Thesis (MScAgric)--University of Stellenbosch, 2009. / Bibliography. Also available via the Internet.

Thesis (Th. M.)--Grace Theological Seminary, 1983. / Abstract. Includes bibliographical references (leaves 104-112).

Thesis (MPhil)--University of Stellenbosch, 2005 / Bibliography. Also available via the Internet.

Thesis (MSc)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Thesis (Th. M.)--Grace Theological Seminary, 1983. / Abstract. Includes bibliographical references (leaves 104-112).

Thesis (MScFoodSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Twenty five commercial Chenin blanc wines produced solely from bush vine vineyards and including three vintages, three styles and five production areas, were sourced for this study. Descriptive sensory analysis (DSA) and chemical analyses including GC-FID (gas chromatography fitted with a flame ionisation detector) and FTMIR (Fourier transform mid-infrared) spectroscopy were employed to establish the sensory and chemical characteristics, whereas consumer tests were conducted to determine consumer perception and liking of bush vine Chenin blanc wines. DSA (a profiling technique) was also compared to the sorting task (a classification technique) with a description assignment to evaluate the sorting task’s ability to profile wines. According to the results of DSA, the wines separated into two groups. One group associated with sensory attributes which can be considered indicative of the Fresh and Fruity Chenin blanc style. The other group associated with sensory attributes which can be considered indicative of the Rich and Ripe style of Chenin blanc. No separation between the wooded and unwooded Rich and Ripe styles was apparent. According to the results of the chemical analyses, the wines also separated into two groups. This separation seemed to be caused by vintage and the chemical changes associated with ageing as the wines from the youngest vintage (2010) was strongly associated with high levels of esters and malic acid. The older wines were situated farthest away from these attributes indicating low concentrations. When comparing the results from the sorting task and DSA, it could be seen that similar wine style groupings formed, indicating that DSA can also be regarded as an effective tool when categorising wines. The differences in the positioning of some of the wines and attributes on the DSA multivariate plots and the sorting task plots could be attributed to the difference in panels used. The sorting task was conducted using an expert panel with persons illustrating significant technical knowledge of Chenin blanc wines. Experience, exposure and technical knowledge tend to establish a common language amongst wine experts which could have caused the expert panel to perceive some wines differently when comparing the results of the latter panel to that of the trained panel. DSA was found to remain the most effective method for establishing a comprehensive sensory profile. Consumer analyses showed that regular white wine drinkers prefer the unwooded styles (Fresh and Fruity and Rich and Ripe unwooded) of Chenin blanc more than the wooded style. It was also found that consumers with a higher level of objective wine knowledge tend to associate the terms ‘bush vine’ and ‘old bush vine’ with the Rich and Ripe style of Chenin blanc, whereas consumers with a lower level of objective wine knowledge associated ‘old bush vine’ with the Fresh and Fruity style. Since all the wines used in the consumer analysis were produced from old bush vines, it is evident that consumer education on the impact of bush vine training system and vine age on wine quality is needed. Better understanding of these principles could lead to elevated product appraisals and consumer satisfaction. / AFRIKAANSE OPSOMMING: Vyf en twintig kommersiële Chenin blanc wyne, uitsluitlik van bosstok wingerde geproduseer, is bekom vir hierdie studie. Die wyne het drie style, drie oesjare en vyf produksiestreke ingesluit. Beskrywende sensoriese analise (BSA) en chemiese analises, wat GC-FID (gas chromatografie gekoppel met vlam-ioniserende deteksie) en FTMIR (Fourier-transformering mid-infrarooi) spektroskopie insluit, is uitgevoer om onderskeidelik die sensoriese en chemiese eienskappe van die wyne te bepaal. Verbruikerstoetse is ook uitgevoer om verbruikerspersepsie en -voorkeure vir bosstok Chenin blanc wyne te bepaal. BSA (‘n profilerings tegniek) was ook vergelyk met ‘n sorterings taak (‘n klassifikasie tegniek) met ‘n beskrywings opdrag, primêr om die sorterings taak se vermoë om wyne te profileer te ondersoek. Volgens die resultate van BSA, het die wyne in twee groepe verdeel. Een groep het met die sensoriese eienskappe wat op ‘n Vars-en-Vrugtige-styl dui, geassosieër. Die ander groep het met sensoriese eienskappe geassosieër wat met die Volrond-styl verband hou. Geen verdeling tussen die gehoute en ongehoute wyne binne die Volrond-styl was sigbaar nie. Volgens die resultate van die