Thesis (MSc)--Stellenbosch University, 2002. / Full text to be digitised and attached to bibliographic record. / ENGLISH ABSTRACT: The winemaking process constitutes a unique ecological niche that involves the interaction
of yeasts, lactic acid bacteria and acetic acid bacteria. Saccharomyces cerevisiae has
established its importance as a wine yeast and also proven itself as a reliable starter
culture organism. Its primary role is to convert the grape sugar into alcohol and, secondly,
its metabolic activities result in the production of higher alcohols, fatty acids and esters,
which are important flavour and aroma compounds that are essential for consistent and
predictable wine quality. There is a growing consumer demand for wine containing lower
levels of alcohol and chemical preservatives.
Glucose oxidase (GOX) has received considerable research interest regarding its
potential application in the wine industry to reduce alcohol levels and as a biocontrol
agent. Several physical processes are used for the removal or reduction of alcohol in wine
and some of them are sometimes used in combination. These processes tend to involve
expensive equipment and can be intensive from a processing point of view. An alternative
approach was introduced with the concept of treating grape must with GOX to reduce the
glucose content of the must, and therefore produce a wine with a reduced alcohol content
after fermentation. Due to the demanding nature of modern winemaking practices and
sophisticated wine markets, there is an ever-growing quest for specialised wine yeast
strains possessing a wide range of optimised, improved or novel oenological properties.
The first and main objective of this study was to genetically engineer wine yeasts to
produce wine with a reduced alcohol content. In order to do this, the structural glucose
oxidase (gox) gene of Aspergillus niger was cloned into an integration vector (Ylp5)
containing the yeast mating pheromone a-factor secretion signal (MFa1 s) and the
phosphoglycerate kinase 1 gene promoter and terminator (PGK1PT). This PGK1p-MFa1sgox-
PGKh gene cassette (designated GOX1) was introduced into a laboratory strain of
S. cerevisiae (~1278). Results obtained indicated the production of biologically active
glucose oxidase and showed that it is secreted into the culture medium. This would mean
that the enzyme will convert the glucose to gluconic acid in the medium before the yeast
cells are able to metabolise the glucose to ethanol. Microvinifications performed with
Chardonnay grapes showed that the laboratory yeast starter cultures transformed with
GOX1 were indeed able to reduce the total amount of alcohol in the finished product.
The second objective of this study involved the potential application of GOX as a
biocontrol agent. Screening was performed for wine spoilage microorganisms, such as
acetic acid bacteria and lactic acid bacteria, using plate assays. The wine spoilage
microorganisms tested formed different sized inhibition zones, indicating varying degrees
of inhibition. The inhibition of some of the wine spoilage microorganisms was confirmed
under a scanning electron microscope. The total collapse of the bacterial cell wáll could be
seen and might be explained by the fact that a final product of the GOX enzymatic reaction
is hydrogen peroxide (H202). The produced H202 leads to hyperbaric oxygen toxicity, a
result of the peroxidation of the membrane lipid, and a strong oxidising effect on the
bacterial cell, which is the cause of the destruction of basic molecular structures, such as
nucleic acids and cell proteins.
In this exciting age of molecular yeast genetics and modern biotechnology, this
study could pave the way for the development of wine yeast starter culture strains for the
production of wine with a lower alcohol content and reduced levels of chemical preservatives, such as sulphur dioxide. The use of genetically modified organisms (GMOs)
within the wine industry is a limiting factor at present and credible means must be found to
effectively address the concerns of traditionalists within the wine industry and the negative
overreaction by some consumer groups. There is a vast potential benefit to the wine
consumer and industry alike and the first recombinant wine products therefore should
unmistakably demonstrate safe products free of potentially harmful compounds, and have
organoleptic, hygienic and economic advantages for both the wine producer and
consumer. / AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n ekologiese interaksie tussen gis, asynsuurbakterieë en
melksuurbakterieë. Saccharomyces cerevisiae het homself alreeds bewys as 'n belangrike
en betroubare inisiëringsgis in wyn. Die hoofdoel van die gis is om druifsuikers na etanol
om te skakel. Tweedens lei die gis se metaboliese aktiwiteite tot die produksie van hoër
alkohole, vetsure en esters, wat tot die konsekwente voorspelbare smaak en
aromaverbindings in herhaalbare kwaliteit wyn bydra. Daar is 'n toenemende aanvraag na
wyne met 'n laer alkoholinhoud en minder preserveermiddels.
