Thesis (PhD (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009. / In many industrial fermentation processes, Saccharomyces cerevisiae yeast should ideally meet two partially conflicting demands. During fermentation a high suspended yeast count is of paramount importance to maintain a rapid fermentation rate, whilst efficient flocculation should ideally be initiated only on completion of the primary alcoholic fermentation, so as to enhance product clarification and recovery. Most commercial wine yeast strains are non-flocculent, probably because this trait was counter-selected to avoid fermentation problems. In this study, we assessed molecular strategies to optimise the flocculation behaviour of non-flocculent laboratory and wine yeast strains. For this purpose, the chromosomal copies of three dominant flocculation genes, FLO1, FLO5 and FLO11, of a non-flocculent S. cerevisiae laboratory strain (FY23) and two commercial wine yeast strains (BM45 and VIN13) were placed under the transcriptional control of the stationary phase-inducible promoters of the S. cerevisiae ADH2 or HSP30 genes. Under standard laboratory media and culture conditions, all six promoter-gene combinations resulted in specific flocculation behaviours in terms of timing and intensity. The data show that the strategy resulted in the expected and stable expression patterns of these genes in both laboratory and industrial wine yeast strains. Most importantly, the data confirm that inducible expression of the native FLO1 and FLO5 open reading frames, albeit to varying degrees, are responsible for a quantifiable cell-cell adhesion phenotype that can be characterized as a Flo1 flocculation phenotype. On the other hand, we found that inducible expression of the native FLO11 ORF under these conditions resulted in flor/biofilm formation and invasive growth phenotypes. However, the specific impact of the expression of individual dominant FLO genes with regard to characteristics such as flocculation efficiency, cell wall hydrophobicity, biofilm formation and substrate adhesion properties showed significant differences between the commercial strains as well as between commercial and laboratory strains. These adhesion phenotype differences may at least in part be attributed to wine yeast FLO gene open reading frames containing significantly smaller intragenic repeat regions than laboratory strains. The data show that the ADH2 regulatory sequences employed in this study were unsuitable for the purpose of driving FLO gene expression under wine-making conditions. However, HSP30p-based FLO1 and FLO5 wine yeast transformants displayed similar flocculent phenotypes under both synthetic and authentic red wine-making conditions, and the intensities of these phenotypes were closely aligned to those observed under nutrient-rich YEPD conditions. The fermentation activities of HSP30p-based transgenic yeast strains were indistinguishable from that of their parental host wine yeast strains. The chemical composition of wines obtained using transgenic yeast strains were similar to those produced by parental strains. The BM45-derived HSP30p-FLO5 transformant in particular was capable of generating compacted or ‘caked’ lees fractions, thereby providing a distinct separation of the fermented wine product and lees fractions. Furthermore, in this study we report a novel FLO11 induced flocculation phenotype that seems to exclusively develop under authentic red wine-making conditions. This strong FLO11 flocculation phenotype was not wine yeast strain dependant, possessed both Ca2+-dependant and Ca2+-independent flocculation characteristics and was insensitive to inhibition by both glucose and mannose. A distinct advantage of this unique FLO11 phenotype was highlighted in its ability to dramatically promote faster lees settling rates. Moreover, wines produced by HSP30p-FLO11 wine yeast transformants were significantly less turbid than those produced by their wild type parental strains. The benefit of this attractive property is it facilitates simpler and faster recovery of wines and also promotes greater volume recovery of the wine product.

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The production of glycerol by Saccharomyces cerevisiae under anaerobic conditions is essential for maintaining the intracellular redox balance thereby allowing continuous energy generation through conversion of sugars into ethanol. In addition, glycerol can act as an osmolyte and is synthesized to maintain turgor pressure under hyperosmotic conditions. The production of ethanol from sugars can be a redox-neutral process, where the NAD+ (nicotinamide adenine dinucleotide) that is consumed during the glycolytic conversion of glyceraldehyde-3-phosphate to pyruvate is later regenerated by the reduction of acetaldehyde to ethanol. However, in particular the redirection of metabolic flux of pyruvate to biomass formation leads to excess NADH formation. The intracellular redox balance in these conditions is then primarily maintained through formation of glycerol which is control by two main enzymes, namely Gpd1p and Gpd2p. Deletion of the genes coding for these two proteins leads to accumulation of NADH and renders the cells incapable of maintaining their fermentative ability and growth under anaerobic conditions. The goal of this study was to investigate the growth, fermentative ability and metabolite synthesis of various gpd1Δgpd2Δ double mutant (DM) strains in which the redox balancing potential was partially restored through expression of native or heterologous genes. Strains were constructed by introducing alternative NADH oxidizing pathways or manipulating existing pathways to favour the oxidation of excess NADH. More specifically, the modifications included (i) sorbitol formation; (ii) establishing a pathway for propane-1,2-diol formation; and (iii) increasing ethanol formation. Apart from genetically manipulating the gpd1Δgpd2Δ double mutant, the addition of pyruvate during growth was also investigated. The experiments were carried out under oxygen limited conditions in a high sugar medium and the fermented product was analyzed for total sugar consumed, biomass and primary and secondary metabolites formed by the different strains. The relationships between sugar consumption, growth and metabolite production by different strains were investigated by comparing the data generated from the different strains by using multivariate data analysis tools. Analysis of the pathways involved in the production of primary (acids, ethanol and other metabolites) and secondary metabolites (aroma compounds) were also carried out in order to establish flux modification in comparison to the wild type (WT) strain. The results revealed that these manipulations