chemiese analises, het die wyne ook in twee groepe verdeel. Die verdeling blyk asof dit veroorsaak is deur oesjaar en die chemiese veranderinge wat met wynveroudering gepaard gaan. Wyne van die jongste oesjaar (2010) het ‘n sterk verband met hoë vlakke van esters en appelsuur getoon. Die ouer wyne was verder weg van hierdie eienskappe geleë, wat op laer ester en appelsuur konsentrasies dui. Wanneer die meerveranderlike resultate van die sorterings taak (met en sonder die aanduiding van sensoriese eienskappe) en dit van BSA vergelyk word, kon soortgelyke groeperings gesien word. Dit is ‘n aanduiding dat BSA ook wyne effektief kan kategoriseer. Die verskil in posisionering van sommige wyne tussen die BSA en sorterings taak resultate, kan toegeskryf word aan die verskillende panele wat gebruik is om die tegnieke uit te voer. ‘n Deskundige paneel (wynkenners) is gebruik om die sortingstaak uit te voer. Ervaring, blootstelling en tegniese kennis is geneig om te lei tot die vestiging van ‘n gemeenskaplike taal onder wynkenners. Hierdie gemeenskaplike taal kan as rede aangevoer word vir die uiteenlopende analise van sommige wyne wanneer die resultate van die deskundige paneel met dié van die opgeleide paneel (in BSA gebruik) vergelyk word. Dit is gevind dat BSA, wanneer ‘n omvattende sensoriese profiel bepaal moet word, die mees effektiefste metode bly. Verbruikerstoetse het getoon dat gereelde witwyn-verbruikers die ongehoute Chenin blanc style (Vars-en-Vrugtig en ongehoute Volrond) bo die gehoute styl verkies. Dit is ook bepaal dat verbruikers met ‘n hoër vlak van objektiewe wynkennis neig om die terme ‘bosstok’ en ‘ou bosstok’ met die Volrond-styl van Chenin blanc te assosieer, terwyl verbruikers met ‘n laer vlak van objektiewe wynkennis die term ‘ou bosstok’ met die Vars-en-Vrugtige Chenin blanc styl assosieër. Aangesien al die wyne wat in die verbruikerstoetse ingesluit is van ou bosstok wingerde geproduseer is, is dit duidelik dat verbruikeropvoeding insake die effek van die gebruik van bosstokke en ou wingerdstokke op wynkwaliteit noodsaaklik is. ‘n Beter begrip van hierdie beginsels sal lei tot verhoogde produkwaardasie, asook ‘n toename in verbruikertevredenheid.

Thesis (MSc Food Sc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: South Africa is a developing country that relies on its agricultural sector as a main source of overall economic welfare. Development does not only give rise to new technology and new products but also results in increased amounts of liquid and solid waste. Generally, the production of wine is considered an environmentally friendly process, but significant amounts of natural resources and organic amendments are necessary, while generating large amounts of liquid and solid wastes. Anaerobic digestion (AD) is an attractive and proven treatment option for both liquid and solid wastes as valuable products and depollution can be obtained. AD of liquid waste results in an effluent and biogas, while anaerobic composting of solid waste results in an organic amendment, leachate and biogas. The overall objective of this study was to investigate the operational feasibility of the cotreatment of leachate produced during the anaerobic composting (AnC) of grape skins in an upflow anaerobic sludge blanket (UASB) reactor while treating winery wastewater. This first aim of this study was to investigate the efficiency of the anaerobic composting of grape skins. Laboratoryscaled digesters (1L) were utilised as anaerobic composting units. The most important operational parameters were identified (pH, moisture content and inoculum (size, ratio, composition)) in order to produce a pH stable, odour free compost in 21 days. Experimental studies highlighted the importance of shredding waste as well as the addition of calcium oxide and green waste to increase the initial pH of the composting mixture. After optimising a 50% (m.m-1) cow manure inoculum, lower inoculum concentrations (10, 15 and 25% (m.m-1)) were investigated to make the process more economically viable. A 10% (m.m-1) anaerobic compost (AC) inoculum was found to produce the most favourable results in terms of pH stabilisation and leachate generation. A 50% (m.m-1) moisture level performed the best by attaining a pH > 6.5 on day 6 and having the highest end pH (7.65) on day 21, while white and red grape skins in an equal ratio were found to generate a higher end pH. With all these optimum parameters in place (shredded waste, green waste, CaO, inoculum, moisture, grape skins), a compost with a final pH (7.09), moisture (58%), nitrogen (2.25%), phosphorous (0.22%) and potassium content (1.7%) was obtained. The optimised parameters were scaled-up (1:10) by using polyvinyl chloride anaerobic digesters (20 L) to suit the operational requirements of the AnC process and also produced a stable compost within 21 days. The second aim of this study was to investigate the combined anaerobic digestion of winery wastewater (WWW) and leachate obtained