Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Monoterpene alcohols (monoterpenols) play an important role in the flavour and aroma of grapes and wine. This is especially applicable to wines of a muscat variety, but these flavour compounds are also present in other non-muscat grape varieties, where they supplement other varietal flavours and aromas. These monoterpenols can be found in grapes and wine as free, volatile and odorous molecules, as well as in flavourless, nonvolatile glycosidic complexes. These complexes most often occur as 6-0-a-L-arabinofuranosyl-p-D-glucopyranosides (vicianosides), 6-0-P-D-xylopyranosyl- P-D-gluco-pyranosides (primverosides), 6-0-P-D-glucopyranosyl-p-D-glucopyranosides (gentio-biosides ), 6-0-a-L -rhamnopyra nosyl-p-D-g lucopyra nos ides (rutinos ides), or 6-0-p-D-apiofuranosyl-p-D-glucopyranosides of mainly linalool, geraniol, nerol, a-terpineol and hotrienol. These precursors are, however, hydrolyzed only to a limited extent by endogenous glycosidases during the fermentation process, as they exhibit very low activity in wine conditions. The monoterpenols can be released from their sugar moieties by one of two methods: either an acid or an enzymatic hydrolysis. The enzymatic hydrolysis mechanism is fully understood, and the process functions in two successive steps: firstly, depending on the precursor, the glycosidic linkage is cleaved by an a-L-arabinofuranosidase, an a-L-rhamnosidase, a p-D-xylosidase, or a p-D-apiosidase. The second step involves the liberation of the monoterpene alcohol by a p-glucosidase. This enzymatic hydrolysis does not influence the intrinsic aromatic characteristics of the wine, as opposed to acid hydrolysis. As the endogenous grape glycosides of Vitis vinifera and the yeast Saccharomyces cerevisiae show very low activity towards these aromatic precursors during the handling of the juice and winemaking processes, the focus has increasingly fallen on introducing exogenous p-glucosidases to wines and juices. Genes encoding p-glucosidases and a-L-arabinofuranosidases have been cloned from various organisms, including bacteria, fungi and yeasts. However, the activities and properties of these enzymes are not always suitable for exploitation under winemaking conditions, where a low pH, low temperatures, and high ethanol and glucose concentrations prevail. A genetically engineered wine yeast strain of S. cerevisiae that expresses glycosidases that are active in these conditions would be useful in improving the flavour and aroma of wines, thereby adding to the complexity and value of the wine. Two p-glucosidase genes, BGL 1 and BGL2 from Saccharomycopsis fibufigera, were subcloned into two Escherichia coli-yeast shuttle vectors. A dominant selectable marker gene (SMR1) was also inserted onto these plasmids. These plasmids were designated pBGL 1 (containing the BGL 1 gene) and pBGL2 (containing the BGL2 gene) respectively. Introduction of the two plasmids into two strains of S. cerevisiae then followed. A laboratory strain, L1278, was transformed to confirm the effective secretion of the expressed protein. An industrial yeast strain, VIN13, was subsequently transformed by making use of the selectable marker (resistance against sulfometuron). Enzyme assays with the synthetic substrate p-nitrophenol-j3-D-glucopyranoside (pNPG) were performed to determine the activity of the j3-glucosidases over a period of days, as well as at certain temperatures and pH values. The stability of the enzymes was also investigated. These recombinant yeasts were able to degrade the pNPG efficiently. They showed promising results concerning pH optima, with a substantial amount of activity found at the pH levels as found in the wine environment. There was also a slight increase in specific activity at lower temperatures. The recombinant yeast strains were also tested in smallscale fermentations. Three wines were made, of which two were from white cultivars (Chenin blanc and GewOrtztraminer) and one from red (Pinotage). Results obtained from micro-extraction from the finished wines showed that the terpenol content did increase, although this was not the only wine component influenced. Other flavour compounds also showed increases, especially the esters. This also played a role in the flavour increase in the wine. Future work would include optimizing the available results. This would