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

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Glutathione (GSH), a tripeptide consisting of glutamate, cysteine and glycine, is the most ubiquitous non‐protein intracellular thiol in a large variety of organisms, including plants, animals and fungi. The thiol moiety of the cysteine residue confers unique redox and nucleophilic properties. In plant cells, GSH fulfils an indispensible role in the antioxidant system, sulphur metabolism and detoxification of xenobiotics. Upon grape crushing, GSH is extracted into the juice where it exerts several protective effects during the vinification process. In must, it reacts with oxidized phenolic compounds to form the colourless grape reaction product (GRP) which limits must browning to a certain extent. During wine ageing, GSH impedes the decrease of important aroma compounds, including certain esters, terpenes and volatile thiols, while at the same time preventing the development of atypical ageing off‐flavours. GSH may also inhibit the yellowing of wine during ageing. It is thus evident that elevated GSH levels in wine, in particular white wine which is more sensitive to oxidation, may be highly valuable for wine quality. The reductive crushing and pressing of white grape varieties, which limits oxidation and the consequent incorporation of GSH into GRP, promotes higher GSH levels in the juice. The reductive handling of juice also limits the formation of oxidized glutathione (GSSG). However, during alcoholic fermentation and maturation, levels generally decrease as a result of assimilation by the yeast Saccharomyces cerevisiae, and inevitable oxidation that takes place during the vinification process. The principal focus of this study was to gain a better understanding of the fate of glutathione during alcoholic fermentation and to establish whether certain oenological applications could result in elevated wine GSH levels. The application studied, included choice of yeast strain, extended lees contact, nitrogen supplementation and supplementation with glutathione enriched inactive dry yeast preparations (GHS‐IDYs). In addition, the need for a rapid analytical method for the simultaneous quantification of both GSH and GSSG in must and wine which does not involve derivatization or require extensive sample preparation, led to the development of a novel UPLC‐MS/MS method. The method was also employed to determine intracellular GSH and GSSG contents of the yeast S. cerevisiae and was studied for the first time in winemaking conditions. It was shown that the GSH levels fluctuated during alcoholic fermentation, suggesting the uptake and release by yeast. At the end of alcoholic fermentation, levels were generally lower than those initially present in grape juice, but in some cases, concentration increases were also observed. This finding indicates that, in some cases, endogenously‐produced GSH may be secreted into must during alcoholic fermentation, contributing to higher GSH levels in wine. Albeit small, significant differences in GSH content could be seen in wines fermented with different yeast strains, implying that yeast strain may to a certain extent influence wine GSH levels. While the effects of lees ageing and nitrogen supplementation seem to be insignificant in contributing to higher GSH levels in wine, the supplementation of must with GSH‐IDYs could result in increased wine GSH levels, provided the supplementation is done early during fermentation. This study has broadened our knowledge of several oenological factors, influencing GSH levels in wine and provided a new baseline for future research studies. / AFRIKAANSE OPSOMMING: Glutatioon (GSH), ’n tripeptied bestaande uit glutamaat, sisteïen en glisien, is die mees algemene nieproteïenagtige intrasellulêre tiool in ‘n wye verskeidenheid organismes, insluitende plante, diere en fungi. Die tioolfunksiegedeelte van die sisteïenresidu verleen unieke redoks‐ en nukleofiliese eienskappe. GSH vervul ‘n onmisbare rol in die antioksidantsisteem, swaelmetabolisme en die ontgiftiging van xenobiotika in plantselle. Tydens die maal van druiwe word glutatioon in die sap geëkstraeer waar dit verskeie beskermende effekte tydens die wynbereidingsproses uitoefen. GSH reageer met geöksideerde fenoliese verbindings om die kleurlose druifreaksieproduk (DRP) te vorm wat die verbruining van mos in ‘n sekere mate beperk. GSH verminder ook die afname van belangrike aromaverbindings tydens wynveroudering, insluitende sekere esters, terpene en vlugtige tiole, terwyl dit terselfdertyd die vorming van atipiese verouderingswangeure belemmer. So ook kan GSH die vergeling van wyn tydens veroudering inhibeer. Dit is dus voor die hand liggend that verhoogde GSH‐vlakke in wyn, in die besonder witwyn, wat meer oksidasie‐sensitief is, van waarde kan wees vir wynkwaliteit. Die reduktiewe maal en pers van witdruifvariëteite wat oksidasie en gevolglike inkorporasie van GSH in DRP beperk, bevorder hoër GSH‐vlakke in sap. So ook beperk die reduktiewe behandeling van sap die vorming van geoksideerde glutatioon (GSSG). Gedurende alkoholiese gisting en veroudering neem GSH‐vlakke egter af as gevolg van assimilasie deur die gis, Saccaromyces cerevisiae, asook onvermydelike oksidasie wat gedurende die wynbereidingsproses plaasvind. Die hooffokus van die studie was om ‘n beter begrip van die lot van glutatioon tydens alkoholiese gisting te verkry en om vas te stel of sekere wynkundige praktyke verhoogde GSH‐vlakke in wyn tot gevolg kan hê. Die studie het gisraskeuse, verlengde gismoerkontak, stikstofaanvulling en aanvulling met glutatioon‐verrykte, onaktiewe droëgis ingesluit. Daarbenewens het die behoefte aan ‘n vinnige analitiese metode vir die gelyktydige kwantifisering van sowel GSH as GSSG in mos en wyn wat nie derivatisering of uitgebreide monstervoorbereiding vereis nie, gelei tot die ontwikkeling van ‘n nuwe UPLC‐MS/MS metode. Hierdie metode is ook gebruik om die intrasellulêre GSH‐ en GSSG‐inhoud van die gis S. cerevisiae te bepaal wat vir die eerste keer in wynbereiding bestudeer is. Daar is bewys dat GSH‐vlakke tydens alkolholiese gisting fluktueer, wat dui op die opname en vrystelling daarvan deur die gis. Die vlakke aan die einde van alkoholiese gisting was oor die algemeen laer as vlakke aanvanklik teenwoordig in die sap. In sommige gevalle is konsentrasietoenames egter ook waargeneem. Hierdie bevinding dui daarop dat intrasellulêr‐vervaardige GSH, in sommige gevalle, in die mos uitgeskei kan word, wat tot hoër GSH‐vlakke in wyn lei. Klein, dog beduidende verskille in GSHinhoud is waargeneem in wyne wat met verskillende gisrasse berei is, wat daarop dui dat gisras in ‘n sekere mate die GSH‐vlakke in wyn kan beïnvloed. Alhoewel die effek van gismoerveroudering en stikstofaanvulling onbeduidend is, kan die aanvulling van mos met glutatioon‐verrykte, onaktiewe droëgis tot verhoogde GSH‐vlakke in wyn lei, mits die aanvulling vroeg tydens alkoholiese gisting gedoen word. Hierdie studie verbreed ons kennis van verskeie wynkundige praktyke wat GSH‐vlakke in wyn beïnvloed en vorm ‘n nuwe basis vir toekomstige navorsingstudies.