from the anaerobic composting of grape skins in an upflow anaerobic sludge blanket (UASB). This involved the operation of a 2.3 L laboratory-scale UASB reactor for 205 days. The reactor successfully co-treated WWW and leachate at ca. 8.5 kgCOD.m-3d-1 with a final chemical oxygen demand (COD) reduction of over 90%, a stable reactor effluent pH (7.61) and alkalinity (3 281 CaCO3 mg.L-1). This study showed the feasibility for the combined treatment of liquid and solid waste from the winemaking process. Although the legal limits for reactor effluent disposal onto land was not met, significant reduction in COD concentrations were achieved, whilst producing a soil amendment that could potentially result in cost savings for chemical fertilisers. The benefits related to using anaerobic bioconversion as a treatment option for liquid and solid waste could possibly be advantageous to the wine industry as an environmental control technology, by converting liquid and solid waste into valuable resources. / AFRIKAANSE OPSOMMING: Suid-Afrika is 'n ontwikkelende land wat staatmaak op sy landbousektor as 'n hoofbron van algehele ekonomiese welstand. Ontwikkeling gee nie net aanleiding tot nuwe tegnologie en nuwe produkte nie, maar lei ook tot die verhoogde bydrae van vloeistof sowel as vaste afval. Oor die algemeen, word die produksie van wyn beskou as 'n omgewingsvriendelike proses, maar aansienlike hoeveelhede natuurlike hulpbronne en organiese kunsbemesting word benodig, terwyl groot hoeveelhede vloeistof en vaste afval gegenereer word. Anaërobiese vertering (AV) is 'n aantreklike en bewese behandelingsopsie vir beide vloeistof en vaste afval aangesien waardevolle produkte en suiwering verkry kan word. AV van vloeistowwe lewer uitvloeisel sowel as biogas, terwyl anaërobiese kompostering van vaste afval 'n organiese kunsbemesting, loog en biogas lewer. Die oorhoofse doel van hierdie studie was om die operasionele doeltreffendheid van die mede-behandeling van loog wat gegenereer word tydens die anaërobiese kompostering (AnK) van druiwe doppe in 'n opvloei-anaërobiese-slykkombers (OAS) reaktor terwyl kelderafvalwater behandel word, te ondersoek. Die eerste mikpunt van hierdie studie was om die doeltreffendheid van die anaërobiese komposteringsproses van druiwe doppe te ondersoek. Laboratorium-skaal verteerders (1L) is gebruik as anaërobiese komposteringseenhede. Die belangrikste operasionele parameters is geïdentifiseer (pH, voginhoud en inokulum (grootte, verhouding, samestelling)) om ‘n 'n pH-stabiele, reukvrye kompos te produseer in 21 dae. Die belangrikheid van gesnipperde afval asook die byvoeging van kalsiumoksied en groen afval om die aanvanklike pH van die komposmengsel te verhoog, is deur eksperimentele studies beklemtoom. Na die optimering van 'n 50% (m.m-1) koeimis inokulum, is laer inokulum konsentrasies (10, 15 en 25% (m.m-1)) geondersoek om die proses meer ekonomies uitvoerbaar te maak. Daar is gevind dat ‘n 10% (m.m-1) anaërobiese kompos (AK) inokulum die mees gunstige resultate lewer in terme van pH stabilisering en loog generering. ‘n 50% (m.m-1) vloeistof vlak het die beste presteer deur 'n pH> 6.5 te bereik teen Dag 6 asook die hoogste eind pH (7.65) teen Dag 21, terwyl wit en rooi druiwe doppe in dieselfde verhouding gevind is om ‘n hoër eind pH te genereer. Met al hierdie optimum parameters in plek (gesnipperde afval, groen afval, kalsiumoksied, inokulum, vog, druiwe doppe) is 'n kompos met 'n finale pH (7.09), vog (58%), stikstof (2.25%), fosfor (0.22%) en kalium inhoud (1.7%) verkry. Die optimale parameters is opgeskaal (1:10) deur gebruik te maak van polivinielchloried anaërobiese verteerders (20 L) om aan die operasionele vereistes van die AnK proses te voldoen en ook om 'n stabiele kompos binne 21 dae te produseer. Die tweede mikpunt van hierdie studie was om die gekombineerde anaërobiese vertering van kelderafvalwater en loog, verkry vanaf die anaërobiese kompos van druiwe doppe in 'n OAS reaktor, te ondersoek. Dit het die bedryf van 'n 2.3 L laboratorium-skaal OAS reaktor vir 205 dae ingesluit. Die reaktor het kelderafwater en loog suksesvol behandel by ongeveer 8.5 kgCSV.m-3d-1 met 'n finale chemiese suurstof vereiste (CSV) vermindering van meer as 90%, 'n stabiele reaktor uitvloeisel pH (7.61) en alkaliniteit (3 281 CaCO3mg.L-1). Hierdie studie het die uitvoerbaarheid van die gekombineerde behandeling van vloeistof en vaste afval van die wynmaakproses getoon. Alhoewel die wetlike vereistes van die reaktor uitvloeisel vir storting op grond nie bereik is nie, is ‘n beduidende vermindering in CSV konsentrasies bereik, asook die vervaardiging van kunsbemesting wat die potensiële aankoopkoste van chemiese kunsmis kan verminder. Die voordele verbonde aan die gebruik van anaërobiese bio-omskakeling as 'n behandelingsopsie vir vloeistof en vaste afval kan moontlik voordelig wees vir die wynbedryf as 'n omgewingsbeheerende tegnologie deur om vloeistof en vaste afval om te skakel na waardevolle bronne.