improved the fermentative capacity of the gpd1Δgpd2Δ double mutant, suggesting a partial recovery of NAD+ regeneration ability, albeit not to the extent of the WT strain. As expected a significant correlation was found between sugar consumption and ethanol and biomass formation. Ethanol yields but not final concentrations were increased by the genetic manipulations. Sorbitol by DM(srlD) and DM(SOR1) strains and propane-1,2-diol by DM(gldA, GRE3, mgsA) strain were formed in significant amounts although at lower molar yields than glycerol. Furthermore, by genetic manipulation the yield of secondary metabolites (isobutanol, isoamyl alcohol, 2-phenyl ethanol and isobutyric acid) was increased whereas the ethyl acetate concentration and yield decreased. The results indicate that aroma compound properties of wine yeasts could be favourably changed by manipulating the glycerol synthesizing pathway. The addition of pyruvate during the growth of gpd1Δgpd2Δ double mutant contributes to excess NADH re-oxidation through additional ethanol formation. / AFRIKAANSE OPSOMMING: Die produksie van gliserol deur Saccharomyces cerevisiae onder anaërobiese toestande is noodsaaklik vir die onderhouding van die intrasellulêre redoksbalans en maak dus ononderbroke energie-ontwikkeling tydens die omsetting van suikers in etanol moontlik. Daarbenewens kan gliserol as ‘n osmoliet optree en word dit gesintetiseer om turgordruk onder hiperosmotiese toestande te onderhou. Die produksie van etanol uit suikers kan ‘n redoksneutrale proses wees, waar die NAD+ (nikotinamiedadenien-dinukleotied) wat tydens die glikolitiese omskakeling van gliseraldehied-3-fosfaat na piruvaat verbruik word, later deur die reduksie van asetaldehied na etanol regenereer word. Die nasending van die metaboliese vloeiing van piruvaat na biomassavorming lei egter na ‘n oormaat NADH-vorming. Onder hierdie toestande word die intrasellulêre redoksbalans dan hoofsaaklik deur die vorming van gliserol onderhou. Laasgenoemde word veral deur twee ensieme beheer, naamlik Gpd1p en Gpd2p. Die delesie van die gene wat vir hierdie twee proteïene enkodeer, lei tot ‘n akkumulasie van NADH en veroorsaak dat die selle nie hulle gistingsvermoë en groei onder anaërobiese toestande kan onderhou nie. Die doelwit van hierdie studie was om die groei, gistingsvermoë en metabolietsintese van verskeie gpd1Δgpd2Δ dubbelmutant (DM) rasse te ondersoek waarin die redoksbalanseringspotensiaal gedeeltelik herstel is deur die uitdrukking van inheemse of heteroloë gene. Rasse is gekonstrueer deur alternatiewe NADH-oksiderende weë in te voer of deur bestaande weë te manipuleer om die oksidasie van oormaat NADH te bevoordeel. Meer spesifiek het die modifikasies die volgende ingesluit: (i) sorbitolvorming; (ii) die vestiging van ‘n weg vir propaan-1,2-diol-vorming; en (iii) die verhoging van etanolvorming. Buiten die genetiese manipulering van die gpd1Δgpd2Δ dubbelmutant, is die byvoeging van piruvaat tydens groei ook ondersoek. Die eksperimente is onder suurstofbeperkte toestande in ‘n hoë-suiker medium uitgevoer en die gegiste produk is ondersoek vir totale suikerverbruik, biomassa en primêre en sekondêre metaboliete wat deur die verskillende rasse gevorm is. Die verhoudings tussen suikerverbruik, groei en metabolietproduksie deur die verskillende rasse is ondersoek deur die data wat deur die verskillende rasse gegeneer is deur middel van meerveranderlike data-analise te vergelyk. Analise van die weë wat in die produksie van primêre (sure, etanol en ander metaboliete) en sekondêre metaboliete (aromaverbindings) betrokke is, is ook uitgevoer om die verandering in vloei te bepaal in vergelyking met die wildetipe (WT) ras. Die resultate het gewys dat hierdie manipulasies die gistingsvermoë van die gpd1Δgpd2Δ-dubbelmutant verbeter het, wat ‘n gedeeltelike herstel van NAD+- regenerasievermoë voorstel, hoewel nie tot dieselfde mate as in die WT-ras nie. Soos verwag, is ‘n beduidende korrelasie tussen suikerverbruik en etanol- en biomassavorming gevind. Etanolopbrengs is deur genetiese manipulasies verhoog, maar nie die finale konsentrasies van etanol nie. Sorbitol is in beduidende hoeveelhede deur die DM(srlD) en DM(SOR1)-rasse gevorm en propaan-1,2-diol deur die DM(gldA, GRE3, mgsA) -rasse, hoewel teen laer molare opbrengste as gliserol. Verder is die opbrengs van sekondêre metaboliete (isobutanol, iso-amielalkohol, 2-fenieletanol en isobottersuur) deur genetiese manipulasie verhoog, terwyl die etielasetaatkonsentrasie en -opbreng verlaag is. Die resultate dui aan dat die aromaverbindingseienskappe van wyngiste voordelig verander kan word deur die gliserolsintetiseringsweg te manipuleer. Die byvoeging van piruvaat tydens die groei van die gpd1Δgpd2Δ-dubbelmutant dra by tot uitermate NADH-reoksidasie tydens die bykomende vorming van etanol.

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Wine fermentation with a yeast strain able to degrade grape cell polysaccharides can result in improved processability and an increase in wine quality by improving extraction of essential compounds from the grapes during the maceration stage. Pectin is the only important cell wall polysaccharide that can be degraded by wild-type Saccharomyces cerevisiae strains. Pectin is degraded by a polygalacturonase (PG) encoded by the PGU1 gene (ORF YJR153W). Only certain S. cerevisiae strains can degrade pectin and PG activity is thus strain specific. The lack of activity in certain strains has been attributed to a number of factors: (1) the complete absence of the PGU1 gene, (2) the PGU1 gene is present but the allele is dysfunctional and (3) the PGU1 gene is present but not transcribed. The lack in transcription has been shown to be due to the gene having a dysfunctional promoter or to regulatory differences between strains. Results published in the literature are contradictory. The primary aim of this investigation was to clarify the regulation of PG activity in S. cerevisiae and to determine why there are differences in PG activity between different strains. Regulation of PG activity between several wine and laboratory strains with varying PG activities was compared by looking at the sequence of the PGU1 gene and its promoter as well as transcription levels of this gene and its main transcription factors, TEC1 and STE12. In order to identify regulatory factors influencing PG activity, the S. cerevisiae genome was screened for activators and inhibitors of PG activity. Fourteen inhibitors and two activators of PG activity were identified during this screen. Real-time PCR analysis showed that the PG activity is regulated by transcription of the PGU1 gene. A linear relationship was demonstrated between PGU1 and its two transcription factors TEC1 and STE12. Some of the genes identified as