Thesis (MscAgric (Viticulture and Oenology))--Stellenbosch University, 2008. / The South African table grape industry is under great pressure to produce table grapes of the best quality for the export markets. Quality defects such as poor colour, inadequate berry firmness, browning and soft tissue breakdown cause great losses in export. The firmness of table grapes is one of the major factors determining the eating quality of grapes. Consumers prefer grapes with a firmer flesh above those with soft flesh. Firmer berries are commonly accepted to have better eating quality and longer cold storage capacity. Factors that promote and maintain berry firmness are only speculated about; therefore producers cannot effectively control the development of firmer berries by managerial practises or by applying specific sprays. The study was done on Redglobe and two Waltham Cross clones (the firmer Clone 8 and softer Clone 13). The aim of this study was two-fold. Firstly the cellular and ultracellular differences between the tissues of firm and soft berries were determined. The effect of gibberellic acid (GA3), synthetic cytokinin (CPPU) and bunch applied calcium sprays on the cellular and ultracellular structure of berry tissues were also under investigation. Secondly, the effects of GA3, CPPU and bunch directed calcium sprays on berry firmness, eating quality and storage capacity were determined. To determine the cellular and ultracelular structure of berry tissues, light microscope (LM) and transmission electron (TEM) studies were done. In order to investigate the effect of different sprays on berry firmness, 20 mg/L GA3 (GA3 treatment) was applied at 10mm average berry size; 20 mg/L GA3 plus 3 mg/L CPPU (CPPU treatment) was applied at 10 mm average berry size; and a mixture of 8 L/ha Stopit® and 5 L/ha Caltrac® (calcium treatment) was applied directly to the bunches every two weeks from berry set till veraison for the calcium treatments. The control received no plant bioregulators (PBR’s). The treatments were the same for both cultivars. Grapes were stored three weeks at 0 °C and one week at 10 °C after which it was evaluated for loose berries, botrytis infections, rachis browning and berry split. Afterwards it was tasted by an independent tasting panel. Firm berries were found to have an opaque coloured flesh while soft berries had a gel-like translucent flesh. For berries with normal firmness, the opaque flesh is limited to the outer mesocarp of the berry. Extremely firm berries’ whole mesocarp consisted of the opaque coloured flesh while soft berries’ mesocarp consisted of mostly the gel-like translucent flesh with, in some cases, a very thin layer of opaque flesh just under the skin. Berry firmness was not related to cell size as the cell size of the tissues in the firm and soft berries were identical. Cell shape seems to play an important role in berry firmness. The cells in the opaque coloured flesh of the outer mesocarp are more turgid and oval than those in the gel-like flesh of the inner mesocarp. Berry firmness is therefore determined by the thickness of the outer mesocarp with the opaque coloured flesh that contains turgid cells. The thickness of cell walls between the different tissues did not differ. There was however a difference between the cell contents and the plasmalemmas of the inner and outer mesocarp. The plasmalemma and tonoplast of the outer mesocarp cells was more intact than those of the inner mesocarp. The membranes in the inner mesocarp are more subtracted form the cell wall than in the outer mesocarp. Both the PBR’s and calcium treatments cause a delay in sugar accumulation in the case of Redglobe and Waltham Cross. The CPPU treatment results in significantly bigger and firmer berries for both cultivars. In the case of Redglobe, this treatment cause bigger cells in the outer mesocarp suggesting a correlation between berry firmness and cell size. In the case of Waltham Cross, however, cell size did not play a role in berry size and firmness; instead the rate of cell division earlier in berry development. The CPPU treatment was the only treatment that maintains berry firmness during cold storage for Redglobe while GA3 and CPPU did so in the case of Waltham Cross. PBR’s seems to have no effect on cell wall thickness. In the case of Redglobe, the calcium treatments resulted in significantly thinner cell walls, but this can not be explained. Calcium and GA3 treatments had a negative effect on grape quality after cold storage of both Redglobe and Waltham Cross. The Waltham Cross CPPU treatment results in better taste and colour as observed by the tasting panel, while in the case of Redglobe, the tasting panel preferred the control. It is found that the use of CPPU in combination with GA3 had the best effect on the eating quality, storage capacity, berry size and firmness. When a producer decides to use the CPPU treatment in order to improve berry firmness, he must realize that it can cause delayed ripening which can affect the export of the fruit.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2006. / The assessments of grape and wine quality are complex issues and the wine industry needs more objective analysis of grape and wine quality. The standard quality assessment protocol for grading grapes at most wine cellars in South Africa is based on viticultural practices and the determination of chemical parameters such as ºBrix, pH and titratable acidity (TA). Grape juice indices calculated by formulae such as ºBrix/pH, TA/pH, ºBrix/TA, ºBrix x (pH)2 have been used in the past but these approaches have had limited success. It was shown that the total anthocyanins and total phenolics of red grapes correlate with wine quality and provide additional objective measures of grape quality. Most methods for the quantification of total anthocyanins and total phenolics are complex and time consuming and therefore not easily implemented in the routine laboratory environment. Fourier transform infrared spectroscopy (FT-IR) is widely used in South African laboratories for the routine quantification of wine and grape parameters but the commercial calibration models supplied for the quantification of grape total anthocyanins and phenolics are not satisfactory. The focus of this study was to develop new FT-IR calibration models for the quantification of total anthocyanins and phenolics of grapes and to use the generated data during a preliminary evaluation of the implementation of these parameters as part of the grape quality control protocol at a commercial winery in South Africa. The potential of Fourier transform infrared spectroscopy (FT-IR) for the rapid quantification of total anthocyanins and total phenolics in red grapes was investigated and evaluated for prediction accuracy with independent validation sets. The design of calibration sets aimed at capturing most of the variation due to vintage (2004 and 2005), cultivar (Cabernet Sauvignon, Merlot, Pinotage, and Shiraz) and sugar concentration. Best prediction accuracies were obtained for calibration sets using grapes from a single vintage or cultivar or approximately the same sugar concentration. The highest prediction accuracies were obtained for total anthocyanins calibration sets of grapes with sugar concentrations ≥ 23.5ºBrix (SEP = 0.13 mg/g; R2 validation set = 0.77) and for total phenolics calibration sets of grapes with sugar concentrations < 23.5ºBrix (SEP = 0.13 OD280/g; R2 validation set = 0.74). Strong correlations were found between the spectral data and the total anthocyanins (SEP = 0.12 mg/g; R2 validation set = 0.84) and total phenolics concentration data (SEP = 0.10 OD280/g; R2 validation set = 0.76) for 2005 Merlot calibration sets indicating that the FT-IR spectra captured most of the variation. Overall the RPD (ratio of the standard deviation of the reference data to the standard error of prediction) values of all calibration models were below 3 indicating that calibration models are fit for screening purposes. Spectroscopic absorbance at 280 nm is not specific enough for the quantification of total phenolics and the use of an alternative reference method such as high performance liquid chromatography (HPLC) will be considered in the future. Principal component analysis (PCA) revealed that the major sources of variation in the FT-IR spectra of grapes could be ascribed to vintage and grape sugar concentration and this had an effect on the accuracy of the analytical data generated when using FT-IR spectroscopy. This report is the first to our knowledge where FT-IR has been used for the quantification of total anthocyanins and phenolics of grapes. The evaluation of the reference laboratory protocol for the quantification of total anthocyanins and total phenolics in grapes were evaluated in Chapter 4 and emphasized the importance of meticulous laboratory practices to obtain reliable reference data for calibration purposes. This large scale investigation of the total anthocyanins and phenolics concentrations in grapes is the first of its kind in South Africa and a quantitative database containing analytical data of the anthocyanins and total phenolics