entail the addition of another glycosidic enzyme, such as a-L-arabinofuranosidase, to the genome of the wine yeast to aid the further breakdown of glycosidic bonds. The cloning or engineering of a j3-glucosidase enzyme that is more active at low temperatures would also yield better results and release even more of the aroma of the wine. / AFRIKAANSE OPSOMMING: Monoterpeenalkohole (monoterpenole) speel 'n belangrike rol in die geur en aroma van druiwe en wyn. Dit is veral van toepassing op wyne van Muskaat-varieteite, maar hierdie geurkomponente is ook teenwoordig in ander nie-Muskaat druifsoorte, waar dit bydra tot die varieteitsqeur en aroma. Hierdie monoterpenole kom voor in druiwe as vry, vlugtige en aromatiese molekules, of as geurlose, nie-vlugtige glikosidies-gebonde komplekse. Hierdie komplekse is meestal in die vorm van 6-0-a-L-arabinofuranosiel-~-D-glukopiranosiede, 6- O-~-D-xilopiranosiel-~-D-glukopiranosiede (primverosiede), 6-0-~-D-glukopiranosiel-~-Dglukopiranosiede (gentiobiosiede), 6-0-a-L-ramno-pyranosiel-~-D-glukopiranosiede (rutinosiede), of 6-0-~-D-apiofuranosiel-~-D-glukopirano-siede van hoofsaaklik linalool, geraniol, nerol, a-terpineol en hotrienol. Hierdie geurvoorlopers word egter slegs tot In beperkte mate tydens die proses van fermentasie deur die endogene glikosidase ensieme gehidroliseer, aangesien hulle baie min aktiwiteit toon onder wynbereidingstoestande. Die monoterpenole kan op een van twee wyses van hul suikermolekules vrygestel word: 'n suurhidrolise, of ensiematiese hidrolise. Die ensiematiese hidroliseproses word baie goed begryp en behels twee opeenvolgende stappe: eerstens, afhangende van die aard van die voorloper, word die glikosidiese verbinding deur In a-L-arabinofuranosidase, In a-L-ramnosidase, In ~-D-xilosidase, of 'n ~-D-apiosidase gebreek. In die tweede stap word die monoterpeenalkohol deur In ~-glukosidase vrygestel. Hierdie ensiematiese afbraakproses verander nie die intrinsieke aromatiese kenmerke van die wyn, soos wat met suurhidrolise die geval is nie. Omdat die endogene glikosidases van Vitis vinifera en die van die gis Saccharomyces cerevisiae baie lae aktiwiteit ten opsigte van die aromatiese voorlopers gedurende die hantering van die druiwesap en wynmaakprosesse toon, val die fokus al hoe meer op die inkorporering van eksogene ~-glukosidases in wyn en sappe. Gene wat vir ~- glukosidases en a-L-arabinofuranosidases kodeer, is al vanuit verskeie organismes gekloneer, insluitende bakteriee, fungi en giste. Die aktiwiteite en kenmerke van hierdie ensieme is egter nie altyd wenslik vir hul gebruik in wyn nie, aangesien dit In omgewing is met 'n lae pH, lae temperatuur, en hoe etanolvlakke en glukose-konsentrasies. In geneties veranderde wyngis van S. cerevisiae wat in staat is om glikosidases uit te druk wat onder hierdie kondisies aktief is, sal baie handig te pas kom in die verbetering van die geur en aroma van wyne, om daardeur die kompleksiteit en die waarde van die wyn te verhoog. Twee ~-glukosidasegene, BGL 1 en BGL2 vanaf die gis Saccharomycopsis fibuligera , is in twee afsonderlike Esccherichia coli-gis-pendelplasmiede gesubkloneer. In Dominante selekteerbare merkergeen (SMR1) is ook in hierdie plasmiede gekloneer. Hierdie plasmiede word onderskeidelik pBGL 1 (met die BGL 1-geen) en pBGL2 (bevattende die BGL2-geen) genoem. Hierdie twee plasmiede is hierna apart na twee rasse van S. cerevisiae getransformeer. Eerstens is 'n laboratoriumras, L1278, getransformeer om te bevestig dat effektiewe sekresie en uitdrukking van die proteTen plaasvind. Hierna is 'n industriele gisras, VIN13, getransformeer deur gebruik te maak van die selektiewe merker (bestandheid teen sulfometuron). Ensiem-bepalings met behulp van die sintetiese substraat p-nitrofeniel-p-O-glukopiranosied (pNPG) is gedoen om die aktiwiteit van die p-glukosidqses oor 'n aantal dae te bepaal, asook om die aktiwiteit by sekere temperature en pH-vlakke te meet. Die stabiliteit van die ensieme is ook bepaal. Hierdie rekombinante giste was in staat om pNPG effektief af te breek. Hulle het belowende resultate betreffende die pH-optima getoon, met 'n aansienlike hoeveelheid aktiwiteit by die pH-vlakke soos dit in die wynomgewing voorkom. Daar was ook 'n effense verhoging in die ensieme se aktiwiteite by laer temperature. Die rekombinante gisrasse is ook in kleinskaalse wynfermentasies gebruik. Drie verskillende wyne is gemaak, waarvan twee wit kultivars was (Chenin blanc en GewOrtztraminer) en een 'n rooi kultivar (Pinotage). Resultate wat deur die mikro-ekstraksie van die voltooide wyne verkry is, het getoon dat die terpenolinhoud wei verhoog het, alhoewel dit nie die enigste wynkomponente was wat beinvloed is nie. Ander geurkomponente het ook 'n verhoging in konsentrasie getoon, veral die esters. Hierdie verbindings het ook 'n rol in die verhoging van geur in die wyne gespeel. Toekomstige werk sal die beskikbare resultate verder optimaliseer. Dit sal insluit die byvoeging van nog 'n glikosidiese ensiem, soos a.