutelfaktor was ten opsigte van die uitwerking wat GOX op VIN13-D1-
fermentasies in vergelyking met VIN13-fermentasies. VIN13-D1 effek manifestasies
is die beste waargeneem op grafieke wat PLS1-diskriminantanalise-data bevat.
Verder het PLS1-diskriminantanalise ook aangetoon dat van die ‘groepe’ wat
gebruik was tydens die analise, die FT-MIR-komponente en die GC-komponente
beter resultate gelewer het. Die GC-komponente het hulle verder daartoe geleen
om tussen die verskillende kultivars en VIN13/VIN13-D1-fermentasies te diskrimineer.
Daar kan dus met sekerheid gesê word dat die grootste invloed in VIN13-D1
wyne sigbaar is in die aromakomponent, maar omdat daar wel ook variasies sigbaar
was in die MIR-komponente, dat die smaakkomponente ook beïnvloed was.
Die aktiwiteit van GOX in druiwesap is verder ondersoek deur gebruik te maak
van kleinskaalse fermentasies in bioreaktors. Daar is bevestig dat die VIN13-D1-
geproduseerde GOX biologies-aktief was tydens aerobiese kondisies, onaktief was
tydens anaerobiese kondisies, en onmiddelik geaktiveer kon word wanneer ’n anaerobiese
fermentasie aerobies gemaak word (gesimuleerde mikro-oksigenasie). Hierdie
verskillende fermentasies dui daarop dat glukose-oksidase inderdaat aktief is in
druiwesap, en dat suurstof ’n sleutelfaktor is tydens die aktivering van die ensiem.
Met behulp van ’n vereenvoudigde model kon aangetoon word dat tydens ideale
toestande dit wel moontlik is om die alkoholvlakke te verlaag na ’n voorafbepaalde
konsentrasie vir die bereiding van lae-alkohol wyne.
Hierdie studie verskaf verdere insig hoe om ’n GOX-produserende wyngis gedurende die wynmaakproses vir die verlaging van die alkoholvlakke te benut. Verder
is dit duidelik dat suurstof van kardinale belang is vir die aktivering van die glukoseoksidase-
ensiem en dat ’n tegniek soos mikro-oksigenasie ’n belangrike rol in hierdie
verband tydens die wynmaakproses sou kon speel.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/4008 |
| Date | 03 1900 |
| Creators | Malherbe, Daniel Francois |
| Contributors | Van Rensburg, P., Pretorius, I. S., Du Toit, M., Divol, B., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology, 2010. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 210 p. : ill. |
| Rights | Stellenbosch University |
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