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Grapevine is constantly under attack from a wide variety of pathogens including viruses, bacteria and fungi. In order to ensure survival, the grapevine has developed a vast array of defense mechanisms to combat invading organisms. A key element of this disease resistance is the production of phytoalexins, of which resveratrol is the primary component. The synthesis of resveratrol, together with other structural and biochemical defense mechanisms equips the plant to combat a number of pathogens resulting in the production of healthy grapes for the vinification of top quality wine. As part of the active disease response resveratrol is synthesised de novo in the berry skin at the site of infection, on recognition of the pathogen. Here it is able to limit the damage caused by the pathogen as well as preventing it from spreading. This gives the plant the opportunity to initiate its systemic acquired resistance thereby protecting the rest of the plant and preventing secondary infections. The fermentation of red wine on the grape skins allows for the extraction of resveratrol from the skin into the wine. Red wines therefore have a significantly higher concentration of resveratrol than white varieties, which contain little or no resveratrol at all. It is for this reason that the moderate consumption of wine, in particular red wine, is synonymous with a healthy lifestyle. The antioxidant and anti-inflammatory activities of resveratrol are important contributors to the cardiovascular benefits derived from the consumption of red wine. It now seems, however, that significant cardiovascular protection is derived from the synergistic action of resveratrol, the polyphenols and the alcohol in wine. With the wholesomeness of any food or beverage being of extreme importance, the aim of this project was to manipulate wine yeast to produce resveratrol during fermentation. This required the introduction of an entire metabolic pathway, by integrating plant genes into the yeast. Resveratrol synthase utilises three malonyl-CoA and one pcoumaroyl- CoA molecules to produce one molecule of resveratrol, Saccharomyces cerevisiae produces malonyl-CoA but no p-coumaroyl-CoA. Therefore, the following genes were obtained to enable yeast to produce p-coumaroyl-CoA: PAL, encoding phenylalanine ammonia-lyase to convert phenylalanine into cinnamic acid; C4H, encoding cinnamate-4- hydroxlyase to convert cinnamic acid into p-coumaric acid; and 4CL9 or 4CL216 encoding CoA-ligases to convert the p-coumaric acid into p-coumaroyl-CoA. To attain high-level expression, the genes were subcloned under the control of the phosphoglycerate kinase gene (PGK1) promoter and terminator. Due to integration problems with these expression cassettes and the fact that the yeast was able to consume p-coumaric acid, the 4CL9, 4CL216 and Vst1 (encoding resveratrol synthase) genes were subcloned under the control of the alcohol dehydrogenase (ADH2) and PGK1 promoters into episomal plasmids, respectively. A laboratory yeast strain containing both the Vst1 and 4CL9, or the Vst1 and 4CL216 genes was evaluated for its ability to utilise p-coumaric acid and produce resveratrol. Northem analysis confirmed that the Vst1, 4CL9 and 4CL216 genes were transcribed and over-expressed compared to the control strain. The transformants expressing the CoA-ligase genes utilised the p-coumaric acid faster than the control, although it was not possible to determine whether p-coumaroyl-CoA was produced. No resveratrol was produced under the assay conditions used. The results indicated that the yeast is unable to produce active resveratrol synthase, which is required to catalyse the final reaction in the production of resveratrol. Posttranslational modification, such as overglycosylation and disulphide formation, of the heterologous protein in yeast has been indicated as the possible reason for the lack of enzyme activity. This introduces an exciting area of research for the development of biotechnological tools with the ability to increase the production of active heterologous proteins in yeast. / AFRIKAANSE OPSOMMING: Wingerde word voortdurend deur 'n groot verskeidenheid patogene, insluitende virusse, bakteriee en swamme, aangeval. Ten einde oorlewing te verseker, het die wingerdstok In wye reeks verdedigingsmeganismes ontwikkel om weerstand te bied teen indringerorganismes. 