Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The polysaccharides that are present in wine originate from the grapes, the fungi that
grow on the grapes and from other microorganisms that come into contact with the
must during winemaking. The grape-derived polysaccharides of most concern in
winemaking are pectin, glucan and xylan that can be enzymatically degraded by
pectinases, glucanases and xylanases, respectively. These are the main structural
polysaccharides of the cell wall of the grape cell. Degradation of the cell walls will
result in the separation and rupture of the grape cells, and cell wall-bound
compounds will be released into the must. Treating the must with pectinase and
macerating enzyme preparations can result in an increase in free-flow juice, an
improvement in must clarification and filtration, and an increased extraction of
phenols and tannins. The tannins that are extracted polymerise with anthocyanins in
red wine during ageing, resulting in increased colour intensity and stability. Wine
aroma is also influenced by enzyme treatment. The degradation of the cell wall
contributes to the release of glycosidically-bound terpene or alcohol precursors from
the berries. The hydrolysis of these precursors during fermentation can result in an
improvement in aroma. It can thus be seen that it is possible to improve wine quality
and processing by supplementing the endogenous enzymes that are present in the
fermentation with commercial enzyme preparations. Commercial enzymes are
typically crude fungal preparations.
The majority of commercial pectinase and glucanase preparations are derived
from Aspergillus and Trichoderma, respectively. Since the endogenous
polysaccharase activity of Saccharomyces cerevisiae is very limited, the
heterologous expression of specific polysaccharase genes in an industrial yeast
strain can improve the winemaking process, resulting in a higher quality wine without
the addition of expensive commercial enzyme preparations. Since only the desired
enzymes are secreted by the recombinant strain, there will be no undesired sideactivities,
which can be detrimental to wine quality. Several pectinase-, glucanaseand
xylanase-encoding genes, cloned from a variety of organisms, have been
expressed successfully in laboratory strains of S. cerevisiae. Attempts have also
been made to construct industrial wine yeast strains that express these
polysaccharase genes and secrete the encoded enzymes. Fermentation with some
of these strains resulted in a decrease in total phenolics and turbidity, an increase in
juice extraction, and alterations in the colour and aromatic profile of the resulting
wines.
In this study, four polysaccharide-degrading, recombinant wine yeast strains were
constructed. The endo-β-1,4-xylanase gene, XYN2, and the endo-β-1,4-glucanase
gene, end1, were previously cloned from the soft rot fungus Trichoderma reesei and
the rumen bacterium Butyrivibrio fibrisolvens, respectively. These genes were
subcloned into different expression cassettes which were used to construct the four
integration plasmids. The recombinant plasmids contained the following gene
cassettes: TEF1P-XYN2-ADH2T (plasmid pDLG29) ADH1P- MFα1S -end1-TRP5T (plasmid pDLG30) ADH1P-MFα1S-end1-TRP5T and
ADH2P-XYN2-ADH2T (plasmid pDLG33), ADH1P-MFα1S-end1-TRP5T and YG100PXYN2-
ADH2T (plasmid pDLG39). These four plasmids were then separately
integrated into the ILV2 locus of the commercial wine yeast strain S. cerevisiae
VIN13. Wine was made with the four strains constructed in this study, a pectolytic
strain, VIN13[pPPK], a glucanase- and xylanase-secreting strain, VIN13[pEX], an
untransformed VIN13 strain, and an untransformed strain with the addition of the
commercial enzyme preparation Rapidase EX Colour. Microvinification experiments
were carried out on Pinot noir, Ruby Cabernet and Muscat d’Alexandria wines.
Fermentation with the polysaccharide-degrading strains resulted in significant
improvements in juice extraction, colour intensity and stability, and in alterations in
the aromatic profiles of the wines produced.