-L-arabinofuranosidase, tot die genoom van die wyngis, om verdere afbraak van glikosidiese verbindings teweeg te bring. Die klonering of verandering van 'n p-glukosidase-ensiem met verhoogde aktiwiteit by laer temperature sal ook beter resultate toon en meer geur in die wyn kan vrystel.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Brandy production is a multi-step process that involves grape harvesting, base wine fermentation, distillation, wood maturation and blending. Within each of these production process steps there are a number of factors that can influence the composition and resultant quality of the base wine, unaged and wood matured distillates. These factors include geographic and climatic features of the origin of grapes used, viticultural practices, grape maturity, grape variety, vintage variation, vinification techniques, storage of the base wine prior to distillation, distillation technique, age and origin of oak wood used for maturation and barrel toasting levels. The composition of flavour is extremely complex in wine and distilled beverages such as brandy. A multitude of compounds can take part in the formation of flavour, and it is rare that a particular compound, that is solely responsible for nuances of a specific flavour, is identified. Thus, taking brandy production factors and the nature of aroma and flavour into account, it is clear that thorough understanding of the complexities affecting the perception of quality in brandy is difficult. In commercial brandy production, standard operating procedures do not allow for the separate distillation of brandy base wines from different producers and mixing of base wines from different regions and producers thus occurs. This makes it difficult to determine whether the quality determination of the base wine is in fact an indication of the quality of the resultant distillate after the required period of wood maturation. Therefore, the aim of this study is to determine whether there is any merit in storing and distilling brandy base wines from different producers and regions separately. This was done by firstly determining the demographic and production factors that influence brandy base wine composition. Secondly, the influence of brandy base wine composition on the quality of the respective unaged potstill distillates as well as the ultimate style and quality of the threeyear old wood matured potstill distillates was determined. For this purpose, four potstills with a capacity of 2000 L each were isolated and used for commercial scale distillations of 33 and 25 brandy base wines in 1999 and 2000, respectively, at the DisteIl distillery in Worcester. The experimental outlay used in the study closely emulated Distell's standard operating procedures for commercial brandy production. Chenin blanc and Colombar are the two most popular grape varietals used in the making of brandy base wine in South Africa. Due to the complexity of the brandy production process, the first part of the study only focussed on South African young Chenin blanc wines, with a predominantly fermentation derived aroma. The concentration of iso-amyl acetate, hexyl acetate, ethyl caprylate, ethyl caprate, 2-phenethyl acetate and octanoic acid was significantly higher in wines awarded gold and silver medals and decreased significantly with subsequent decreases in quality categories. Ethyl lactate exhibited the opposite pattern. A quality predictor model based on the behaviour of 21 volatile compounds quantified in these wines had a resonable prediction accuracy when having to predict the quality of wines made in the same vintages as it had been trained on. It was significantly poorer when tested on a completely different vintage of wines to the ones it had been trained on. The number