concentrations of 692 grape samples representing a wide range of grape maturities of Vitis vinifera cultivars Cabernet Sauvignon, Merlot, Pinotage and Shiraz from the 2004 and 2005 vintages was established based on the reference values. The data were used in a preliminary investigation of the implementation of total anthocyanins and total phenolics concentrations as part of grape quality classification at a commercial South African winery (Chapter 5). The results showed that the total anthocyanins and total phenolics concentration in grapes increased with increasing grape maturity (measured as sugar concentration). ANOVA and post-hoc analysis (Bonferroni testing) revealed significant differences between the total anthocyanins and total phenolics concentrations of the four Vitis vinifera cultivars investigated. Grapes harvested earlier in the season had significantly higher (p≤0.05) total anthocyanins and total phenolics concentrations than grapes harvested later in the season. This implies that grapes harvested earlier in the harvest season could produce wines with higher quality. Grapes from regularly irrigated vineyards had lower total anthocyanins and total phenolics concentrations compared to dryland vineyards. The current grape grading system in use at the industrial cellar did not correlate well with the total anthocyanins and total phenolics concentrations of grapes which highlighted the need for the inclusion of more objective measures during grape grading. The information captured in the database can be used as a starting point to establish profiles of the typical anthocyanins and total phenolics of South African grapes and data from more vintages should be included and continually updated. These findings highlight the important contribution of the results obtained in this preliminary study for the incorporation of total anthocyanins and phenolics concentrations as objective parameters of grape quality. Finally multivariate data analysis of the FT-IR spectra revealed important information regarding factors (both physical and chemical) that contribute to the variation of the spectra. The main variation between the 2004 and 2005 samples can probably be interpreted in terms of the water content of the samples.

Thesis (MscAgric (Viticulture and Oenology))--University of Stellenbosch, 2006. / Under certain South African conditions, Redglobe develops a colour that is too dark and thus unacceptable for the Far Eastern markets. These markets require a pink colour instead of a dark red colour. The cultivation of grapes with an acceptable colour involves amongst other, canopy management practices. This generally includes the removal of leaves and/or lateral shoots. Hereby, the leaf area and the microclimatic conditions in the canopy are altered. The aim of this study was to test the usefulness of leaf and lateral shoot removal at different defoliation times after anthesis in order to obtain a pink coloured Redglobe crop. Other quality aspects, namely total soluble solids (TSS), total titratable acidity (TTA), berry mass and total yield, were also evaluated. A canopy management trial was conducted on six year old Redglobe vines with moderate vigour. The treatment design was a 2 x 3 x 4 factorial and involved two leaf removal (L) levels (L0 = 0% leaf removal; L33 = 33% leaf removal) in combination with three lateral shoot removal (LS) levels (LS0 = 0 % lateral shoot removal; LS50 = 50% lateral shoot removal; LS100 = 100% lateral shoot removal). Four defoliation times (DT) were selected: 36 (pea berry size), 69 (véraison), 76 (one week after véraison) and 83 (two weeks after véraison) days after anthesis (DAA). A total of 24 treatment combinations, replicated in four blocks, were applied. Generally, treatment combinations involving 33% leaf removal lowered the main shoot leaf area. Likewise, the lateral shoot leaf area was decreased by increasing levels of lateral shoot removal at any defoliation time. As expected, 33% leaf removal applied in combination with any level of lateral shoot removal, always resulted in a lower total vine leaf area compared to where 0% leaf removal was part of the treatment combination. Compensation reactions occurred and in this regard the main shoot leaf size increased due to 33% leaf removal applied at 1 week after véraison and 2 weeks after véraison. Treatment combinations involving lateral shoot removal increased the ratio of main shoot leaf area to the total leaf area. On the other hand, the main shoot leaf area percentage was lowered by the application of 33% leaf removal at 2 weeks after véraison compared to no leaf removal at the same defoliation time. It can therefore be assumed that the contribution of lateral shoot leaves to grape composition might have increased in cases where the main shoot leaf area was lowered at a later stage (e.g. 2 weeks after véraison). The bunches were visually evaluated and divided into classes from dark (class one) to light (class nine). This visual bunch evaluation showed that the mean bunch colour was in class three (lighter than class two) due to the defoliation time. The lateral shoot removal x leaf removal interaction resulted in a mean bunch colour that was in classes 2 and 3. However, within these classes, there was a tendency that bunch colour decreased for defoliation times later than pea berry size. The lateral shoot removal x leaf removal interactions showed that bunch colour was darker when the treatment combinations involved 0% leaf removal. The percentage of bunches with the desired colour was increased by application of the treatments at véraison, compared to the other defoliation times, and also with 50% lateral shoot removal and 100% lateral shoot removal compared to 0% lateral shoot removal. Biochemical analyses confirmed that increased levels of lateral shoot removal generally lowered the anthocyanin concentration regardless of defoliation time. A similar effect on TSS was observed, i.e. from véraison onwards, the application of 50% lateral shoot removal and 100% lateral shoot removal tended to lower TSS. The effect of these levels of lateral shoot removal at véraison was significant. The role of the lateral shoots in colour development and sugar accumulation is therefore emphasized. Furthermore, the special role that lateral shoots also play in berry development is illustrated in that berry mass tended to decrease when 100% lateral shoot removal in combination with 33% leaf removal and 100% lateral shoot removal in combination with 0% leaf removal were applied at véraison. This, together with the positive relationship obtained between grape colour and the lateral shoot leaf area:fruit mass ratio, accentuates the role of active leaf area during the ripening period. The possible effect of the microclimatic light environment on colour must also be considered. However, although the light intensity increased with increased levels of LS, the colour that was obtained was probably not associated with the differences in light intensity. It was found that it is possible to manipulate the colour of Redglobe grapes with defoliation treatments. However, the treatments that have a decreasing effect on grape colour also affected other quality parameters like TSS and berry size negatively. Although, it is possible to reduce the colour of Redglobe through the application of leaf and lateral shoot removal at different defoliation times, the question arises whether the treatment combinations used in this study are worthwhile to pursue because the mean bunch colour that was obtained was still too dark. However, it was possible to increase the percentage of bunches with the desired colour. Therefore, if such treatments are applied, it must be approached cautiously, keeping in mind that assimilate supply has to be sustained throughout the ripening period.