Subject to the approval by the regulatory authorities and eventual consumer
acceptance of the use of genetically modified organisms (GMOs) in fermented foods
and beverages, it might be required that the GM status of the yeast that is used
appears on the label. Currently, there is no robust technique available with which the
use of GM yeast can be revealed in a finished wine because the yeast cells and their
DNA are removed from or denatured in the wine during filtration and processing. One
way with which the undeclared use of a GM yeast in winemaking could be exposed
would be to compare the chemical profile of a suspect wine with that of non-GM wine.
In order to explore this concept further, a secondary aim of this study was to
investigate whether Fourier Transformation Infra Red (FT-IR) spectroscopy coupled
with multivariate data analysis could distinguish between wines fermented with
transgenic and non-transgenic yeast strains, or between wines fermented with
different transgenic strains. The results showed that this method could be used to
classify wines fermented with different yeast strains if fermentation with the strain
resulted in a unique chemical profile in the resulting wine. This was a preliminary
study and these findings were summarised as an addendum to the thesis. / AFRIKAANSE OPSOMMING: Die polisakkariede wat in wyn teenwoordig is, is afkomstig van die druiwe, die
swamme wat op die druiwe groei en vanaf ander mikroörganismes wat tydens die
wynmaakproses met die mos in aanraking kom. Die belangrikste druifpolisakkariede
in wynbereiding is pektien, glukaan en xilaan, wat onderskeidelik deur pektinases,
glukanases en xilanases afgebreek kan word. Hierdie is die vernaamste strukturele
polisakkariede van ‘n druifsel se selwand. Die afbreking van die selwande veroorsaak
dat die druifselle skei en skeur, met die gevolg dat die selwandgebonde verbindings
in die mos vrygelaat word. Die behandeling van die mos met pektinase en
versappingsensiempreparate kan tot ʼn toename in vry-afloopsap lei, sowel as ʼn
verbetering in mosverheldering en -filtrasie en ʼn verhoogde ekstraksie van fenole en
tanniene. Die tanniene wat geëkstraheer word, polimeriseer in rooiwyn tydens
veroudering, en dit lei tot verhoogde kleurintensiteit en -stabiliteit. Wynaroma word
ook deur ensiembehandeling beïnvloed. Die afbreking van die druifselwand dra by tot
die vrylating van glikosidiesgebonde terpeen- en alkoholvoorlopers uit die korrels. Die
hidrolise van hierdie voorlopers tydens gisting kan lei tot ʼn verbetering van die
aroma. Dit is dus duidelik dat dit moontlik is om wynkwaliteit en wynbereiding te
verbeter deur die endogene ensieme wat in die gisting teenwoordig is met
kommersiële ensiempreparate te supplementeer.
Kommersiële ensiempreparate is tipies ongesuiwerde swampreparate. Die
meerderheid kommersiële pektinase- en glukanasepreparate word onderskeidelik
vanaf Aspergillus en Trichoderma verkry. Aangesien die endogene polisakkaraseaktiwiteit
van Saccharomyces cerevisiae baie beperk is, kan die heteroloë uitdrukking
van spesifieke polisakkarase-gene in ʼn industriële gisras die wynbereidingsproses
verbeter en lei tot ʼn hoër kwaliteit wyn sonder die byvoeging van duur kommersiële
ensiempreparate. Omdat die verkose ensieme deur die rekombinante ras uitgeskei
word, sal daar geen ongewenste newe-effekte teenwoordig wees wat ʼn nadelige
effek op wynkwaliteit kan hê nie. Verskeie mikrobiese gene wat vir pektinases,
glukanases en xilanases kodeer, is reeds voorheen uit ‘n wye verskeidenheid van
organismes gekloneer en suksesvol in laboratoriumrasse van S. cerevisiae uitgedruk.
Pogings is ook aangewend om industriële wyngisrasse te konstrueer wat hierdie
polisakkarasegene uitdruk en hul enkodeerde ensieme uitskei. Gisting met sommige
van hierdie rekombinante gisrasse het gelei tot ʼn afname in totale fenoliese
verbindings en troebelheid, ʼn verhoging in sapekstraksie, en veranderings in die
kleur en aromatiese profiel van die gevolglike wyne.