of vintages, number of wines per vintage and the number of compounds quantified for the model influence its prediction accuracy. Twenty-seven volatile compounds were quantified in 33 and 25 brandy base wines, their unaged and three year old distillates from 1999 and 2000, respectively. ANOVA and CART analysis showed that vintage, region, harvest time, choice of cultivar and yeast strain can have a significant influence on the volatile compound composition of brandy base wines, their unaged and three year old distillates. These factors as well as the volatile compound composition were also found to influence the sensory quality of these products. Base wines, unaged and three year old distillates originating from the De Dooms region, which predominantly cultivates table grapes, were of significantly lower quality than those from the remaining regions. Products made from grapes harvested early in the season were of significantly higher quality. The volatile aroma compound composition was found to differ significantly between the 1999 and 2000 base wines and distillates, irrespective of the exclusion of those samples that had undergone partial or complete malolactic fermentation. Consequently, quality indicating compounds may vary from vintage to vintage. The relationship between the quality of brandy base wines and the concentration of n-butanol, iso-amyl acetate, ethyl lactate, ethyl caprylate, octanoic- and decanoic acid was the same as that reported in young Chen in blanc wines in this study. In unaged distillates, increased levels of ethyl lactate also exert a negative influence on distillate quality. Iso-amyl acetate, hexyl acetate, ethyl caproate, ethyl caprylate, n-butanol, octanoic acid, ethyl caprate and decanoic acid showed some positive correlation, whilst iso-butanol, ethyl lactate, acetic acid, acetaldehyde and ethyl acetate showed a significant negative correlation to three-year old distillate quality. Sensory descriptive analysis on selected good, average and poor quality distillates using the South African brandy aroma wheel showed that there are small differences in profile between the good and average distillates, there were however significant differences between the good and poor quality distillate profiles throughout maturation. After three years of wood maturation, the aroma profile of poor quality distillates can be characterised by prominent herbaceous and woody aromas, which are more intense than the fruity aromas. Good quality distillates contained characteristically intense fruity aromas. Volatile compound concentration differences were noted during the course of and after three years of wood maturation and in barrels of varying ages. Distillates matured in new block barrels exhibited significant differences in volatile and wood compound composition after three years when compared to remaining barrels used. The style classification of the three-year old potstill distillate was influenced by demographic and production factors and volatile compound composition, but not by the sensory quality of the distillates. In summary, vintage, region, cultivar, harvest time and choice of yeast strain have a significant influence on the volatile composition of brandy base wines, their unaged and three year old potstill distillates, which in turn affects the sensory quality of these products. These effects cannot be viewed in isolation as they jointly exert an influence on the composition and quality of these products. From a commercial perspective, this study has provided a valuable indication as to which production and demographic factors can influence the quality and style of potstill brandy. Thus, future brandy base wine intake should, as far as possible, take place in such a manner to allow base wines originating from the same cultivar or region or harvest time or combination thereof (and to a lesser extent yeast strain) to be received simultaneously at the distillery for distillation. / AFRIKAANSE OPSOMMING: Brandewyn produksie is 'n veelvoudige proses wat die volgende insluit: pars van druiwe, basiswyn fermentasie, verstoking, houtveroudering en versnyding. Binne hierdie stappe is verskeie faktore wat 'n invloed op die samestelling en resultate van die basiswyn, onverouderde en verouderde distillaat kan hê. Hierdie faktore sluit die volgende in: geografiese ligging, klimaat toestande van die streek van herkoms, wingerdboupraktyke, graad van rypheid, druifvarieteit, wynoes variasie, wynbou tegnieke, opberging van basiswyn voor distillasie, distillasie tegnieke, ouderdom en oorsprong van akkerhout vir die veroudering, asook tot watter mate die vat gerooster is. Die samestelling van geur in wyn en gedistilleerde produkte soos brandewyn is uiters kompleks. 