'n Belangrike faktor in hierdie weerstand teen siektes is die produksie van fitoaleksiene, waarvan resveratrol die hoofkomponent is. Oeur die sintese van resveratrol, asook ander strukturele en biochemiese verdedigingsmeganismes, word die plant toegerus om weerstand te kan bied teen In hele aantal patogene ten einde gesonde druiwe te produseer wat gebruik kan word vir die vinifikasie van topgehalte wyn. As deel van die aktiewe reaksie teen siektes, word resveratrol de novo in die dop van die korrel by die plek van infeksie gesintetiseer sodra 'n patogeen herken word. Hier kan dit die skade deur die patogeen veroorsaak, beperk en verhoed dat dit versprei. Oit gee aan die plant die geleentheid om sy sistemies-verworwe weerstand te inisieer, en daardeur die res van die plant te beskerm, sowel as sekondere infeksies te verhoed. Die fermentasie van rooiwyn op die druifdoppe maak voorsiening vir die ekstraksie van resveratrol uit die dop na die wyn. Die konsentrasie van resveratrol in rooiwyn is dus beduidend hoer as in die wit varietelte, wat geen of baie min resveratrol bevat. Oit is dan juis die rede waarom die matige inname van wyn, veral rooi wyn, gesien word as In integrale deel van 'n gesonde leefwyse. Resveratrol se aktiwiteit as antioksidant en antiinflammatoriese middel lewer In belangrike bydrae tot die kardiovaskulere voordele wat verkry word uit die inname van rooiwyn. Oit blyk egter nou dat die beduidende kardiovaskulere beskerming gesetel is in die sinergistiese werking van resve ratro I, die polifenole en die alkohol in wyn. Aangesien die heilsaamheid van enige voedsel of drank van die uiterste belang is, was dit die doel van hierdie projek om wyngis te manipuleer ten einde tydens die fermentasieproses resveratrol te produseer. Hiervoor moes 'n volledige metaboliese pad daargestel word deur plantgene in die gis te inkorporeer. Resveratrol-sintase maak gebruik van drie maloniel-KoA-molekules en een p-kumarotel-Kos-molekule om een molekule resveratrol te produseer. Saccharomyces cerevisiae produseer maloniel-KoA, maar nie p-kumaroiel-Kcs, nie. Oie volgende gene is dus aangewend om die gis in staat te stel om p-kumarolel-Koe, te produseer: PAL, wat fenielalanien-ammoniak-liase enkodeer om fenielalanien om te sit na kaneelsuur; C4H, wat sinnamaat-4-hidroksliase enkodeer om kaneelsuur om te sit na p-kumaarsuur; en 4CL9 of 4CL216 wat KoA-ligases enkodeer om p-kumaarsuur om te sit na p-kumarolel-Kos, Om hoevlak-uitdrukking te verkry, is die gene gesubkloneer onder beheer van die fosfogliseraat-kinase-geen(PGK1)- promotor en -terminator. As gevolg van integrasieprobleme met hierdie uitdrukkingskassette en die feit dat die gis die p-kumaarsuur kon verteer, is die 4CL9-, 4CL216- en Vst1- (wat resveratrol-sintase enkodeer) gene na episomale plasmiede gesubkloneer onder beheer van die alkohol-dehidrogenase(ADH2)- en PGK1-promotors onderskeidelik. 'n Laboratorium-gisstam wat 6f beide die Vst1-geen en die 4CL9-geen, 6f die Vst1-geen en die 4CL216-geen bevat het, is geevalueer vir die verrnoe om pkumaarsuur te benut en resveratrol te produseer. Noordelike klad analises het bevestig dat die Vst1-, 4CL9- en 4CL216-gene getranskribeer en ooruitgedruk was in vergelyking met die kontrole-stam. Die transformante wat die KoA-ligases uitgedruk het, het die pkumaarsuur vinniger benut as wat die kontrole dit gedoen het, alhoewel dit nie moontlik was om vas te stel of o-kurnarotel-Kos, geproduseer is nie. Met die essai-kondisies wat gebruik is, is geen resveratroI geproduseer nie. Die resultate het daarop gedui dat die gis nie daartoe in staat is om aktiewe resveratrol-sintase, wat nodig is vir die katalise van die finale reaksie in die produksie van resveratrol, te produseer nie. Naomsettingsmodifikasies van die heteroloe protelen in die gis, soos oor-glikosilasie en disulfiedvorming, is aangewys as die moontlike rede vir die gebrek aan ensiemaktiwiteit. Dit stel In opwindende veld vir verdere navorsing voor, naamlik die ontwikkeling van biotegnologiese middele met die vermoe om die produksie van aktiewe heteroloe protelene in gis te verhoog.