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The total sale of brandy for 2007 in South Africa was R 7 300 000 000 and local statistics indicate that brandy is by far the most purchased spirit beverage. Sales of brandy even outweigh the total sales for whisky and the forecast for the estimated sales of brandy in the next five years is said to increase by 25%. It is therefore crucial to investigate those factors that influence the production of brandy as better understanding and control of these processes leads to the production of a brandy that is consistent and of premium quality. Many factors influence the final outcome of distillates. Of these factors, the distillation technique, the apparatus used for the purpose of distillation together with the low wine is of utmost importance as they influence the sensory profile and the chemical composition of the distillate. The effect of different variations of pot still designs on the chemical composition and the sensory profile of the resultant distillate was investigated. Five different Pot still variations were used and varied with regards to the design of their pot still head and swans neck apparatus. Two low wines were used for the purpose of distillations and were both from 2007 vintage. GCFID was used to identify the volatile compounds found in the distillates and together with Quantitative Descriptive Analysis (QDA) a profile of the distillates was produced which was used to differentiate between the different pot still variations and their effect on the final product. The data generated from the QDA sessions was subjected to Principal Component Analysis (PCA) and together with the chemical analysis a correlation between certain compounds and sensory attributes were found in the distillates. Distillate samples were also subjected to a sensory style classification system and were classified accordingly. The chemical composition of the two low wines prior to distillations differed significantly from each other with low wine one containing a larger amount of total esters and carbonyl compounds whilst low wine two contained a larger amount of total higher alcohols and acids. The distillates of low wine one also contained over all larger amounts of total esters and in the case of the distillates of low wine two, they contained larger amounts of higher alcohols and acids than low wine one. Variation one was based on the Alambic Charentais method of pot still design and it was found that only variation one influenced the chemical composition and the sensory profile of the distillates. This variation produced a distillate with a lower amount of total esters and more specifically ethyl acetate as well containing a lower intensity of the fruit and sweet associated caramel aromas and flavours. The esters, ethyl acetate and the ethyl esters of the long chained fatty acids were found to correlate with the sensory attributes known as fruit associated aroma, soapy aroma, and spicy aroma and therefore indicated that these compounds are responsible for these attributes. There were no correlations found between the chemical compounds, sensory attributes and sensory style classifications in the distillates of both low wine one and two. It was shown that the addition of certain esters, carbonyl compounds, higher alcohols and acids in specific ratios could alter the sensory classification of the distillates. Therefore the chemical composition and the sensory characteristics of distillates are largely dependent on the chemical composition of the low wine prior to distillation rather than the pot still design. Therefore, with further research it could be possible to predict the outcome of the chemical composition of the distillates by analyzing the chemical compounds found in the low wine prior to distillation. / AFRIKAANSE OPSOMMING: Die totale verkope aan brandewyn vir 2007 in Suid Afrika beloop R7 300 000 000 en statistiek wys dat brandewyn by verre die mees gesogte spiritus drank is. Verkope van brandewyn is selfs meer as die verkope van whisky en die voorspelling is dat die verkope van brandewyn met 25% gaan vermeerder in die volgende vyf jaar. Dit is dus belangrik om die faktore te ondersoek wat die produksie van brandewyn beïnvloed om sodoende die verstokingsproses te verstaan en te kontroleer om ‘n konsekwente kwaliteitsproduk op die mark te plaas. Baie faktore beïnvloed die finale produk. Faktore soos die distillasie tegnieke, die apperate wat gebruik word vir distillasie tesame met die rabatspiritus is van die uiterste belang aangesien dit die sensoriese profiel en die chemiese samestelling van die distillaat beïnvloed. Die effek van die verskillende variasies van potketelhelms op die chemiese samestelling van die distillate word ondersoek. Vyf verskillende helms met variasies in die swaannek ontwerp was gebruik. Twee verskillende rabatspiritus, van die 2007 oesjaar, was gebruik vir distillasie. GC-FID was gebruik om die vlugtige komponente van die distillate mee vas te stel. Kwantitatiewe Beskrywende Analise (QDA) is gebruik om ‘n profiel van die distillate op te stel wat weer gebruik is om te differensieer tussen die verskillende potketelhelm variasies en hulle effek op die finale produk. Die data wat deur die QDA sessies gegenereer was, is in die Vernaamste Komponent Analise (PCA) ingevoer en tesame met die chemiese analise is ‘n korrelasie tussen sekere komponente en die sensoriese analise van die distillate gevind. Distillaat monsters was ook aan sensoriese styl van klassifikasie onderwerp en is as volg daarvan geklassifiseer. Die chemiese samestelling van die twee rabatspiritus voor finale distillasie het betekenisvol van mekaar verskil ten opsigte daarvan dat die eerste rabatspiritus het hoë konsentrasies esters en karboniel verbindings gehad terwyl die tweede rabatspiritus meer hoë konsentrasies van sure en hoër alkohole gehad het. Die distillaat van die eerste rabatspiritus het ook hoë konsentrasies esters en karboniel verbindings gehad terwyl die distillaat van die tweede rabatspiritus weer hoë konsentrasies van sure en hoër alkohole gehad het. Variasie een is gebaseer op die Alambic Charentais van potketel ontwerp en daar is ook gevind dat hierdie variasie die enigste een was wat die chemiese samestelling en die sensoriese profiel van die distillate beïnvloed het. Hierdie variasie het ‘n distillaat geproduseer wat lae konsentrasies van totale esters, veral etielasetaat, sowel as laer intensiteit van vrugtige en soet geassosieerde karamel aromas en geure. Die esters, etielasetaat en etiel esters van die lang ketting vetsure, is gevind dat dit goed korreleer met die sensoriese eienskappe wat geassosieer word met vrugtige aromas, spesery-agtige aromas en seperige aromas. Daar is geen korrelasie gevind tussen die chemiese verbindings, sensoriese eienskappe en sensoriese styl van klassifikasie van distillate een en twee nie. Dit was ook bewys dat byvoeging van esters, karboniel verbindings, sure en hoër alkohole, in spesifieke verhoudings, die sensoriese eienskappe kan verander. Dus is die chemiese samestelling en sensoriese eienskappe van die distillate grootliks afhanklik van die chemiese samestelling van die rabatspiritus, voor die tweede distillasie, as wat dit afhanklik is van die potketelhelm ontwerp. Gevolglik, met verdere v navorsing, is dit moontlik om die uitkoms van die chemiese samestelling van die distillaat te voorspel deur die analise van die chemiese verbindings van die rabatspiritus te ontleed.