In hierdie studie is vier polisakkaried-afbrekende, rekombinante wyngisrasse
gekonstrueer. Die endo-β-1,4-xilanasegeen, XYN2, en die endo-β-1,4-
glukanasegeen, end1, is voorheen reeds onderskeidelik vanaf die sagte vrotswam,
Trichoderma reesei, en die rumenbakterium, Butyrivibrio fibrisolvens, gekloneer.
Hierdie gene is in vier integrasieplasmiede in verskillende ekspressiekassette
gesubkloneer. Die plasmiede het die volgende geenkassette bevat: TEF1P-XYN2-
ADH2T (plasmied pDLG29) ADH1P- MFα1S -end1-TRP5T (plasmied pDLG30) ADH1PMFα1S-
end1-TRP5T and ADH2P-XYN2-ADH2T (plasmied pDLG33), ADH1P-MFα1S end1-TRP5T and YG100P-XYN2-ADH2T (plasmied pDLG39). Hierdie vier plasmiede
is toe afsonderlik in die ILV2-lokus van die kommersiële wyngisras, S. cerevisiae VIN
13, geïntegreer. Wyn is met hierdie vier gekonstrueerde gisrasse gemaak, die
pektolitiese gisras, VIN13[pPPK], die glukanase- en xilanase-afskeidende gisras,
VIN13[pEX], die ongetransformeerde VIN13-ras, en met ʼn ongetransformeerde
VIN13 gis waarby die kommersiële ensiempreparaat, Rapidase EX Colour, bygevoeg
is. Mikro-wynbereidingseksperimente is op Pinot noir-, Ruby Cabernet- en Muscat
D’Alexandria wyne uitgevoer. Gisting met die polisakkaried-afbrekende gisrasse het
gelei tot ʼn noemenswaardige verbetering in sapekstraksie, kleurintensiteit en
kleurstabiliteit, asook in veranderinge in die aromatiese profiele van die
geproduseerde wyne.
Indien die gebruik van geneties gemodifiseerde organismes (GMOs) in
gefermenteerde voedsel en drank deur die reguleringsowerhede goedgekeur en
uiteindelik deur die verbruiker aanvaar sou word, sou dit vereis kon word dat die GMstatus
van die wyngisgis op die etiket van die wynbottel aangebring word. Verpligte
etikettering van GM-wyn sal metodes vereis waarmee die ‘nalentskap’ van GMgisselle
in die finale produk geïdentifiseer en gemoniteer kan word. Tans is daar
geen robuuste tegnieke beskikbaar waarmee die gebruik van GM-giste openbaar kan
word nie, aangesien die gisselle en hul DNA tydens filtrasie en prosessering
verwyder word. Een wyse waarop die onverklaarde gebruik van ‘n GM-gis in
wynbereiding blootgestel sou kno word, is om die chemiese profiel van die verdagte
wyn met dié van ‘n nie-GM-wyn te vergelyk. Ten einde hierdie konsep verder te
ondersoek was ‘n sekondêre doelwit van hierdie studie om te bepaal of FT-IR
(Fourier-transformasie-infrarooi) spektroskopie tesame met meervariante dataanalise
gebruik kan word om te onderskei tussen wyne wat met transgeniese en nietransgeniese
gisrasse gegis is, of tussen wyne wat met verskillende transgeniese
rasse gegis is. Die resultate het aangedui dat hierdie metode gebruik kan word om
wyne wat met verskillende gisrasse gegis is, te klassifiseer indien die betrokke gisras
ʼn unieke chemiese profiel in die uiteindelike wyn veroorsaak het. Dit was egter ʼn
voorlopige ondersoek en is as ʼn byvoegsel tot die tesis geskryf.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/16382 |
| Date | 12 1900 |
| Creators | Louw, Campbell (Campbell Trout) |
| Contributors | Van Rensburg, P., Pretorius, I.S., University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | vi, 97 leaves : ill. |
| Rights | University of Stellenbosch |
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