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The production of wine is a complex process, which involves the conversion of sugar in grape must to ethanol, carbon dioxide and other byproducts. The principal organism in winemaking is yeast, of which Saccharomyces cerevisiae is the most important due to its ability to survive winemaking conditions, its GRAS (Generally Regarded As Safe) status and the favourable flavours it imparts during the winemaking process. However, due to the demands of the consumer and the emergence of sophisticated wine markets, a demand is developing for specialised yeast strains with enhanced and new oenological properties. For these reasons, research into the contribution of wine yeast to the aroma bouquet as well the influence of wine or brandy maturation in wood on the aroma bouquet is important for consumer demands to be met. The fruity aroma of wine is associated with esters, which are produced during the alcoholic fermentation by yeast. Important acetate esters in wine and brandy are ethyl acetate, which has a fruity, solvent-like aroma, and isoamyl acetate, which has a banana-like aroma. These esters are produced through the action of acetyltransferases (AATases), which catalyse the reaction between a higher alcohol and acyl Coenzyme A. Esters are mainly a product of alcoholic fermentation. However, their concentration changes during wood maturation and it has been found that the concentration of acetate esters can increase during the maturation period. In this study, the aim was to investigate the influence of AATase I and AATase II, which are encoded by the ATF1 and ATF2 genes respectively, on the aroma bouquet of wine and brandy. Therefore, the first objective of this study was to clone the ATF2 gene from a commercial wine yeast strain and to overexpress this gene in a commercial wine yeast strain and in a wine yeast strain that already has the A TF1 gene overexpressed. Disruption cassettes were also designed in order to disrupt the ATF1 and ATF2 genes in a commercial wine yeast strain. The resultant recombinant wine yeast strains were used for the production of wine and brandy. GC analyses and tasting trials were conducted to determine the effect of the overexpression or disruption of these genes on the aroma bouquet of wine. The results obtained indicated that there are differences in the aroma bouquet of wine and brandy when changes are made in gene expression. The results indicated that the A TF1 gene plays a large role in the production of ethyl and isoamyl acetate. When this gene was overexpressed, the level of ethyl acetate was 5.6-fold more than that of the control and the level of isoamyl acetate was 3.5-fold higher than that of the control. However, no increase in ethyl acetate or isoamyl acetate was observed when the A TF2 gene was overexpressed. An increase in 2-phenylethyl acetate and diethyl succinate was observed in brandy, although there was a decrease in total ester concentration. A decrease in acetic acid was also observed in the brandy produced, which could be an indication of ester production. Similarly, no increase in ethyl acetate or isoamyl acetate was observed in the wine or brandy produced when both the ATF1 and ATF2 genes were overexpressed in a single yeast. Once again, a marked decrease was observed in acetic acid concentration in both the wine and brandy. In conclusion, it is clear that changes in gene expression can change the aroma profile of wine or brandy. However, the role of the ATF2 gene still remains unclear and further studies are needed to clarify its role in yeast. Future studies involving the effect of wood maturation on ester concentration will also be of importance, so that the winemaker or distiller can make a product that suits the ever-changing market. / AFRIKAANSE OPSOMMING: Die produksie van wyn is 'n komplekse proses wat die omskakeling van die suiker in mos tot etanol, koolstofdioksied en ander byprodukte tot gevolg het. Die hooforganisme betrokke in die wynmaakproses is gis, waarvan Saccharomyces cerevisiae as een van die belangrikste geag word as gevolg van die vermoë daarvan om onder die wynfermentasietoestande te kan oorleef, die "GRAS"-status (Generally Regarded As Safe) daarvan en die invloed wat dit op die aroma van die uiteindelike produk het weens die werking daarvan gedurende alkoholiese fermentasie. Die behoefte aan wyn met nuwe, verbeterde eienskappe het die vraag na meer gespesialiseerde gisrasse deur beide die verbruiker en nuwe wynmarkte gedurende die afgelope paar jaar drasties laat toeneem. Dit is om dié redes dat navorsing oor die bydrae van wyngis en houtveroudering tot die aroma van beide wyn en brandewyn so belangrik geag word. Die vrugtige aroma van wyn word geassosieer met die esters wat gedurende die alkoholiese fermentasie deur gis gevorm word. Die belangrikste asetaatesters in wyn en brandewyn is etielasetaat, wat vir 'n oplosmiddelagtige, vrugtige aroma bekend is, en isoamielasetaat, wat 'n piesangaroma