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The table grape industry employ a wide range of viticultural management practices in order to produce the high quality grapes demanded by the export market. A common contributor to degrading the quality of table grapes is the occurrence of berry split, which not only has an unattractive visual effect, but also increases the berries’ susceptibility to infection by spoilage organisms. A number of environmental conditions such as rainfall and humidity, and/or agricultural practices, such as irrigation, and high density canopies, can lead to higher plant cell water content. This in turn, can increase the potential of berry split to occur. To date, the main method of berry split prevention has been the management of plant water status by; (i) regulating irrigation withdrawal times, and (ii) covering of canopies if rainfall is predicted prior to harvest. The aim of this study was to determine the effect that irrigation frequency, as induced by irrigation withdrawals; as well as boron (B) and calcium (Ca) treatments, applied as bunch directed sprays, have on pre- and post-harvest berry split. To this end, a newly released late ripening, white seedless cultivar, SoutherngrapeOne was chosen as a model cultivar as it has a high susceptibility to berry split. SoutherngrapeOne vines were subsequently subjected to a range of irrigation frequencies based on typical irrigation scheduling used in the table grape industry, which comprised of a low, medium and high frequency. The low frequency was duplicated in order to demonstrate the effect that a heavy irrigation, just before harvest may have on berry split. These treatments were further subdivided to investigate the effect that B and Ca may have on berry split. For the B treatment, four Solubor1 bunch directed sprays were applied from 8mm berry size to véraison. The Ca treatment consisted of Stopit R 2 and Caltrac R 3 bunch directed sprays applied over the same period. In addition, a combination of the B and Ca treatment were applied to investigate any possible interaction. To account for the effect of water as solvent in the B and Ca treatments, and the spraying effect, pure water as treatment was also evaluated. Control vines received no sprays. The applied irrigation treatments resulted in different plant water status conditions. Separate applications of B and Ca treatments resulted in a decrease of B and Ca content in the flesh respectively. The control and combination treatment, of B and Ca resulted in the same of B and Ca content in the flesh. Furthermore, none of the applied treatments resulted in an increase of either B or Ca content in the berry skin. It was found that the medium frequency irrigation resulted in the best irrigation strategy to prevent pre-harvest berry split. Surprisingly, all the subtreatments: B, Ca, and combination of B and Ca, resulted in an increased incidence of pre-harvest berry split when compared to the control group for the 2006/07 season. However, in the 2007/08 season only the B treatment resulted in an increase of pre-harvest berry split. Concerning post-cold-storage physiological disorders, Ca treatments appear to have reduced berry drop, but increased decay. In the 2006/07 season, the B treatment resulted in reduced post-cold-storage berry split, whereas Btreatment in the 2007/08 season had no effect. Both B and Ca should be considered as having the potential to increase the appearance of hairline cracking. Calcium treatment also led to an increase in decay which may have been as a result of the splitting it contributed to. Low frequency irrigation recieving irrigation before harvest was found to result in browner stems. Low irrigation frequencies decreased the cell size of the berry skin. The Ca treatment gave rise to thicker (weaker) cell walls, this morphological change may be responsible for the physiological disorders it caused. From these findings, it can be deduced that poorly managed irrigation, together with unnecessary application of B and/or Ca may result in an increase of berry split and other physiological disorders, with subsequent financial losses for the producer. / AFRIKAANSE OPSOMMING; Die tafeldruifindustrie maak gebruik van ’n wye reeks wingerdkundige praktyke ten einde die hoë gehalte druiwe te produseer wat die uitvoermark vereis. Korrelbars is ’n algemene verskynsel wat afbreek maak tot die gehalte van tafeldruiwe. Behalwe dat voorkoms van die druiwe benaadeel word, verhoog dit ook in vatbaarheid vir infeksie deur verrottingsveroorsakende swamme. Hoë reënval en humiditeit, sowel as wingerdkundige praktyke soos besproeiing en hoë lowerdigtheid, wat kan lei tot verhoogde waterstatus in plante, kan lei tot ’n toename in korrelbars. Daar word hoofsaaklik van twee metodes gebruik gemaak om korrelbars te beheer, naamlik die bestuur van plantwaterstatus deur; (i) beheer van besproeiingsontrekkingstye en (ii) bedekking van lowers indien reën voorspel word voor oestyd. Die doel van hierdie studie was om vas te stel wat die invloed van besproeiings frekwensies sowel as trosgerigte boor (B) en kalsium (Ca), spuitbehandelings, op voor- en na-oes korrelbars het. Die onlangs vrygestelde laat rypwordende, wit, pitlose kultivar, SoutherngrapeOne is gebruik, aangesien dit hoogs gevoelig is vir korrelbars. Stokke is aan verskillende besproeiings intervalle, soos tipies gebruiklik in die tafeldruifindustrie, blootgestel. Hierdie intervalle bestaan uit n’ lae, medium en hoë besproeiings frekwensie. Die lae besproeiings frekwensie is herhaal ten einde die invloed van besproeiing net voor oestyd op korrelbars te ondersoek. Die invloed van B- en Ca-behandeling op korrelbars is ook ondersoek. Vir die B-behandeling is vier Solubor1 trosgerigte spuite aangewend vanaf 8mm korrelgrootte tot deurslaan. Vir die Ca-behandeling is Stopit R 2 en Caltrac R 3 as trosgerigte spuite oor dieselfde tyd toegedien. Kombinasiebehandelings is ook aangewend om enige interaksie tussen B en Ca te ondersoek. Waterbehandelings is ook toegedien om die invloed van water as oplosmiddel van B- en Ca-behandelings sowel as die spuit-effek te ondersoek. Kontrole stokke is ook ingesluit en het geen spuitebehandeling ontvang nie. Die besproeiingsbehandelings het verskillende plantwater toestande tot gevolg gehad, B- en Ca-behandelings het gelei tot ’n afname in B- en Cainhoud in die vleis onderskeidelik. Die kombinasie en kontrole behandelings het eenderse hoeveelhede B en Ca in die vleis tot gevolg gehad. Geen van die aangewende behandelings gelei tot ’n toename in B- en Ca-inhoud in die dop nie. Die resultate toon dat medium besproeiings frekwensie die beste besproeiingsstrategie is om voor-oes korrelbars te voorkom. In vergelyking met die kontrole-behandeling in 2006/07, het B, Ca en die kombinasie van B en Ca, ’n toename in voor-oes korrelbars tot gevolg gehad. In die 2007/08 seisoen het slegs die B-toediening egter tot ’n toename in voor-oes korrelbars gelei. Kalsium behandelings het ’n afname in los-korrels, maar ’n verhoging in korrelbars tot gevolg gehad. In 2006/07, het B-toediening tot ’n afname in korrelbars na koelopberging gelei, maar in die 2007/08 seisoen het dit geen effek gehad nie. Beide B- en Ca-toediening het die potensiaal om haarlyn barste te veroorsaak. Kalsium toediening het bederf verhoog wat moontlik aan die hoër bars wat dit induseer toegeskryf kan word. Lae besproeiings frekwensie, het bruiner stingels veroorsaak, en ook gelei tot ’n afname van selgrootte in die dop. Die Ca-toediening het aanleiding gegee tot dikker selwande in die dop. Hierdie anatomiese veranderinge kan moontlik die rede wees vir die verhoging in fisiologise afwykings. Van hierdie bevindinge kan ons aflei dat swak bestuur van besproeiing, sowel as die onnodige aanwending van B en/of Ca, kan aanleiding gee tot ’n toename in korrelbars en ander fisiologiese afwykings, en dus finansiële verliese vir die produsent inhou.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Cabernet Sauvignon is the most planted red cultivar in South Africa and the second most planted red cultivar in the Olifants River region. The cultivar is prone to vigorous growth with low yields. Excessive irrigation could accentuate these cultivar characteristics. Considering the foregoing, the aim of the study was to describe how Cabernet Sauvignon will react to climate, soil type (texture) and irrigation within the Lower Olifants River wine region to enable growers to make the right decisions regarding long term as well as short term cultivation practices. This study is part of a project carried out by the ARC Infruitec-Nietvoorbij at Stellenbosch to determine the effects of soil type and atmospheric conditions on yield and wine quality of Cabernet Sauvignon in different grape growing regions of South Africa. Similar studies are being carried out in the Orange River, Stellenbosch and Swartland regions of South Africa. The Lower Olifants River region could be divided into three climatic regions. Furthermore, two climatic regions is evident regarding the formation of grape wine colour and aromas. Proximity to the Atlantic Ocean would play an important role in a cultivar