inhibitors of PGU1 transcription are involved in gene silencing by Telomere Position Effect (TPE) indicating that PGU1 is possibly silenced due to its subtelomeric location within 25 kb from the right telomere of chromosome X. Moving the PGU1 gene with its native regulatory machinery to a different position away from its telomere resulted in an increase in PGU1 transcription and PG activity, demonstrating the epigenetic influence on PGU1 regulation. Results from this study suggested that the strain related difference in PGU1 expression occurs at an epigenetic level, with steric hindrance preventing RNA polymerase access to the PGU1 promoter and thus inhibiting transcription of this gene in some strains. Understanding regulation of PG activity can potentially lead to the development of more effective strategies to improve PG degradation by S. cerevisiae. The genetic model describing regulation of PGU1 transcription was extended by this study and a novel mechanism of regulation of PG activity was identified. The secondary aim of this study written as an addendum to this thesis, focussed on degradation of another grape cell wall polysaccharide xylan by recombinant strains of S. cerevisiae. These strains were enabled to degrade this polysaccharide through heterologous expression of novel xylanase encoding genes from various origins. Xylanase activity of the recombinant strains generated was compared. Overexpressing the complete gene xynA of Ruminococcus flavefaciens, the functional domain xynAa or the functional domain xynAc within optimal conditions for these enzymes all conferred very low xylanase activity to S. cerevisiae, with xynAc resulting in the highest xylanase activity. Since overexpression of the R. flavefaciens xynA gene yielded very low activity under optimal conditions activity in wine making conditions would be negligible. The genes XYN2 and XYN4 from Trichoderma reesei and Aspergillus niger respectively yielded higher levels of activity. According to these results, only the expression of XYN2 and XYN4 could have a potential effect on wine An effective strategy for improving pectin degradation can in future potentially be combined with heterologous expression of a xylanase encoding gene in S. cerevisiae in order to engineer a wine yeast strain with improved polysaccharase abilities. / AFRIKAANSE OPSOMMING: Gisting van druiwe met polisakkaried-afbrekende gisrasse kan lei tot ‘n verbetering in wyn prosessering en tot die produksie van hoër kwaliteit wyne deur die ekstraksie van belangrike wynkomponente uit druifselle te verbeter. Pektien is die hoof komponent van die druifselwand wat deur wilde tipe Saccharomyces cerevisiae giste afgebreek kan word en word afgebreek deur ‘n poligalaktoronase (PG) wat deur die PGU1 (YJR153W) geen gekodeer word. Slegs spesifieke gisrasse kan pektien afbreek en die ensiem aktiwiteit is dus ras-spesifiek. Die gebrek aan PG aktiwiteit in sekere rasse is al omskryf as gevolg van die afwesigheid van die geen, die teenwoordigheid van ‘n nie-funksionele alleel of dat die geen wat teenwoordig is nie uitgedruk word nie. Transkripsie is al bewys om nie plaas te vind nie a.g.v. die teenwoordigheid van ‘n nie-funksionele promotor of a.g.v. ‘n verskil in regulering van transkripsie tussen rasse. Sommige studies wat PG regulering ondersoek het, het teenstrydige resultate verkry. Die hoofdoel van hierdie studie was om PG regulering te ondersoek en te bepaal waarom daar verskille in PG aktiwiteit tussen verskillende gisrasse voorkom. Regulering van PG aktiwiteit is ondersoek tussen wyn en laboratorium gisrasse met wisselende vlakke van PG aktiwiteit deur die DNS volgorde van die PGU1 geen en sy promotor, so wel as die DNS volgorde van die geen se hoof transkripsie faktore TEC1 en STE12 te bepaal. Om reguleerders van PG aktiwiteit te identifiseer is die genoom van die gis S. cerevisiae ondersoek om faktore te identifiseer wat PG aktiwiteit aktiveer of inhibeer. “Real-time PCR” het bewys dat PG aktiwiteit gereguleer word deur transkripsie van die PGU1 geen en dat daar ‘n lineêre verhouding tussen die transkripsie van die PGU1 geen en sy twee hoof transkripsie faktore TEC1 en STE12 bestaan. Sommige van die gene wat geïdentifiseer is as inhibeerders van PG aktiwiteit is voorheen bewys om betrokke te wees by die inhibering van transkripsie deur middel van die telomeer posisie effek, dit dui daarop dat transkripsie van die PGU1 geen moontlik geïnhibeer word as gevolg van die geen se subtelomeriese posisie binne 25 kb vanaf die regter telomeer van chromosoom X. Die PGU1 geen is met sy natuurlike regulerings elemente na ‘n ander posisie in die genoom, weg van sy naaste telomeer geskuif, die verandering in posisie van die geen het gelei tot ‘n toename in PG aktiwiteit en transkripsie van die PGU1 geen en het dus bewys regulering word beïnvloed deur ‘n epigenetiese effek. Die resultate van hierdie studie het daarop gedui dat die verskil in transkripsie van die PGU1 geen plaasvind op ‘n epigenetiese vlak waartydens die chromatien struktuur toegang van die RNA polimerase tot die PGU1 geen voorkom en dus word transkripsie van die geen sodoende in sommige rasse voorkom. Die tweede doelwit van hierdie studie het gefokus op die afbraak van ‘n ander komponent van die druif selwand, xilaan, deur S. cerevisiae. Hierdie navorsing vorm ‘n addendum aan die tesis en Xylanase aktiwiteit van verskeie rekombinante rasse is in hierdie studie vergelyk. Baie lae xylanase aktiwiteit is verleen aan rekombinante giste wat die volledige xynA geen gekloneer van die bakteriee Ruminococcus flavefaciens, asook twee aktiewe domeins van die geen, domein xynAa en domein xynAc uitdruk. Van die voorafgenoemde giste het die uitdrukking van die domein xynAc die rekombinante gis ras met die hoogste aktiwiteit tot gevolg gehad. Ooruitdrukking van die gene XYN2 en XYN4 wat gekloneer is van die fungi Trichoderma reesei en Aspergillus niger onderskeidelik, het beide gisrasse wat oor hoë vlakke van xylanase aktiwiteit beskik tot gevolg gehad. Hierdie resultate dui dus daarop dat van die gene ondersoek in die studie, slegs XYN2 en XYN4 potensiaal het om xylanase aktiwiteit van wyngiste te verbeter. Deur die regulering van PG aktiwiteit te bestudeer kan meer effektiewe strategieë potensieel ontwikkel word om PG aktiwiteit in S. cerevisiae te verbeter. Hierdie studie het die genetiese model wat PG regulering omskryf uitgebrei deur ‘n nuwe meganisme van regulering van toepassing op PGU1 te identifiseer. As ons die regulering van die PGU1 goed verstaan kan dit in die toekoms gekombineer word met ‘n effektiewe strategie om ‘n gis aan te pas om xylaan af te breek, om sodoende ‘n wyngis geneties te verbeter om beide xylaan en pektien te kan afbreek.

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.