'n Menigte samestellings kan deel vorm van die inhoud van die geur, en dit is raar dat 'n bepaalde verbinding alleenlik verantwoordelik is vir 'n kenmerkende geur. Weens die verskeidenheid van produksie faktore, aroma en smaak is volledige kennis van brandewyn ingewikkeld en kompliseerd om te verstaan. In kommersiële brandewynproduksie, maak standaard werksprosedures nie voorsiening vir aparte distillasie van basiswyn van verskillende produsente nie, en die vermenging van basiswyn van verskillende streke en produsente kom voor. Dit maak dit moeilik om te bepaal of die kwaliteit van die basiswyn of die distillaat na die neergelegde periode van houtveroudering, in werklikheid 'n beslissende indikasie op die kwaliteit van die verouderde produk is. Daarom is een van die doele van hierdie studie om te bepaal of daar meriete in is om brandewyn basiswyn van die verskillende produsente en streke apart te berg en te verstook. Dit is gedoen deur eerstens, die effek van demografiese en produksie faktore op die samestelling van brandewyn basiswyn te bepaal. Daarna, om die invloed van brandewyn basiswynsamestelling op die kwaliteit van onderskeidelik die onverouderde distillate sowel as die uiteindelike styl en kwaliteit van die 3 jaar oue houtverouderde potketel distillate, te bepaal. Vir hierdie doel, is vier potketels met 'n kapasiteit van 2000 liters elk geïsoleer en vir kommersiële skaal distillasies van 33 en 25 brandewyn basiswyne in 1999 en 2000, ondeskeidelik, by DisteIl Distillerdery, Worcester verstook. Die eksperimentele uitleg wat in hierdie studie gebruik is, ewenaar DisteIl se standaard verstokingsprosedures vir kommersiële brandewynproduksie. Chenin blanc en Colombar is die twee mees populêre druifvarieteite vir die maak van brandewyn basiswyn in Suid-Afrika. As gevolg van die kompleksiteit van die brandewyn produksieproses, is die eerste gedeelte van die studie gefokus op die Suid-Afrikaanse jong Chenin blanc wyne, met 'n oorheersende fermentasie geur. Die konsentrasie van iso-amyl asetaat, heksiel asetaat, etiel kaprilaat, etiel kapraat, 2-phenetiel asetaat en oktanoë suur was aansienlik hoër in wyne wat met goud en silwer medaljes bekroon is en neem aansienlik af met afname in kwaliteit kategorieë. Etiel laktaat vertoon die teenoorgestelde patroon. 'n Kwaliteit voorspellersmodel, gebaseer op die gedrag van 21 vlugtige komponente was opgestel. Die model het redelike akkuraatheid getoon op wyne van dieselfde oesjaar as waarop oorspronklik opgelei. Die model se voorspellingsakkuraatheid was aansienlik swakker toe dit vergelyk getoets is met wyne van 'n nuwe oesjaar. Die hoeveelheid oesjare, hoeveelheid wyne per oesjaar en die hoeveelheid verbindings gebruik in hierdie model sal die voorspellingsakkuraatheid beïnvloed. Sewe-en-twintig vlugtige komponente was gekwantifiseer in 33 en 25 brandewyn basiswyne, die onverouderde en 3 jaar oue distillate vanaf 1999 en 2000, onderskeidelik. Variasie ontledings (ANOVA) en klassifikasie en regressie ontledings (CART) ontledings wys dat oesjaar, streek, parstyd, keuse van kultivar en gisrasse In aansienlike invloed op die vlugtige samestelling van brandewyn basiswyne asook hul onverouderde en 3 jaar distillate het. Hierdie faktore, sowel as die vlugtige verbinding samestellings, beinvloed ook bevind die sensoriese kwaliteit van hierdie produkte. Basiswyne, onverouderde en 3 jaar oue distillate wat vanuit die De Doorns streek, wat hoofsaaklik tafeldruif kultivars is, is van In swakker kwaliteit as die ander streke. Produkte wat gemaak is van druiwe wat vroeg in die seisoen gepars is, was van beter kwaliteit. Die vlugtige geurkomponentsamestellings het tussen die basiswyne en distillate van die 1999 en 2000 oesjare verskil, ongeag of party van die monsters gedeeltelike of volledige appelmelksuurgisting ondergaan het. Gevolglik, die konsentrasie van kwaliteitaanwysende komponente mag van seisoen tot seisoen verskil. Die verhouding tussen die kwaliteit van die brandewyn basiswyne en die konsentrasie van n-butanol, iso-amiel