establishment policy. The variation in stem water potential (ΨS) could be related to soil water status expressed in terms of matric potential (ΨM). In the case of sandy soils, ΨS decreased substantially more as the ΨM decreased compared to the sandy loam soils. The reason could be that the unsaturated hydraulic conductivity in sandy soils decreased more rapidly as the ΨM decreased compared to the heavier soils. Thus could explain why the grapevines in the sandy soils experienced more water stress than the ones in the sandy loam soils at a given ΨM. Climate had a strong influence on grapevine water status with grapevines nearer to the ocean experiencing less water stress compared to the ones further inland. This was especially true for grapevines in the sandy soils. Vegetative growth and yield of grapevines in the sandy soils were more sensitive to water deficits compared to the ones in the sandy loam soils. For deficit irrigated grapevines in the sandy soils, vegetative growth and yield decreased by ca. 30% when ca. 55% less water was applied from flowering to harvest. Yield reduction were ca. 15% with no or very little influence on vegetative growth with ca. 80% reduction in water applied from flowering to harvest for grapevines in the sandy loam soils. The influence of soil texture on wine quality and style were evident under intensive irrigation as well as over different climatic regions. Overall sensorial potential wine quality of grapevines in sandy soils tended to be higher compared to the ones in the sandy loam soils. Deficit irrigation tended to increase wine colour intensity, irrespective of soil texture. Furthermore, deficit irrigation in sandy loam soils tended to increase wine fullness and the berry characteristics of the wine. Berry characteristics of wines from the sandy soils tended to be higher compared to the ones from the sandy loam soils. Too severe water deficits in sandy soils could be detrimental to wine quality. Climate tended to have an influence on wine style of grapevines in the sandy soils with wines produced further away from the ocean tended to have higher berry characteristics. Irrigation management could be a powerful tool to manipulate the grapevine in sandy soils. For grapevines the sandy loam soils in addition to regulated deficit irrigation, additional canopy management practices could be needed to improve wine quality. / AFRIKAANSE OPSOMMING: Cabernet Sauvignon is die mees aangeplante rooidruif kultivar in die Suid-Afrikaanse wynbedryf. In die Olifantsriver streek is dit naas Shiraz, die tweede mees aangeplante rooidruif kultivar. Cabernet Sauvignon is bekend as ‘n groeikragtige skaamdraer. Indien oorbesproei word, kan hierdie potensiële nadelige eienskappe nog meer na vore tree. Die doel van die studie is om die invloed van die klimaat, grond en besproeiing op Cabernet Sauvignon se vegetatiewe groei, produksie en wyngehalte in die Benede Olifantsrivier streek te bepaal. Hierdie inligting kan produsente help om ingeligte kortsowel as langtermyn besluite te maak rakende die verbouing van Cabernet Sauvignon. Hierdie studie vorm deel van ‘n breër studie in die Suid-Afrikaanse wynbedryf, gedryf deur die Landbou Navorsingsraad (LNR) Infruitec-Nietvoorbij, Stellenbosch om die invloed van atmosferiese toestande en grond op die produksie en wyngehalte van Cabernet Sauvignon te bepaal. Soortgelyke projekte word uitgevoer in die Oranjerivier, Stellenbosch en Swartland wynstreke. Die Benede Olifantsrivier streek kan verdeel word in drie klimaatstreke op grond van temperatuurdata. In terme van die ontwikkeling van druifkleur en aromas, kan die streek verdeel word in twee klimaatstreke. Die afstand vanaf die Atlantiese Oseaan kan ‘n belangrike rol speel in die ontwikkeling van ‘n kultivarriglynplan vir die streek. Grondwaterstatus, uitgedruk as die matrikspotensiaal (ΨM), kan aanleiding gee tot variasie in middag blaarwaterpotensiaal (ΨS) lesings. Die ΨS van die sand gronde verlaag vinniger soos die ΨM verlaag invergelyke met die sandleem gronde. Dit kan moontlik wees as gevolg van die verskil in die grond onversadigde hidroliese konduktiwiteit. Sand gronde se hidroliese konduktiwiteit verlaag vinniger soos die ΨM verlaag, invergelyke met sandleem gronde. Dit verklaar waarom wingerde in sand gronde by dieselfde ΨM, meer waterspanning ondervind as wingerde in sandleem gronde. Klimaat het ‘n invloed op die waterstatus van die wingerdstok. Wingerde nader aan die see het minder waterspanning ondervind invergelyke met wingerde wat verder in die binneland geleë is. Dit was veral die geval met wingerde in die sand gronde. Vegetatiewe groei en produksie van wingerde in die sand gronde is meer sensitief vir waterspanning as wingerde in die sandleem gronde. Tekortbesproeiing in die sand gronde het die groei asook produksie met ongeveer 30% verlaag deur ongeveer 55% minder water toe te dien vanaf blom tot oes. In teenstelling daarmee is die produksie van wingerde in die sandleem gronde met ongeveer 15% verlaag met geen tot baie min verlaging in die groeikrag. Ongeveer 80% minder water is toegedien vanaf blom tot oes. Grondtekstuur kan wyngehalte en -styl beïnvloed ten spyte van intensiewe besproeiing en klimaatsverskille. Sensoriese potensiële wyngehalte van wingerde in die sand gronde was beter invergelyke met dié van die sandleem gronde. Die wyne vanaf die sand gronde het ook geneig om oor meer bessie intensiteit te beskik as wyne vanaf die sandleem gronde. Tekortbesproeiing neig om die wynkleur intensiteit te verhoog, ongeag van grondtekstuur. Tekortbesproeiing in die sandleem gronde kan ook die volheid van die wyne verbeter, asook die bessie intensiteit van die wyn verhoog. Te hoë waterspanning in die sand gronde kan wyngehalte nadelig beïnvloed. Klimaat kan ook die wynstyl vanaf sand gronde beïnvloed met wyne verder vanaf die see wat oor meer bessie intensiteit beskik as wyne nader aan die see. Beheerde tekortbesproeiing kan as ‘n kragtige hulpmiddel gebruik word om wingerde in die sand gronde te manipuleer. Vir wingerde in die sandleem gronde, addisioneel tot beheerde tekortbesproeiing en normale loofbestuurspraktyke, kan ekstra loofbestuurspraktyke bv. die verwydering van sylootlote, dalk nodig wees om wyngehalte te verbeter.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Within-vineyard variability in vigour and water status commonly occurs in South African vineyards. Different soil types found over short distances are probably the main cause of vigour variability, while differences in grapevine water status are commonly induced by lateral water flow in the vineyard, blocked irrigation emitters and differences in soil water-holding capacity. These factors can cause heterogeneous ripening and differences in fruit quality between different parts of the vineyard, an aspect that needs to be avoided as far as possible in order to produce quality wines. Measurements of carbon isotope discrimination (CID) have proved to be a tool to assess grapevine physiology in order to study the effects of environmental parameters on leaf carbon dioxide (CO2) gas exchange and stomatal conductance (gs). Grapevine water deficit stress/strain in reaction to these environmental conditions can then be determined by observing the amount of 13C absorbed by plant material after discrimination of 13C has taken place, and this is influenced by the grapevine stress condition and can indicate water-use efficiency. In this study, the variability of grapevine water status and vigour was determined in order to quantify these parameters in different parts of the vineyard. Two separate trials were conducted, the first at Wellington, South Africa, where different irrigation regimes resulted in variability in grapevine water status between plots. The second trial was at Stellenbosch, South Africa, where plots were divided among different vigour classes and irrigation was applied in different quantities for different irrigation treatments. Within-vineyard variability in water status (Wellington and Stellenbosch) and vigour (Stellenbosch) were then quantified and the effects on some grapevine physiological parameters and berry composition were measured. The