Thesis (PhDAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Stuck alcoholic fermentations are a major enological problem for the international winemaking industry. Incomplete wine fermentations are frequently characterized by high residual fructose concentrations and the near-absence of residual glucose, a fact that is due to the glucophilic character of the wine yeast Saccharomyces cerevisiae. Wines with high contents of post fermentation sugar are very susceptible for microbial spoilage since residual fructose and/or glucose can be metabolized by bacteria and yeast to undesired by-products such as volatile acid and off-flavours, resulting in wine spoilage and considerable economic losses. It has been reported that stuck fermentations are usually caused by several synergistically acting inhibition factors, and the glucose to fructose ratio (GFR) is thought to play an important role in this context. This study is aimed at contributing towards a better understanding of this industrial problem, and at finding industrially applicable solutions. In a first part, this study describes the isolation of two appropriate strains of the fructophilic yeast Zygosaccharomyces bailii from the natural microflora of grapevine, followed by trials in small scale test fermentations using stuck industrial fermentations as model media. These experiments were expanded to also investigate large scale industrial fermentations. As a result, a strategy for the treatment of stuck fermentations was developed and successfully applied in several wineries with fermentation problems. This methodology represents an entirely novel and industrially applicable solution to high residual fructose levels. In a second part, the data contributes to elucidating the molecular nature of the fructophilic phenotype of Z. bailii by characterizing some of the genes and proteins that may be responsible for the fructophilic character. In particular, the investigation focused on the first two steps of hexose metabolism, the transport of sugar into the cell by permeases and sugar phosphorylation by hexokinases, which combined are thought to be primarily responsible for sugar preference. One result of this study was Fructoferm W3©, a dry yeast product which is commercially available. Fructoferm W3 was awarded with the innovation medal for enological products at Intervitis/Interfructa, Stuttgart, Germany in 2007. / AFRIKAANSE OPSOMMING: Die voorkoms van steek alkoholiese fermentasies is ‘n ernstige problem in die internasionale wyn industrie. Onvolledige fermentasies word dikwels gekenmerk deur hoë residuele fruktose konsentrasies en die veitlike afwesigheid van residuele glukose. Die kenmerke kan meestal toegeskryf word aan die glukofilliese kakakter van die wyngis Saccharomyces cerevisiae. Wyne met ‘n hoë suiker inhoud na die afloop van fermentasie is vatbaar vir mikrobiese bederf aangesien residuele fruktose en/of glukose gemetaboliseer kan word deur bakterië en gis om ongewenste byprodukte soos vlugtige sure en bygeure te vorm wat kan lei tot wyn bederf en aansienlike ekonomies verlies. Dit is vasgestel dat steek fermentasies gewoonlik veroorsaak word deur verskeie sinergisties werkende inhibisie faktore, waartoe die glukose/fruktose verhouding ‘n noemenswaardiege bydrae lewer. Die mikpunt van hierdie studie was om ‘n bydrae te lewer tot die begrip van steek fermentasies en die daarstelling van moontlike industriële oplossings. Die eerste deel van die werk beskryf die isolasie van twee rasse van die gis Zygosaccharomyces baillie uit die natuurlike wingerd mikroflora, gevolg deur steekproewe in die vorm van kelinskaalse fermentasies met steek industriële fermentasies gebruik as model media. Hierdie ekserimente is vervolgens uitgebrei om grootskaalse industriële steek fermentasies te bestudeer. Die uitkoms van hierdie werk het gelei tot die ontwikkeling van ‘n strategie vir die behandeling van steek fermentasies wat susksesvol toegepas is in verskeie wynmakerye. Die metodiek bring ‘n nuwe en industrieel toepasbare oplossing vir hoë residuele fruktose vlakke. Die data aangebied in die tweede afdeling dra by tot die verheldering van die molekulêre natuur van die fruktofilliese fenotipe van Z. baillie deur die tipering van gene en protiëne wat moontlik verantwoordelik is vir die fruktofilliese karakter van die gis. Die ondersoek het spesifiek op die eerste twee stappe van heksose metabolisme, naamlik die invoer van suiker in die sel deur permeases en suiker fosforilering deur heksokinases, gekonsentreer. Die kombinasie van die twee prosesse is vermoedelik verantwoordelik vir die regulering van suiker voorkeur. ‘n Gevolg van die studie was die ontwikkeling van ‘n droë gisproduk, Fructferm W3©, wat kommersieel beskikbaar gestel is. Fructoferm W3 is in 2007 toegeken met die innovasie medalje vir wynkundige produkte by Intervittis/Interfructa in Stuttgart, Duitsland.

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae senses change within its environment and responds through specific adaptive cellular programmes, in particular by modifying gene expression. Many adaptive changes affect the physico-chemical properties of the cell wall, and several mechanisms that specifically affect the expression levels of genes that encode for cell wall components have been described previously. Cell wall modification directly impacts on general cell wall properties and cell-cell and cell-surface interactions. Many of these properties have been directly linked to families of cell wall proteins referred to as adhesins. In particular members of the Flocculation (FLO) gene family have been shown to play a crucial role in adhesion phenotypes. Flo11p functions in a variety of phenotypes including agar invasion, plastic adhesion and the formation of pseudohyphae, “flor” and “mats”, whereas Flo1p appears to control flocculation. The regulation of FLO11 expression is well documented and is mainly controlled by the mitogen activated protein kinase (MAPK) and cyclic AMP protein kinase A (cAMP-PKA) signalling cascades. Genetic analysis shows that Mss11p acts downstream and is central to these pathways, and furthermore interacts with the cAMP-PKA component Flo8p to activate transcription. In this study we further explore additional gene targets of Flo8p and Mss11p, as well as their regulation and their impact on cell wall characteristics and associated adhesion phenotypes. Our analysis shows that Mss11p is also required for FLO1 expression, and functions together with Flo8p to control many Flo-dependent adhesion phenotypes. Genome-wide gene expression analysis further reveals that altered Mss11p levels leads to the change in the expression of various cell membrane and cell wall genes, notably AQY2 and members of the DAN and TIR gene families. Further genetic analysis indicates that adhesion phenotypes display an almost exclusive dependence on FLO gene expression. We also demonstrate that these phenotypes require Flo10p and are thus dependent on the specific balance of Flo proteins in the cell wall. The analysis of signalling deletion mutants show that regulation of FLO10 shares signalling components with FLO11, but that the two genes are differentially regulated. Unlike FLO11, FLO10 transcription also does not display an absolute requirement for Mss11p but rather for the MAPK component Ste12p. Whole genome expression analysis were also performed on strains with altered levels of Flo8p which were compared with the above mentioned transcriptome data set. This analysis shows that Flo8p and Mss11p co-regulate the FLO genes, as well as AQY2 and TIR3, but also have significant unique gene targets. The combination of transcriptome data with current information concerning