asetaat, etiel laktaat, etiel kaprilaat, oktanoë- en dekanoë suur was dieselfde soos gerapporteer oor die jong Chenin blanc wyne. In onverouderde distillate, het verhoogde vlakke van etiel laktaat In negatiewe invloed op distillaat kwaliteit veroorsaak. Iso-amiel asetaat, heksiel asetaat, etiel kaproaat, etiel kaprilaat, n-butanol, oktanoë suur, etiel kapraat en dekanoë suur In positiewe korrelasie, terwyl iso-butanol, etiel laktaat, asynsuur, asetaldehied en etiel asetaat betekenisvolle negatiewe korrelasie met 3jaar oue distillaat kwaliteit bewys. Beskrywende sensoriewe analises op geselekteerde, middelmatige en swak kwaliteit distillate (gedoen deur gebruik making van die Suid-Afrikaanse Brandewyn Aromawiel) wys dat daar klein verskille tussen die profiele van goeie en middelmatige distillate is. Daar is egter aansienlike verskille tussen goeie en swak kwaliteit distillataat profiele regdeur die drie jaar verouderingsperiode. Na 3 jaar van houtveroudering kan die geur profiel van swak kwaliteit distillate uitgeken word aan kruid- en houtagtige geure, wat meer intens as die vrugagtige geure is. Goeie kwaliteit distillate het intense vrugagtige-aroma eienskappe. Vlugtige verbinding konsentrasie verskille is waargeneem gedurende en na die 3jaar houtverouderingsperiode in vate wat van ouderdomme verskil. Distillate wat in nuwe blokkies vate verouder is, vertoon aansienlike verskille in vlugtige en houtkomponent samestellings na 3 jaar wanneer dit met die, wat in die oorblywende ouer vate verouder is, vergelyk word. Die styl klassifikasie van die 3 jaar oue potketeldistillaat was deur demografiese en produksie faktore sowel as vlugtige verbinding samestellings beïnvloed, maar nie met die sensoriese kwaliteit van die distillate nie. In opsomming, oesjaar, streek, kultivar, parstyd en keuse van gisrasse het 'n aansienlike invloed op die vlugtige komponent samestelling van die brandewyn basiswyne, onverouderde en 3 jaar oue potketel distillate, wat weer op hul beurt die sensoriese kwaliteit van die onderskeidelike produkte beïnvloed. Die effek van hierdie faktore kan nie in isolasie gemeet word nie, aangesien dit gesamentlik die kwaliteit van die produkte beïnvloed. Van 'n kommersiële perspektief het hierdie studie 'n waardevolle indikasie gelewer van hoe produksie en demografiese faktore die kwaliteit en styl van potketelbrandewyn kan beïnvloed. Daarom word voorgestel dat toekomstige brandewyn basiswyn innames, sover as moontlik, voorsiening moet maak om wyne van dieselfde kultivar, uit dieselfde streek, parsseisoen inaggeneem, of kombinasie daarvan, met 'n mindermate die gisrasse in aanmerking geneem, gelyktydig vir verstoking by die distilleerderye ontvang word sodat hierdie wyne apart van wyne met ander bogenoemde variasies verstook en verouder kan word.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: No single chemical constituent can be accredited with giving wine and brandy their overall aroma and flavour. The aroma and flavour of wine and brandy are rather attributed to a number of chemical constituents reacting together and it is these reactions that give the beverage its character. Certain chemicals within wine and brandy do, however, make larger contributions to the flavour. These include the esters, terpenes and volatile acids, although others also exist. Esters are a large group of volatile compounds with variable aroma and flavour characteristics, including banana-like (isoamyl acetate), apple-like (ethyl caproate) and chemical/solvent-like (ethyl acetate). Esters are produced as secondary metabolites during the conversion of sugar to ethanol and are formed when an alcohol binds with a fatty acid. Chemically, ester metabolism is well documented and understood; however, much work still needs to be done on a genetic level. The yeast strain used during fermentation is one of the most important factors contributing to the type and quantity of esters produced. This is due to differences in genetic makeup. The metabolism of esters is controlled largely on a genetic level, with numerous genes being involved. The alcohol acetyltransferase genes are involved in ester anabolism, whilst esterase genes are involved in ester catabolism. Esterases have a negative effect on the overall level of esters within an alcoholic beverage, as they are capable of reducing the number of esters and are thus capable of altering the