veroorsaak. Die esters word geproduseer deur die werking van asetieltransferases (AATases), wat as katalis in die reaksie tussen 'n hoër alkohol en asetiel-Ko-ensiem A optree. Alhoewel esters hoofsaaklik 'n produk van alkoholiese fermentasie is, wissel die konsentrasie daarvan gedurende houtveroudering. Daar is gevind dat die konsentrasie van die asetaatesters gedurende die verouderingsproses kan verhoog. Die studie het ten doelom die invloed van AATase I en AATase II, wat onderskeidelik deur die ATF1- en ATF2-gene geënkodeer word, op die aroma van wyn en brandewyn te ondersoek. Die eerste doelwit van die studie was vervolgens om die ATF2-geen vanaf 'n kommersiële wyngisras te kloneer en dit daarna te ooruitdruk in 'n kommersiële wyngisras, asook die geen te ooruitdruk in 'n kommersiële wyngisras wat reeds die ATF1-geen ooruitdruk. Disrupsiekassette is ook vir die disrupsie van die ATF1- en ATF2-gene in 'n kommersiële wyngisras ontwerp. Die rekombinante wyngisrasse wat gedurnde die studie gemaak is, is vir die produksie van wyn en brandewyn gebruik. Gas chromatografise-ontledings en sensoriese evaluerings is ook op die wyn en brandewyn uitgevoer. Die resultate van die studie het bewys dat daar wel veranderings plaasvind wanneer 'n verandering in geenuitdrukking gemaak is. Die resultate het weereens bevestig dat die ATF1-geen 'n belangrike rol in die produksie van etiel- en isoamielasetaat speel. Wanneer die ATF1-geen ooruitgedruk is, is die etielasetaatproduksie 5.6 keer meer en die isoamielasetaatproduksie 3.5 keer meer as in die kontrole. Die ooruitdrukking van die ATF2-geen het geen verhoging in etielasetaat of isoamielasetaat of in totale esters in die wyn getoon nie, alhoewel die ras 2.7 keer meer diëtielsuksinaat geproduseer het. In die brandewyn wat geproduseer is met die gisras waarin ATF2 ooruitgedruk is, was daar wel 'n verlaging in die asynsuur, wat 'n aanduiding van estervorming kan wees, alhoewel die totale esters wat geproduseer is minder was as in die kontrole. 'n Verhoging in diëtielsuksinaat en 2-fenielasetaat is ook gevind. Daar is geen verhoging in etiel- of isoamielasetaat getoon wanneer die ATF1- en ATF2-geen saam ooruitgedruk is nie. Die ras het minder totale sure in wyn en brandewyn geproduseer en ook geen verhoging in totale esters getoon nie. Uit die resultate is dit duidelik dat veranderings in geenuitdrukking 'n verandering in die aromaprofiel van wyn en brandewyn kan veroorsaak. Die rol van dié A TF2-geen is nog steeds onduidelik en verdere studies sal moet plaasvind om die rol van die geen te verduidelik. Studies wat konsentreer op die invloed van houtveroudering op esterkonsentrasie is ook belangrik vir die toekoms, want dit sal die wyn- of brandewynmaker meer beheer oor die uiteindelike produk gee en daardeur die wyn- of brandewynmaker help om 'n produk te vervaardig wat sy mark bevredig.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: During wine fermentation, yeasts release extracellular enzymes that significantly impact wine properties. While the extracellular proteins of Saccharomyces cerevisiae have been characterised, those of non-Saccharomyces yeasts remain largely unknown. Most of these enzymes break down sugar polymers or catalyse the liberation of glycosidically-bound molecules. Another category of enzymes of oenological interest is represented by acid proteases that are able to prevent or reduce protein haze, as reported in literature, while simultaneously increasing the assimilable nitrogen content of wine. The liberation of amino acids from peptides and proteins that serve as aroma precursors may also have an indirect effect on wine aroma. In a recent study performed at the Institute for Wine Biotechnology (IWBT), the sequences of two aspartic proteases were retrieved from non-Saccharomyces yeast species isolated from South African wines. The genes, MpAPr1 and CaAPr1, were isolated from two non-Saccharomyces species, Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384, respectively. However, no further characterization was undertaken. This study aimed to clone these two genes into a recombinant bacterial host for expression and purify the corresponding enzymes as a first step toward characterizing their kinetic properties. Considering that some non-Saccharomyces species have been shown to produce more than one acid protease, an additional aim was to identify novel acid proteases within M. pulcherrima IWBT Y1123. Cloning of the genes and transformation of the expression vectors into E. coli