transcription factor (TF) interaction networks reveals the importance of network interaction between Cin5p, Flo8p, Mga1p and Mss11p. From these data we constructed a TF interaction model in which Flo8p acts as the predominantly activating TF component, whereas Mss11p function as a target hub TF, possibly as a mediator- or polymerase II holo-enzyme component. Finally we provide a first report on “mat” formation by an industrial wine yeast strain, and show that by adjusting FLO11 expression in this strain we are able to significantly change this phenotypic behaviour. / AFRIKAANSE OPSOMMING: Die gis Saccharomyces cerevisiae neem veranderinge in sy omgewing waar en reageer daarop deur middel van spesifieke sellulêre programme, in die besonder deur geenuitdrukking aan te pas. Verskeie aanpasbare veranderinge beïnvloed die fisieke, asook chemiese eienskappe van die selwand, en talle meganismes is al beskryf wat die uitdrukkingsvlakke beïnvloed van gene wat vir selwandkomponente kodeer. Die modifikasie van die selwand het ’n direkte impak op selwand-eienskappe, asook die sel-sel- en sel-oppervlak-interaksies. Verskeie van hierdie eienskappe word direk gekoppel aan die selwandproteïenfamilies, wat ook as adhesie-faktore bekend staan. Veral lede van die Flokkulasie (FLO) -geenfamilie het ’n noodsaaklike funksie in adhesie-fenotipes. Flo11p speel ’n rol in verskeie fenotipes, wat insluit die indringende groei van agar, plastiekaanhegting en die vorming van pseudohifes, “flor“ en “matte“, terwyl Flo1p flokkulasie beheer. Die regulering van FLO11-uitdrukking is deeglik gedokumenteerd en dit word hoofsaaklik gereguleer deur die mitogeen-geaktiveerde proteïenkinase (MAPK) en sikliese AMP-proteïenkinase A (cAMP-PKA) seintransduksiekaskades. Genetiese analises toon dat Mss11p stroom-af en sentraal tot hierdie kaskades funksioneer, en dit aktiveer transkripsie deur interaksie met die cAMP-PKA-komponent, Flop8. In hierdie studie word ’n ondersoek gedoen na addisionele teikengene van Flo8p en Mss11p, en hoe hierdie gene gereguleer word, asook hul impak op selwandeienskappe en geassosieerde adhesie-fenotipes. Ons analises toon dat Mss11p ook benodig word vir die ekspressie van FLO1 en dat dit, tesame met Flo8p, beheer uit oefen oor verskeie Flo-afhanklike fenotipes. Genoomwye geenekspressie-analises wys verder daarop dat veranderde Mss11p-vlakke lei tot die aanpassing van die ekspressie van verskeie selmembraan- en selwandgene, naamlik AQY2 asook lede van die DAN- en TIR-geenfamilies. Verdere genetiese analise dui daarop dat adhesie-fenotipes byna eksklusief afhanklik is van FLO-geenekspressie. Daar is verder getoon dat hierdie fenotipes ook Flo10p benodig en dus afhanklik is van die spesifieke balans van Floproteïene in die selwand. Die analise van seintransduksiemutante demonstreer dat FLO10 en FLO11 seintransduksie-komponente deel, maar dat hierdie gene verskillend gereguleer word. Anders as FLO11, toon FLO10 nie ’n absolute noodsaaklikheid vir Mss11p nie, maar eerder vir die MAPK-komponent, Ste12p. Totale genoomekspressie-analises is ook gedoen op gisrasse met aangepaste vlakke van Flo8p en dis vergelyk met bogenoemde transkripsiedatastel. Hierdie analise wys dat Flo8p and Mss11p die FLO-gene, asook AQY2 en TIR3, koreguleer, maar ook beduidende unieke teikengene het. Die kombinasie van transkripsiedata met huidig beskikbare informasie betreffende transkripsiefaktor (TF) -interaksienetwerke dui op die relevansie van netwerkinteraksie tussen Cin5p, Flo8p, Mga1p en Mss11p. Hiervan is daar ’n model opgestel waarin Flo8p in die meeste gevalle as die aktiverende TF-komponent optree, terwyl Mss11p as TF-teiken dien, moontlik as ’n mediator- of polimerase II holoënsiemkomponent. Laatens word daar vir die eerste keer verslag gedoen van ”mat”-vorming deur ’n industriële wyngisras en toon ons verder dat hierdie fenotipe beduidend verander word deur middel van die aanpassing van FLO11-uitdrukking.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: South African red wine is often acknowledged world wide as being full bodied and deep in colour. This is often the result of high temperatures that is experienced during the important growth stages of grapes especially post véraison. In the Robertson area in South Africa however, temperatures often exceeds the range for optimal anthocyanin development during these growth stages. The distinction between grapes being technologically ripe and being ripe on a phenolic level is also accepted as an important determining factor for the perfect time to pick grapes. In co-operative wineries such as Robertson Winery (RW) where grapes are delivered from a large area and different producers, it is difficult to individualise grape blocks when it comes to ripeness level in terms of sugar or phenolic ripeness. In most circumstances a generalised set of parameters for deeming grapes ripe or acceptable for delivery is the best substitute. The levels of these parameters are based on research literature that is available for the area as well as data collected through years of maintaining the vineyards of that area. The grape parameters that are currently being used by RW for ripeness and quality are pH, titratable acidity (TA) and sugar level. In recent years RW in conjunction with the Department of Viticulture and Oenology, Stellenbosch University, decided to investigate more parameters to determine the quality of grapes at the time of harvest. Most importantly for the grape growers this quality is connected to a price point and therefore compensation. Two important quality parameters of red wine are the red colour and mouth feel of wine. Anthocyanin and tannins are respectively connected to these two quality attributes and are both widely accepted as quality indicators. Wine with high anthocyanin and tannin content often originates from grapes with a high colour and phenolic profile. The existence of a correlation between grape and wine anthocyanin and tannin content is therefore the basis of attempting to use these parameters in the grape to predict end wine’s colour and phenolic quantity. Determination of anthocyanin and tannin content of grapes has already become part of some private owned wineries’ standard set of determinations. However, sample preparations, extractions and consumables needed are all factors that need to be reduced to make the measurement and therefore the use of these parameters more viable in a co-operative cellar laboratory, where large volumes of grapes are received during harvest. The first objective of this work was to determine the levels of anthocyanin and tannin in red grapes from different vineyard blocks from the producers of RW from three successive vintages. This would give insight as to what can be seen as a low and high anthocyanin and tannin content for grapes received at the cellar. For this purpose, blocks of the most important red wine cultivars for RW was selected and analysed for these compounds. The ranges and average levels of anthocyanin and tannin content were determined using measurement techniques that could be used by any winery. The average mono flavanol and total colour level of the grapes were found to be lower than those often reported in literature, with total grape flavanols being higher. However, a wide range of values for these compounds were found that correlated with those found in other studies. The possible reasons for differences in levels of occurrence of these compounds were discussed and mostly pertain to differences in cultivar, micro climatic and season. The second objective was to determine the correlation between levels of colour and phenolic compounds in grapes and their corresponding wines. Such correlations