beverage's aroma and flavour profile. The IAH1 and the TIP1 gene products are believed to encode for two such esterases. The objective of this study was to investigate the contribution of the IAH1 and TIP1 genes to the level of esters in both wine and brandy. This was accomplished by using two approaches. Firstly, the above genes were disrupted using a polymerise chain reaction (PCR)-generated disruption cassette homologous to either the IAH1 or the TIP1 gene. These cassettes were integrated into the industrial wine yeast, Saccharomyces cerevisiae strain VIN13. The integrations were verified by Southern blot analysis to produce yeasts VIN13-~IAH1 and VIN13-~TIP1; however, only a single copy of each was disrupted. Secondly, the IAH1 and the TIP1 genes were cloned from S. cerevisiae using PCR into plasmid pj between the phosphoglycerate kinase gene (PGK1) promoter and terminator, producing plasmids pJ-IOE1 and pJ-TOE1. The PGK1 promoter has previously been shown to constitutively express genes at high levels. These new constructs were then used as template for PCR to produce two overexpression cassettes, one for IAH1 and the other for TlP1. These cassettes were integrated into S. cerevisiae VIN13 and verified by Southern blot analysis to produce strains VIN13-IOE1 and VIN13-TOE1. The above yeast strains including VIN13 were used for the production of wines and base wines from Colombard must. Reverse-transcriptase (RT-PCR) confirmed that the VIN13-IOE1 and VIN13-TOE1 strains overexpressed the appropriate gene at a higher level than the control VIN13 strain. The VIN13-AIAH1 disrupted strain showed no difference in expression level to that of the control strain, whilst VIN13-ATIP1 showed lower levels of expression than that of the control strain. VIN13-IOE1 behaved as expected, with a decrease of between 30% and 60% in the total ester level in the wine and base wine respectively, a 30% decrease in the total acid level and no change in the higher alcohol level. The VIN13-AIAH1 strain showed no difference to the control wine, most likely as this strain still expressed the IAH1 gene at levels consistent with the control strain. VIN13-TOE1 behaved in an unexpected manner - instead of hydrolysing esters, it appeared to produce them. This increase in the total ester level was most noticeable during distillation, when a 20% increase took place. Another unexpected occurrence was a large decline in the total acid level, with acetic acid being the most significant contributor, decreasing by up to 78%. This is a very favourable finding, as acetic acid is a known spoilage molecule and is a cause of sluggish/stuck fermentations. VIN13-ATIP1 behaved in an opposite manner to VIN13-TOE1, with higher total acid levels and slightly decreased total ester levels, especially during distillation. Neither affected the total higher alcohol levels. Sensorially, the only significant difference in the wine samples was for the fruity flavour. A panel of judges distinguished that VIN13-TOE1 was fruitier than the other wines, with VIN13-ATIP1 being the least fruity. This study again proves the significant impact that a single gene can have on the chemical makeup of wine and brandy. The relatively simple genetic alteration of an organism can drastically change and improve not only the organoleptic properties of the organism, but its viability as well. These alterations can produce more favourable organisms with more desirable characteristics for the fermenting beverage industry to produce products of higher quality and better suitability. / AFRIKAANSE OPSOMMING: Geen chemiese komponent kan uitgesonderword as die produseerder van aroma en geur in wyn of brandewyn nie. Die aroma en geur van wyn en brandewyn word eerder toegeskryf aan die interaksie tussen 'n groot aantal chemiese komponente om aan die drank sy karakter te gee. Enkele van hierdie chemiese komponente sluit in esters, terpene en vlugtige sure, om maar 'n paar te noem. Esters is "n groot groep van vlugtige verbindings wat beskik oor 'n verskeidenheid van aroma- en geurkenmerke, soos piesangagtig (isoamielasetaat), appelagtig (etielkaproaat) en chemies/oplosmiddelagtig (etielasetaat). Esters word as sekondêre metaboliete geproduseer wanneer suikers na etanolomgeskakel word en word gevorm wanneer "n alkohol met "n vetsuur verbind. Estermetabolisme is chemies goed beskryf en verstaan, maar op "n