were achieved. Optimal conditions for induced expression were established following extensive optimization. Furthermore, while native extraction of the recombinant proteins was unsuccessful, denaturing conditions allowed their recovery, suggesting that the recombinant proteins are encapsulated into inclusion bodies. Recombinant MpAPr1 was purified by using a nickel based column system and mass fingerprinting of the purified enzyme (MpAPr1) confirmed its identity. Purification was followed by refolding experiments, but yielded poor recovery of active enzymes. Unfortunately, recombinant expression of CaAPr1 could not be observed for reasons yet to be elucidated that may include the large sequence dissimilarities between CaAPr1 and MpAPr1. Finally, Southern blot analysis on the genomes of M. pulcherrima IWBT Y1123 and C. apicola IWBT Y1384 revealed that both possess at least one additional protease other than those previously described. Further analysis of the extracellular proteome of M. pulcherrima IWBT Y1123 also confirmed the presence of at least one enzyme able to hydrolyze BSA at a low pH. Unfortunately, mass fingerprinting performed on the entire extracellular proteome and on small groups of proteins thereof did not allow the identification of these enzymes. / AFRIKAANSE OPSOMMING: Gedurende fermentasie van druiwe sap skei gis ekstrasellulêre ensieme af wat ‘n aanmerklike impak op wyn eienskappe het. Terwyl die ekstrasellulêre proteïene vanaf Saccharomyces cerevisiae al gekarakteriseer is, bly die van nie-Saccharomyces spesies grootliks onbekend. Meeste van hierdie ensieme breek suiker polimere af of kataliseer die vrystelling van glikosiediese verbonde molekules. ‘n Ander klas van ensieme wat van belang is vir oenologie word voorgestel deur proteases wat in staat is daartoe om proteïenewaas te verminder, soos voorheen geraporteer is in literatuur, terwyl dit terselfde tyd die assimileerbare stikstof inhoud kan vermeerder. Die vrystelling van aminosure vanaf peptiede en/of proteïene wat as aroma voorlopers dien mag ook ‘n indirekte effek op die wyn se aroma profiel hê. In ‘n onlangse studie wat uitgevoer is by die Instituut vir Wynbiotegnologie (IWBT) was die volgordes van twee aspartiese proteases bepaal vanaf twee nie-Saccharomyces gis spesies wat geisoleer was uit Suid-Afrikaanse wyne. Die gene MpAPr1 en CaAPr1, was afsonderlik geisoleer vanuit twee nie- Saccharomyces giste, Metschnikowia pulcherrima IWBT Y1123 en Candida apicola IWBT Y1384. Egter was daar geen verder karakterisering van hierdie ensieme nie. Die doel van hierdie studie is om die bogenoemde gene in ‘n rekombinante bakteriese gasheer te kloneer vir uitdrukking en suiwering as ‘n eerste stap tot karakterisering van hul kinetiese eienskappe. Om in ag te neem dat sommige nie-Saccharomyces spesies meer as een protease produseer was ‘n aditionele mikpunt om vir nuwe suur proteases te soek binne M. pulcherrima IWBT Y1123. Klonering van hierdie gene en transformasie van die uitdrukkings vektore in E. coli was suksesvol. Optimale kondisies vir die induksie van ekspressie was bevestig na omvattende optimalisering. Verder, terwyl inheemse ekstraksie van die rekombinante proteïene onsuksesvol was, het denatureerende kondisies toegelaat vir suksesvolle ekstraksie, wat voorgestel het dat die rekombinante proteïene geinkapsileer word in inklusie liggame. Rekombinante MpAPr1 was gesuiwer deur gebruik te maak van ‘n niekel gebaseerde kolom sisteem en massa petied fingerafdrukke van die gesuiwerde ensiem (MpAPr1) het die identiteit bevestig. Suiwering was gevolg deur hervouing eksperimente, maar het swak opbrengste gelewer van die aktiewe ensiem. Ongelukkig kon die rekombinante ekspressie van CaAPr1 nie gevisualiseer word nie vir redes wat nog bevestig moet word, maar wat mag behels dat daar groot volgorde veskille tussen MpAPr1 en CaAPr1 kan wees. Uiteindelik was Southern blot hibridiseering analises uitgevoer op die genome van albei M. pulcherrima IWBT Y1123 en C. apicola IWBT Y1384 wat voorgestel het dat albei ten minste een addisionele protease, anders as die wat voorheen geidentifiseer was, bevat. Verder analiese van die ekstrasellulêre proteoom van M. pulcherrima IWBT Y1123 het ook die teenwoordigheid van ten minste een ensiem bevestig wat die vermoë het om BSA te hidroliseer by ‘n lae pH. Ongelukkig het massa peptied vingerafdrukbepaling wat uitgevoer was op die hele ekstrasellulêre proteoom en op klein groepe protein nie identifikasie van hierdie ensieme bevestig nie.