will form the foundation for the use of phenolic content to predict the colour and phenolic potential of the wine and possibly wine quality as well. When the grape and wine colour and phenolic data were correlated for all seasons and cultivars inclusive it was found that grape and wine colour showed better correlations than for instance total phenols and tannins. This was especially true for total colour pigments in red grapes, measured with HPLC, when correlated with certain spectrophotometric analysis of wine colour. Cultivar and season as well as the synergism between the two were further investigated for its role in affecting correlations. When these relationships were further differentiated by season and by cultivar the resulting correlations varied. This work contributed a great deal of information to support the use of grape colour and phenolic compounds for the prediction of end wine colour and phenolic composition. The third objective was to investigate near infrared spectroscopy (FT-NIR) as a viable option to rapidly measured anthocyanins, tannins and total phenolics in red grapes. If proven successfully, this could be employed by a large cellar such as RW. FT-NIR has been used with success on grape extracts and in this instance the focus was to establish a calibration on the grape homogenate itself. Preliminary results showed that FT-NIR could be applied for the use of determination of anthocyanin and tannin levels in red grapes originating from RW. The prediction of total phenols was not found to be as accurate, but this could also be due to the reference method that was used. This work brought some interesting, practical information not only of importance for RW, but all wineries that are concerned with improving the basis on which grape quality is determined. The use of aerial data mapping for indicating areas regarding important grape colour and phenolic parameters was used in this study and is a very visual way of showing the distribution of certain ripeness parameters over a large area. Correlations between the grape and wines of such a large amount of red grape blocks for a specific area have not also been reported in South Africa before. The use of FT-NIR to determine anthocyanins and tannin concentrations in grape homogenates is also novel for its use in South African wineries. This work may assist grape and wine producers as well as analysts on the phenolic and colour profile of grapes and wines from RW. / AFRIKAANSE OPSOMMING: Suid-Afrikaanse rooiwyn word wêreld-wyd geken aan ‘n dieprooi kleur en vol struktuur. Die grootste rede vir hierdie verskynsel is hoë temperature wat ervaar word tydens rypwording en veral na véraison. In die Robertson wynstreek is temperature egter tydens rypwording dikwels vêr bo dit was as optimaal vir antosianien ontwikkeling beskou word. Die gepaste tyd om druiwe te pluk word nie net gedryf deur die tegnologiese rypheidsvlak nie, maar ook deur fenoliese rypheid. In ‘n koöperatiewe kelder omgewing soos Robertson Wynkelder (RW) word ‘n hoë lading druiwe elke dag ontvant vanaf verskillende produsente oor ‘n breë streek. Dit maak dit moeilik om te bepaal watter druiwe werklik beide tegnologies en fenolies ryp is. Die beste manier om hiervoor te vergoed is om ‘n standaard te stel vir ‘n reeks voorafbepaalde parameters. Die vlakke van die gekose parameters is, word bepaal deur navorsinguitsette sowel as die geskiedkunde data wat ingesamel is vanaf elkeen van die bepaalde blokke. Die parameters wat tans in gebruik is by RW om oesdatum en kwaliteit by inname te bepaal is pH, titreerbare suur (TA) en suiker vlak. Die tekortkoming hier is dat kwaliteit van druiwe beswaarlik met slegs hierdie informasie kan bepaal word, maar dat dit die betaling van die produsent by aflewering wesenlik kan beïnvloed. Dit het RW genoop om in samewerking met die Departement van Wingerd en Wynkunde, Universiteit van Stellenbosch nog parameters te ondersoek wat hierdie rypheid- en kwaliteitsbepaling by inname sou kon versterk. Twee belangrike faktore wat kwaliteit van rooiwyn bepaal is die kleur en struktuur. Antosianiene en tanniene is onderskeidelik verantwoordelik vir hierdie kwaliteits eienskappe van wyn. Wyn wat bestempel word as hoog in kleur en tannien inhoud word dikwels verbind met druiwe wat hoog is in hierdie faktore. Die moontlike korrelasie tussen die antosianien en tannien inhoud van druiwe en die wyn wat daarvan berei word is dus die basis waarop die potensiële toepassing van hierdie parameters berus. Die bepaling van antosianien en tannien vlakke word reeds in sommige laboratoriums gedoen. Die monster voorbereidings tyd, ekstraksies, toerusting en verbruikbare items nodig om hierdie tipe analieses te doen is egter hoog. Die analiese van hierdie komponente is meer lewensvatbaar in groot laboratoriums (soos in ‘n koöperatiewe kelder) waar groter volume druiwe ingeneem word gedurende parstyd. Die eerste doelwit van hierdie studie was om te bepaal teen watter vlakke antosianiene en tanniene in druiwe voorkom, spesifiek van die Robertson area. Die het behels ‘n wye verskeidenheid van blokke, verspreid oor die hele streek wat oor 3 seisoene gemonitor is in terme van veral kleur en tanniene maar ook ander belangrike parameters. Die idee hier is om insig te kry rakende watter vlakke bestempel kan word as laag en hoog in terme van antosianien en tanniene vir die Robertson streek. Daarvoor is slegs die mees aangeplantste rooi kultivars gebruik. Die verspreiding en gemiddelde vlakke waarteen antosianien en tanniene voorkom was bepaal deur gebruik te maak van metodes wat as relatief algemeen in laboratoria gebruik word. Die gemiddelde mono-flavonoïed en totale kleur pigment inhoud van die druiwe was laer as van die vlakke wat in die literatuur beskikbaar is, met totale flavanole wat hoër was. Die wyer verspreiding van die waardes het egter beter gekorreleer met die waardes soos beskryf in die literatuur. Die moontlike redes vir die verskillende vlakke word in die studie bespreek en word waarskynlik bepaal deur verskille in kultivar, mikro-klimaat en seisoen. Die tweede doelwit was om te bepaal of daar ‘n korrelasie te vinde is tussen die kleur en tannien inhoud van die druiwe en ooreenstemmende wyne. Sulke tipe korrelasies sal die basis vorm om antosianien en tannien inhoud van wyn reeds in die druiwe fase te kan voorspel. Nadat die ingesamelde druif en wyn data as ‘n geheel beskou was, was dit sigbaar dat die wynkleur parameters beter korrelasies bied as meeste tannien en totale fenole. Dit was veral waar in die geval van totale kleur pigmente soos gemeet met die HPLC teenoor die wynkleur parameters gemeet met spektrofotometriese metodes. Verdere ondersoeke in terme van die impak wat die kultivar en seisoenale kan hê het tot variërende korrelasies gelei.. Hierdie werk het ‘n groot bydrae gelewer om voorspellings van wyn kleur en fenoliese inhoud reeds met sukses vanaf die druif te bepaal. Derdens het die werk fourier transformasie naby infrarooi skandering (FT-NIR) ondersoek as ‘n lewensvatbare metode vir die bepaling van antosianien, tannien en totale fenoliese inhoud van druiwe en wyn. FT-NIR word reeds oor ‘n wye reeks wyne en druiwe ekstraksiemonsters toegepas en die doelwit hier was om druiwe homogenaat as matriks te kalibreer. Voorlopige resultate het bevind dat antosianien en tannien vlakke in druiwe van RW gemeet kan word met die FT-NIR, maar dat die kalibrasie vir totale fenole nog verbeter kan word. Hierdie werk het ‘n wye reeks interessante en prakties bruikbare informasie na vore gebring wat van onskatbare belang is vir RW en ander kelders wat besorgd is oor die verbetering van algemene druifkwaliteit. Geografiese kaarte wat belangrike druifkleur en fenoliese parameters aandui is in hierdie studie gebruik en wys hoe data visueel voorgestel kan word om die geheelindruk van gekose parameters oor ‘n groot area te vergelyk. Korrelasies tussen druiwe en wyn van so ‘n groot hoeveelheid druiwe blokke is nog nooit voorheen in Suid-Afrika getoon nie. Dieselfde geld vir die gebruik van FT-NIR vir die meet van kleur en fenoliese parameters in druiwe homogenate. Hierdie werk kan druiwe- en wynproduseerders sowel as analiste assisteer in terme van die kleur en fenoliese profiel van druiwe en wyn van RW. / Robertson Winery with Mr Bowen Botha as well as THRIP for funding this project