Glukoseoksidase (GOX) het heelwat navorsing in die wynindustrie uitgelok omdat dit
gebruik kan word om die alkoholinhoud in wyn te verlaag, asook as 'n biologiese
beheermiddel kan funksioneer. Daar is reeds sekere fisiese prosesse wat gebruik kan
word om die alkohol in wyn te verwyder of te verminder. Sommige van hierdie prosesse
word soms in kombinasie gebruik. Die nadeel is egter dat hierdie prosesse baie duur en
intensief is, veral ten opsigte van prosessering. 'n Alternatief om die alkoholinhoud van
wyn te verlaag, het egter na vore gekom toe daar voorgestel is om die mos met GOX te
behandel. As gevolg van die veeleisende aard van moderne wynmaakpraktyke en
gesofistikeerde wynmarkte, is daar 'n nimmereindigende soektog na meer
gespesialiseerde wyngisrasse wat 'n wye reeks van geoptimiseerde en verbeterde, en
selfs unieke, wynkundige einskappe bevat.
Die hoofdoelwit van hierdie navorsingsprojek behels die genetiese manipulasie van 'n
gisras sodat dit in staat is om wyn met 'n laer alkoholinhoud te produseer. Om hierdie doel
te verwesentlik, is die strukturele glukoseoksidasegeen (gox) van Aspergillus niger in 'n
integreringsvektor gekloneer. Transkripsie-inisiëring en -terminering is deur
fosfogliseraatkinase-1-promotor en -termineerder (PGK1PT) bewerkstellig. Die a-spesifieke
gisferomoon-a-faktor (MFa1 s) is gebruik om die uitskeiding van GOX uit die gis te
bewerkstellig. Saam vorm bogenoemde die PGK1p-MFals-gox-PGKh-geenkasset, wat
as GOX1 bekend is. GOX1 is na 'n labaratoriumras van S. cerevisiae (:E1278)
getransformeer. Resultate dui aan dat biologies aktiewe GOX geproduseer en uitgeskei
word. Dit beteken dat van die glukose in die medium reeds na glukoonsuur omgesit sal
word voordat die gis dit kan begin benut en alkohol produseer. Kleinskaalse
wynmaakprosesse wat met Chardonnay-druiwe en GOX-produserende labaratoriumgis
uitgevoer is, het inderdaad tot laer alkoholpersentasies gelei.
Die tweede doelwit van die navorsingsprojek was om te bepaal of GOX die potensiaal
as biologiese beheermiddel het. Daar is ondersoek ingestel na sekere
wynbederfsorganismes soos asynsuur- en melksuurbakterieë en die inhibisie van die
organismes is op agarplate gemonitor. Verskillende grade van inhibisie, soos die grootte
van die inhibisiesone, was sigbaar vir die verskillende wynbederfsorganismes wat getoets
is. Die inhibiese van sekere wynbederfsorganismes is ook met behulp van 'n
skandeerelektronmikroskoop bevestig. Die totale ineenstorting van die bakteriële selwand
was sigbaar en kan verklaar word deur die teenwoordigheid van waterstofperoksied
(H202). Laasgenoemde is 'n byproduk van die laaste metaboliese reaksie en staan as 'n
antimikrobiese middel bekend. Die byproduk (H202) gee aanleiding tot hiperbariese
suurstoftoksisiteit, 'n gevolg van die peroksidasie van membraanlipiede en 'n sterk
oksiderende effek t.o.v. die bakteriële selwand. Dit lei tot die vernietiging van die basiese
molekulêre strukture, soos die nukleïensure en selproteïene.
Tydens hierdie opwindende era van molekulêre gisgenetika en biotegnologie kan
hierdie navorsing die fondament lê vir die ontwikkeling van 'n wyngiskultuur wat in staat is
om wyn met 'n laer alkoholinhoud te produseer. Die gebruik van geneties gemanupileerde
organismes (GMO's) in die wynbedryf is egter nog 'n beperkende faktor. 'n
Geloofwaardige manier moet dus gevind word om die bekommernisse van tradisionaliste,
asook die negatiewe oorreaksies van sommige verbruikers, aan te spreek en hok te slaan.
Daar is groot potensiaal en voordele vir beide die verbruiker en industrie. Dit is dus
belangrik dat die eerste rekombinante wynprodukte wat die mark betree, veilig en vry van
potensieel skadelike verbindings is, asook organoleptiese, higiëniese en ekonomiese
voordele toon te opsigte van beide die wynprodusent en gebruiker.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52840 |
| Date | 12 1900 |
| Creators | Malherbe, Daniel Francois |
| Contributors | Pretorius, I. S., Van Rensburg, P., Du Toit, M., Stellenbosch University. Faculty of AgriScience. 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 | 72 pages : illustrations |
| Rights | Stellenbosch University |
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