treatments in the Wellington trial led to differences in grapevine water status, which could be quantified by measurements of stem water potential (SWP) and leaf water potential (LWP). Soil variability also led to differences in grapevine vigour, which were quantified by measurements of pruning mass, leaf area and shoot length. The effect of the variability in grapevine water status on grapevine physiology was assessed by measuring CID, which was the main focus of the study. Other physiological measurements, such as gs and leaf and canopy temperature, were also conducted. The effect of these conditions on grape berry composition was also studied. In the Stellenbosch trial, soil water content, plant water status measurements (SWP, predawn LWP and LWP), physiological measurements (CID and gs) and berry size measurements were used to classify plots into water status treatments (“wet” and “dry” treatments). The effect of vigour differences was analysed separately from these treatments by using pruning mass as a covariate in the statistical analyses. The effect of vigour variability on the measurements was studied by looking at the effect of the covariate on the measurements, while shoot growth rate, shoot length and leaf area measurements were conducted as vegetative growth measurements. Differences in measurements were then studied between the treatments and between the vigour levels of the different plots. In the Wellington trial, plant water status was determined by irrigation, showing increased stress for treatments that received less irrigation. The differences in plant water status then caused differences in grapevine physiology between the treatments, leading to increased gs for increased irrigation. This of course influenced leaf internal CO2 and therefore CID, although CID was also clearly influenced by berry development. Berry size was influenced by irrigation, with larger berries found in wetter treatments, while berry chemical composition was influenced by the irrigation regime, with increased irrigation leading to increased pH and leading to trends showing increased total soluble solids and malic acid, and reduced total and tartaric acid and colour intensity. In the Stellenbosch trial, plots with higher vigour had increased shoot growth rate, longer shoots and increased leaf area, although topping influenced this. Wet treatment vines also showed slightly longer shoots and larger leaf areas. There were differences in soil water content between the wet and dry treatments, and this led to differences in plant water status. Vigour also influenced pre-dawn LWP, especially in the 2007 season, as higher-vigour vines struggled more to rehydrate through the night. Differences in plant water potential led to differences in grapevine physiology, with increased gs for vines from the wet treatment, while higher-vigour vines had slightly increased gs. The differences in gs led to gas exchange differences and therefore differences in CID, meaning that water status and vigour influenced CID. CID measurements illustrated the long term effect of water status on plant physiology, while measurements such as SWP illustrated the short term effects. CID measurements therefore proved to be accumulative over the season, in contrast to SWP measurements that were much more dependent on the current state of grapevine water status. Other physiological measurements showed that wet-treatment vines had higher photosynthetic rates and evapotranspiration and lower leaf temperatures, while higher-vigour vines had slightly increased evapotranspiration and decreased leaf temperatures. Wet-treatment vines had larger berries, while a higher vigour also led to slightly larger berries. Berry composition was influenced by treatment, where wet-treatment vines had increased pH and total soluble solids, while higher-vigour vines had increased juice pH and, in the 2008 season, decreased total soluble solids. Extremely stressed conditions did not show significant effects on plant water potential, but SWP measurements indicated slightly higher stress for the extremely stressed vines and LWP showed slightly less stressed conditions for these vines. Measurements of gs showed slightly lower values for the extremely stressed vines, while measurements of CID showed large significant differences, with the extremely stressed vines having measurements showing high stress. The measurement therefore indicated highly stressed conditions accurately, while other physiological measurements, such as photosynthetic rate, evapotranspiration and leaf temperatures, only showed trends and no significant differences. Measurements of stomatal conductance reacted to plant water status measurements throughout the diurnal measurement days, while CID only reacted slightly with gs changes during these days and was perhaps influenced more by berry chemical composition and development at this early stage of the season. Vigour and water status therefore influenced grapevine physiology, with a more direct effect by water status and an indirect effect by vigour due to microclimatic differences. This also influenced berry composition and therefore quality. In future studies, CID measurements should be done on juice from which organic acids have been removed in order to eliminate the effect of seasonal berry composition on the measurement. Measurements of CID proved to be an integrative, but sensitive, indicator of grapevine stress, especially at the end of the season. It might at best be useful as a post-harvest management tool for producers or grape buyers, especially for irrigation control, as has also been stated by Van Leeuwen et al. (2007). / AFRIKAANSE OPSOMMING: Binne-wingerd variasie in groeikrag en waterstatus is algemeen in Suid-Afrikaanse wingerde. Verskillende grondsoorte wat na aan mekaar voorkom, is seker een van die vernaamste oorsake van variasie in groeikrag, terwyl verskille in wingerdwaterstatus algemeen deur laterale watervloei in die wingerd, verstopte besproeiingspuite en verskille in grond waterhouvermoë geïnduseer word. Hierdie faktore kan aanleiding gee tot heterogene rypwording en verskille in vrugkwaliteit tussen verskillende dele van die wingerd, ‘n aspek wat so ver moontlik vermy moet word om kwaliteitwyne te kan produseer. Die meting van koolstof-isotoopdiskriminasie (KID) is bewys om as gereedskap te kan dien vir die assessering van wingerdfisiologie om die effekte van omgewingsparameters op blaar koolstofdioksied (CO2) - gasuitruiling en stomatale geleiding (gs) te bestudeer. Die stres/stremming as gevolg van ‘n watertekort in die wingerd in reaksie op hierdie omgewingstoestande kan dan bepaal word deur te kyk na hoeveel 13C deur die plantmateriaal geabsorbeer word ná 13C-diskriminasie plaasgevind het, en dít word deur die wingerdstrestoestande beïnvloed en kan ‘n aanduiding verskaf van die doeltreffendheid van waterverbruik. In hierdie studie is die variasie in wingerdwaterstatus en groeikrag bepaal om hierdie parameters in verskillende dele van die wingerd te kwantifiseer. Twee afsonderlike proewe is uitgevoer, die eerste by Wellington, Suid-Afrika, waar verskillende besproeiingsregimes gelei het tot verskille in die wingerdwaterstatus tussen persele. Die tweede proef was by Stellenbosch, Suid-Afrika, waar persele tussen verskillende groeikragklasse verdeel is en besproeiing in verskillende hoeveelhede vir verskillende besproeiingsbehandelings toegepas is. Binne-wingerd variasie in waterstatus (Wellington en Stellenbosch) en groeikrag (Stellenbosch) is toe gekwantifiseer en die effekte op sekere wingerd-fisiologiese parameters en korrelsamestelling is gemeet. Die behandelings in die Wellington-proef het gelei tot verskille in wingerdwaterstatus, wat deur metings van stamwaterpotensiaal (SWP) en blaarwaterpotensiaal (BWP) gekwantifiseer kon word. Grondverskille het ook gelei tot verskille in wingerdgroeikrag, wat deur metings van snoeimassa, blaaroppervlak en lootlengte gekwantifiseer is. Die effek van die variasie in wingerdwaterstatus op wingerdfisiologie is deur metings van KID bepaal wat die hooffokus van hierdie studie was. Ander fisiologiese metings, soos gs en blaar- en