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2009. / The PGU1 gene encodes an endo-polygalacturonase, an enzyme that degrades pectin. Although the presence and function of this gene is well characterized in Saccharomyces cerevisiae, its regulation is very complex and not yet fully understood. Yeast producing a highly active polygalacturonase (PG) during alcoholic fermentation could potentially improve filtration and turbidity and also enhance extraction of certain aroma compounds. This could replace the addition of expensive commercial enzyme preparations that often contain unwanted enzymes. The first objective of this study was to evaluate PGU1 expression in recombinant strains of S. cerevisiae that originally lacked the PGU1 gene. A functional PGU1 gene and its promoter were successfully re-introduced into their native position in the genomes of five wine strains. Three of these strains recovered PG activity while two did not transcribe the gene and subsequently lacked activity. The three strains that recovered activity were used in microvinification experiments to determine the effect of PG-producing yeast on the aroma profile of the wine. No significant differences were observed in the volatile compounds production between the recombinants and their respective wild types, but some tendencies arose, especially for the monoterpene geraniol. The second objective of this study was to analyze the PGU1 gene and promoter from Saccharomyces paradoxus RO88 (a strain that exhibits high PG activity) and to compare it to those of S. cerevisiae S288C in order to identify differences that could potentially be responsible for the difference in their PG activities. Comparison of the gene sequences revealed several amino acid differences, one of which was in the peptide secretion signal. Analyses of the promoters also indicated some potentially important differences. Furthermore, S. cerevisiae strain VIN13, RO88 as well as two interspecies hybrids (all displaying varying PG activities) were compared under winemaking conditions. Clear differences were observed for the production of certain compounds. RO88 and the hybrids produced higher concentrations of certain volatile compounds, although they were not strong fermenters. Two recombinants, each containing a PGU1-overexpressing plasmid (one with the PGU1 gene from S. paradoxus and the other from S. cerevisiae), were also used in vinification to determine the effects of the different PGU1 gene on the aroma profile of the wine. Unfortunately, the plasmids were unstable and lost during the fermentation. Nevertheless, some tendencies were observed that indicated possible higher production of certain compounds by the recombinants compared to their wild types. This study identified that regulation of the PGU1 gene differs between strains with different genetic backgrounds. Certain differences were observed in the PGU1 gene and promoter sequences between S. cerevisiae and S. paradoxus that could potentially be the reason for the difference in their PG activities. From an oenological point of view, the presence of PGU1 in the genome of a fermenting strain tends to increase the aromatic potential of wine. These results provide a good platform for further studies on the PGU1 gene.

Thesis (MScAgric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The production of quality wine is influenced by numerous factors of which grape quality is one of the most important factors. The production of quality wine, however, is not possible without good winemaking techniques and effective quality control. Critical control points (CCP) during the winemaking process must be identified to ensure optimum wine quality. Grape must is a complex medium that contains different micro-organisms which can be either beneficial or negative to wine quality, depending on the physical and chemical conditions that prevail in the must. Yeasts are responsible for alcoholic fermentation, lactic acid bacteria (LAB) for malolactic fermentation (MLF) and acetic acid bacteria (AAB) for the production acetic acid from ethanol. Yeasts and certain LAB can also produce acetic acid and thereby increasing the volatile acidity (VA) of wine. These micro-organisms can influence each other in complex fashions by competing for growth nutrients and by producing inhibitory substances. Most winemakers nowadays use commercial yeast strains to inoculate wine fermentations. This, however, does not assure a problem-free fermentation and cases of stuck and sluggish fermentations are annually reported worldwide. In these or most cases fermentation takes longer than 21 days to complete and the wine contains a residual sugar concentration of more than 4 g/L, which can be utilised by wine spoilage micro-organisms such as certain bacteria and other wild yeasts. Stuck and sluggish fermentations also increase the chances of oxidation due to the absence of the protective CO2 layer on the surface of the wine, which is formed during alcoholic fermentation. Another effect of stuck and sluggish fermentations is that valuable tank space is wasted due to the unexpected time consumption of these fermentation problems. Many factors during the winemaking process can be responsible for stuck and sluggish fermentations. In this thesis the different factors is discussed with the emphasis on the effect of the yeast strain. The way that certain yeast strains influence AAB and LAB numbers during fermentation and MLF through the production of inhibiting by-products such as medium chain fatty acids has not been investigated in detail in the past. Certain fungicides and pesticides that are used in vineyards to control pests (e.g. mildew) contain copper which can be inhibiting to yeast growth and alcoholic fermentation. Legal limits and withholding periods on these sprays are not always strictly obeyed and can lead to stuck and sluggish fermentations. This motivated us to evaluate the growth and fermentation activities of a selection of commercial wine yeasts in the presence of copper levels in the range of maximum legal limits. The effect of these commercial strains on the LAB and AAB numbers during alcoholic fermentation and MLF were also investigated. Our results showed that there was no significant difference on numbers of the AAB obtained from fermentations inoculated with different commercial wine yeast strains. However, with regards to the LAB numbers, one of the strains produced significantly more sulphur dioxide (SO2), which led to the inhibition of MLF in that wine. Our results further indicated which commercial yeast strains were capable of effectively fermenting high sugar musts and which strains were less effective. From the strains tested VIN13, N96 & L2056 were able to utilize fructose more effectively than NT50, RJ11 & D80. We could further distinguish between yeast strains that produced the lowest (VIN13 & RJ11) and the highest (WE372, NT50 & L2056) VA concentrations in must containing high sugar levels. Strains that were more tolerant against high copper levels were also identified. We