genetiese vlak is daar nog heelwat aspekte wat nagevors moet word. Die gisras betrokke gedurende fermentasie word beskou as een van die grootse bydraes tot die tipe en die hoeveelheid esters wat geproduseer word. Dit word toegeskryf aan verskille in die genetiese saamestelling van die gisras. Ester metabolisme word grootliks deur genetiese faktore beheer en verskeie gene is betrokke. Dit is hoofsaaklik die alkoholasetieltransferasegene wat vir esterkatabolisme verantwoordelik is, terwyl die esterasegene vir esteranabolisme verantwoordelik is. Esterases het 'n negatiewe effek op die totale estervlak binne alkoholiese dranke deurdat hulle in staat is om die aantal esters drasties te verminder en sodoende die drank se aroma- en geurprofiel te verander. Daar is voorgestel dat die IAH1- en die TlP1-geen produkte is wat vir twee sulke esterases kodeer. Die doel van hierdie studie was om die IAH1- en die TIP1-gene se bydrae tot die totale estervlak in wyn en brandewyn te ondersoek. Dit is deur twee benaderings uitgevoer. Eerstens is die bogenoemde gene d.m.V. disrupsiekassette wat homoloog aan die IAH1- of die TlP1-gene was, uitgeslaan. Die disrupsiekassette is deur die polimerasekettingreaksie (PKR) geproduseer. Hierdie kassette is in die industriële wyngis, Saccharomyces cerevisiae VIN13, geïntegreer. Die integrasies is deur Southernkladanalise bevestig en het die giste VIN13-~IAH1 en VIN13-~TIP1 gelewer. Net 'n enkele kopie van elke geen is egter uitgeslaan. Tweedens is die IAH1- en TIP1-gene d.m.V. PKR vanaf S. cerevisiae binne in plasmied pJ gekloneer, tussen die fosfogliseraatkinasegeen (PGK1) se promotor en termineerder, om plasmiede pJ-IOE1 en pJ-TOE1 te produseer. Die PGK1-promotor is al tevore geïdentifiseer as "n hoë-vlak konstitutiewe uitdrukker van gene. Hierdie twee nuwe konstrukte het vervolgens gedien as templaat vir PKR om twee ooruitdrukkingskassette, een vir IAH1 en die ander vir TIP1, te produseer. Hierdie kassette is in S. cerevisiae VIN13 geïntegreer en bevestig deur Southernkladanalise. Hierdie integrasies het die giste VIN13-IOE1 en VIN13-TOE1 geproduseer. All die nuwe gisrasse, tesame met VIN13, is gebruik vir die produksie van wyne sowel as rebatwyne vanaf Colombard-mos. Omgekeerde-transkriptase polimerasekettingreaksie (OT-PKR) het bewys dat die VIN13-IOE1 en VIN13-TOE1 rasse die geskikte geen ooruitgedruk het, met hoêr vlakke as van die kontrole VIN13-ras. Dit het ook aangedui dat die VIN13-i\IAH1-ras, waarvan die geen uitgeslaan was, geen verskil in uitdrukking gehad het in vergelyking met die kontroleras nie, terwyl VIN13-i\TIP1 'n lae uitdrukkingsvlak getoon het. VIN13-IOE1 het teen verwagting opgetree, met 'n afname van tussen 30% en 60% in die totale estervlak in beide die wyne en rebatwyne. 'n Afname van 30% in die totale suurvlak, asook geen waarneembare verskil in die hoêr alkoholvlak, in vergelyking met die kontroleras, is ook opgemerk. Die VIN13-i\IAH1-ras het glad nie van die kontroleras verskil nie, heel waarskynlik omdat hierdie ras die IAH1-geen teen dieselfde vlak as die kontroleras kon uitdruk. Die VIN13-TOE1-ras het teen verwagting opgetree deurdat dit esters geproduseer het i.p.v. om esters te hidroliseer. Hierdie toename in die totale estervlak is die meeste waarneembaar tydens distillasie, met tot 'n 20% toename. Nog 'n onverwagte effek was die groot afname in die totale suurvlak. met asynsuur wat die betekenisvolste bydrae gelewer het deurdat dit 'n afname van tot 78% getoon het. Hierdie bevinding is baie voordelig, aangesien asynsuur, 'n bekende bederfmolekuul, veral vir slepende/gestaakte fermentasies verantwoordelik is. VIN13-i\TIP1 het op die teenoorgestelde wyse opgetree as VIN13-TOE1, met 'n hoêr totale suurvlak en 'n klein afname in die totale estervlak. Weereens is dit meer gedurende distillasie waargeneem. Beide rasse het egter geen effek op die hoêr alkoholvlak gehad nie. Die proepaneel het, met betrekking tot die vrugtige geur, een betekenisvolle geurverskil tussen die wyne gevind. VIN13-TOE1 was meer vrugtig as al die ander wyne en VIN13-i\TIP1 was die minste vrugtig. Die studie het weereens bewys dat 'n enkele geen 'n betekenisvolle effek op die chemiese samestelling van wyn en brandewyn kan hê. Die relatief eenvoudige genetiese verandering van 'n organisme kan die organoleptiese eienskappe asook die lewensvatbaarheid van "n organisme, drasties verander en verbeter.