tested six yeast strains in must with added copper (0.25 mM cu2+) in the form of CuSO4 .H2O. Three Cu2+-tolerant (D80, Collection Cepage Cabernet & NT50) yeast strains were distinguished from three less Cu2+-tolerant yeast strains (VIN13, NT112 & RJ11). This study made a valuable contribution in knowledge gained about commercially available wine yeast strains that can ferment effectively under certain stress conditions. Research such as this, where wine yeasts are evaluated to ferment more effectively during strenuous winemaking conditions, will be very beneficial to winemakers. / AFRIKAANSE OPSOMMING: Die produksie van gehalte wyn word deur verskillende faktore beïnvloed waarvan druifkwaliteit seker die belangrikste is. Die produksie van gehalte wyn is egter nie moontlik sonder goeie wynmaaktegnieke en effektiewe kwaliteitsbeheer nie. Kritieke kontrole punte (KKP) tydens die wynmaakproses moet dus geïdentifiseer word om sodoende ‘n verlaging in wynkwaliteit te vermy. Druiwemos het ‘n komplekse mikrobiologiese samestelling en bestaan uit verskillende mikroörganismes wat vooren nadelig vir wynkwaliteit kan wees, afhangende van die fisiese en chemiese toestande wat in die mos bestaan. Giste is verantwoordelik vir alkoholiese fermentasie, melksuurbakterieë (MSB) vir appelmelksuurgisting (AMG) en asynsuurbakterieë (ASB) vir die produksie van asynsuur vanaf etanol. Asynsuur word egter ook deur giste en MSB geproduseer en dra so by tot die vlugtige suurheid (VS) van ‘n wyn. Hierdie mikroörganismes kan mekaar op komplekse wyses beïnvloed deur o.a. te kompeteer vir voedingstowwe asook deur die produksie van inhiberende verbindings. Die meeste wynmakers maak gebruik van kommersiële gisrasse om alkoholiese fermentasies mee uit te voer. Gevalle van sogenaamde slepende en gestaakte alkoholiese fermentasies, waar suiker nie volledig na etanol en CO2 omgeskakel word nie, kom egter nog gereeld in die wynbedryf voor. In sulke gevalle neem die fermentasie gewoonlik langer as 21 dae om te voltooi met ‘n suiker konsentrasie van meer as 4 g/L wat in die wyn oorbly. Dit is nadelig vir wynkwaliteit aangesien dit nie net die kanse vir bederf deur bakterieë en giste verhoog nie, maar ook die kanse vir oksidasie verhoog a.g.v. die verlies van die beskermende CO2 lagie bo-oor die wyn. Hoe sekere gisrasse, ASB en MSB getalle gedurende fermentasie en AMG beïnvloed deur die produksie van inhiberende verbindings soos medium ketting vetsure en SO2, is ook nie baie in die verlede ondersoek nie. Sommige spuitstowwe wat gebruik word in die bekamping van swamsiektes bevat koper wat inhiberend kan wees vir gisgroei en alkoholiese fermentasie. Wetlike maksimum limiete en onthoudingsperiodes op spuitstofresidue word egter nie altyd gehoorsaam nie en kan lei tot slepende en gestaakte fermentasies. Dit het ons gemotiveer om ‘n seleksie van kommersiële gisrasse te evalueer in terme van gisgroei en fermentasie in die teenwoordigheid van kopervlakke naby die maksimum limiet. Ons resultate het gewys dat daar nie noemenswaardige verskille in AAB getalle tydens alkoholiese fermentasie tussen behandelings met verskillende kommersiële gisrasse was nie. Een van die gisrasse het wel noemenswaardig meer SO2 geproduseer wat gelei het tot inhibering van AMG in hierdie wyn. Ons het verder uitgewys watter kommersiële gisrasse instaat is om meer effektief in hoër suiker mos te fermenteer en watter van die rasse minder suksesvol was. Ons het ook rasse geïdentifiseer wat meer weerstandbiedend is teen hoë kopervlakke in mos en sodoende groter kans op ‘n suksesvolle fermentasie sal hê in mos wat koperresidue bevat wat afkomstig is van sekere spuitstowwe. Die effek van die ASB en MSB getalle gedurende fermentasie en AMG is ook ondersoek. Ons resultate het verder gewys watter kommersiële gisrasse instaat was om mos met hoë suikervlakke meer effektief te fermenteer. Vam die gisrasse wat getoets was het VIN13, N96 & L2056 fruktose meer effektief benut as NT50, RJ11 & D80. Ons kon verder onderskei tussen gisrasse wat die laagste (VIN13 & RJ11) en die hoogste (WE372, NT50 & L2056) vlakke van VS produseer in mos met hoë inisiële suikervlakke. Gisrasse wat meer tolerant was teen koperresidue in mos is ook geidentifiseer. Ons het ses gisrasse getoets in mos met bygevoegde koper (0.25 mM Cu2+) in die vorm van CuSO4 .5H2O. Daar is onderskei tussen drie Cu2+-tolerante (D80, Collection Cepage Cabernet & NT50) en drie minder Cu2+-tolerante gisrasse (VIN13, NT112 & RJ11). Hierdie studie lewer ‘n waardevolle bydrae in die invordering van kennis oor kommersieel beskikbare wyngisrasse wat meer effektief sal fermenteer onder sekere streskondisies wat in mos voorkom. Inligting soos hierdie is belangrik om die wynmaker se keuse uit die reeks bestaande kommersiële gisrasse te vergemaklik.

Thesis (Msc (Viticulture and Oenology. Institute for Wine Biotechnology))--University of Stellenbosch, 2009. / Monoterpenes are important aroma compounds in certain grape varieties such as Muscat, Gewürztraminer and Riesling and are present as either odourless, glycosidically bound complexes or as free aromatic monoterpenes. These complexes occur as monoglucosides or, when present as diglycosides, most commonly as 6-O-α-L-arabinofuranosyl-β-D-glucopyranosides of mainly linalool, geraniol, nerol and citronellol. The release of monoterpenes from non-volatile glycosidically bound precursors occurs either by acid hydrolysis or enzymatic hydrolysis. High temperature acid hydrolysis causes a rearrangement of the monoterpene aglycones and a decrease in the aroma and changes in the aromatic characteristics of monoterpenes and is therefore not suitable. Enzymatic hydrolysis does not modify the monoterpene aglycones and can be an efficent method to release potentially volatile monoterpenes. α-L-arabinofuranosidase and β-glucosidase are important enzymes responsible for the liberation of monoterpene alcohols from their glycosides. Glycosidases from Vitis vinifera and Saccharomyces cerevisiae are severely inhibited by winemaking conditions and this leads to unutilized aroma potential, while commercial preparations of aroma liberating enzymes are crude extracts that often have unwanted and unpredictable side effects. It is therefore of interest to investigate alternative measures to release glycosidically bound monoterpenes to increase the floral aroma of wine without side activities that impact negatively on wine. Heterologous α-L-arabinofuranosidases and β-glucosidases have previously been expressed in S. cerevisiae and these studies have evaluated and found increased glycosidic activities against both natural and synthetic substrates. In this study, we expressed the Aspergillus awamori α-L-arabinofuranosidase (AwAbfB) in combination with either the β-glucosidases Bgl2 from Saccharomycopsis fibuligera or the BglA from Aspergillus kawachii in the industrial yeast strain S. cerevisiae VIN13 to facilitate the sequential enzymatic hydrolysis of monoterpene diglycosides. Enzyme assays and GC-FID (Gas Chromatography with a Flame Ionization Detector) results show a significant increase in the amount of free monoterpene concentrations under winemaking conditions in the strain co-expressing the AwAbfB and the Bgl2. The increases in free monoterpene levels obtained were similar to those obtained with a commercial enzyme preparation, LAFAZYM AROM. Sensorial evaluation confirmed the improvement in the wine aroma profile, particularly the floral character. This yeast strain permits a single culture fermentation which improves the sensorial quality and complexity of wine. Further investigations on the factors influencing the stability and reactivity of monoterpenes during alcoholic fermentation are needed.