lowertemperatuur, is ook gedoen. Die effekte van hierdie toestande op die samestelling van die druiwekorrels is ook bestudeer. In die Stellenbosch-proef is grondwaterinhoud, metings van plantwaterstatus (SWP, voorsonopgang SWP en BWP), fisiologiese metings (KID en gs) en metings van korrelgrootte gebruik om die persele in waterstatusbehandelings (“nat” en “droë” behandelings) te verdeel. Die effek van verskille in groeikrag is apart van hierdie behandelings geanaliseer deur snoeimassa as ‘n kovariaat in die statistiese analises te gebruik. Die effek van groeikragvariasie op die metings is bestudeer deur ondersoek in te stel na die effek van die kovariaat op die metings, terwyl lootgroeitempo-, lootlengte- en blaaroppervlakmetings as metings van vegetatiewe groei uitgevoer is. Verskille in metings tussen die behandelings en tussen die groeikragvlakke van die verskillende persele is toe bestudeer. In die Wellington-proef is plantwaterstatus deur besproeiing bepaal, met verhoogde stres in behandelings waar daar minder besproeiing toegedien is. Die verskille in plantwaterstatus het dan verskille in wingerdfisiologie tussen die behandelings veroorsaak, wat gelei het tot ‘n verhoogde gs in die geval van verhoogde besproeiing. Dit het natuurlik ‘n effek op die interne CO2 van die blaar en dus op KID gehad, hoewel KID ook duidelik deur korrelontwikkeling beïnvloed is. Korrelgrootte is deur besproeiing beïnvloed, met groter korrels in die natter behandelings, terwyl die chemiese samestelling van die korrel deur besproeiingsregime beïnvloed is. Verhoogde besproeiing het pH verhoog en gelei na tendense wat verhoogde totale oplosbare vaste stowwe en appelsuur, en verminderde totale suur, wynsteensuur en kleurintensiteit getoon het. In die Stellenbosch-proef het persele met hoër groeikrag ook verhoogde lootgroeitempo, langer lote en verhoogde blaaroppervlak getoon, hoewel dit deur top beïnvloed is. Wingerdstokke van die nat behandeling het ook effe langer lote en groter blaaroppervlakke getoon. Daar was verskille in grondwaterinhoud tussen die nat en droë behandelings en dit het verskille in plantwaterstatus veroorsaak. Groeikrag is ook deur voor-sonopgang BWP beïnvloed, veral in die 2007-seisoen, aangesien stokke met hoër groeikrag meer gesukkel het om in die nag te rehidreer. Verskille in plantwaterpotensiaal het gelei tot verskille in wingerdfisiologie, met ‘n verhoogde gs vir stokke in die nat behandeling, terwyl stokke met hoër groeikrag ‘n effens verhoogde gs getoon het. Die verskille in gs het gelei tot verskille in gasuitruiling en dus verskille in KID, wat beteken dat waterstatus en groeikrag ‘n invloed op KID het. KID was meer verteenwoordigend van die langtermyneffekte van water status op plantfisiologie, terwyl metings soos SWP die korttermyneffekte weerspieël het. KID metings was dus akkumalatief oor die seisoen, terwyl SWP metings meer ‘n weerspieëling was van die huidige toestand van plantwaterpotensiaal. Ander fisiologiese metings het getoon dat stokke in die nat behandeling ‘n hoër fotosintesetempo en evapotranspirasie sowel as laer blaartemperature ondervind het, terwyl die stokke met hoër groeikrag effe verhoogde evapotranspirasie en verminderde blaartemperature getoon het. Stokke in die nat behandeling het groter korrels gehad, terwyl hoër groeikrag ook effens groter korrels veroorsaak het. Korrelsamestelling is deur die behandelings beïnvloed, met stokke in die nat behandeling wat verhoogde pH en totale oplosbare vaste stowwe getoon het, terwyl stokke met hoër groeikrag verhoogde pH van die sap en verminderde totale oplosbare vaste stowwe (laasgenoemde in die 2008-seisoen) gehad het. Uitermate toestande van stres het geen beduidende effekte op plantwaterpotensiaal getoon nie, hoewel SWP-metings effens hoër stres vir die uitermate gestresde wingerde getoon het en BWP effens minder gestresde toestande vir hierdie stokke getoon het. Metings van gs het effens laer waardes vir die uitermate gestresde stokke getoon, terwyl metings van KID groot noemenswaardige verskille getoon het, met die metings vir die uitermate gestresde wingerde wat hoër stres aangedui het. Dié meting het dus hoogs gestresde toestande akkuraat aangedui, terwyl ander fisiologiese metings, soos tempo van fotosintese, evapotranspirasie en blaartemperature net tendense en nie beduidende verskille aangedui het nie. Metings van stomatale geleiding het dwarsdeur die dae waarop daaglikse metings gedoen is op plantwaterstatusmetings gereageer, terwyl KID net effens met gs-veranderinge op hierdie dae gereageer het en moontlik meer deur die chemiese samestelling en ontwikkeling van die korrel in hierdie vroeë stadium van die seisoen beïnvloed is. Groeikrag en waterstatus het dus wingerdfisiologie beïnvloed, met ‘n meer direkte effek deur waterstatus en ‘n indirekte effek deur groeikrag as gevolg van mikroklimaatsverskille. Dit het ook korrelsamestelling en dus kwaliteit beïnvloed. In toekomstige studies moet KID-metings gedoen word op sap waarvan die organiese sure verwyder is om die effek van seisoenale korrelsamestelling op die meting uit te sluit. Metings van KID is getoon om ‘n integrerende, maar gevoelige, aanduider van wingerdstres te wees, veral aan die einde van die seisoen. Dit is ten beste miskien bruikbaar as naoesbestuursgereedskap vir produsente of druiwekopers, veral vir besproeiingsbeheer, soos ook reeds deur Van Leeuwen et al. (2007) aangedui is.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The quality of wine is influenced and determined by various factors, one of which includes the process of malolactic fermentation (MLF). MLF plays an integral role in the flavour and sensory profile of most red wines as well as some white wines like Chardonnay. This process is conducted by lactic acid bacteria (LAB), specifically of the genera Oenococcus, Lactobacillus, Pediococcus and Leuconostoc. Of these, Oenococcus oeni is best adapted to survive in the harsh wine environment. MLF is defined as the conversion of L-malic acid to L-lactic acid and carbon dioxide. The conversion of the dicarboxylic malic acid to the monocarboxylic lactic acid results in a decrease in acidity and an increase in pH, to give a softer mouthfeel and more favourable flavour profile. A further reason for conducting MLF in wine includes the improvement of microbial stability due to the removal of malic acid as a possible substrate for microorganisms. Recently, research focus has shifted to the ability of MLF and LAB to alter the aroma profile of wine via the production and/or modification of certain aroma compounds. In order for wine LAB to conduct MLF, they need to be able to survive the harsh and challenging wine environment. Conditions in South African wines are particularly challenging due to the long, hot ripening seasons resulting in high sugar concentrations which give high ethanol concentrations. Some LAB also struggle to adapt to an environment with high pH and low malic acid concentrations. These factors, combined with the use of sulphur dioxide, cause LAB to struggle in conducting and completing successful MLF. Many of the commercial starter cultures that are currently available contain LAB that have not been isolated from South African wine and are therefore not optimal for use under these challenging wine conditions. Oenococcus oeni is also the single LAB culture present in all commercially available starter cultures. The overriding goal of this study was to create a MLF starter culture containing a mixture of LAB cultures, namely O. oeni and Lactobacillus plantarum, which can successfully convert malic acid to lactic acid, ensure microbial stability, but also make a positive contribution to the wine aroma profile. Lactobacillus plantarum has previously been considered for possible use in a commercial starter culture. The LAB isolates used in this study were selected from the Institute for Wine Biotechnology culture collection as well as isolated from spontaneous MLF. The first objective was to characterise these LAB strains for important traits and for possible use as a MLF starter culture. A total of 23 strains were identified as O. oeni and 19 strains as Lb. plantarum. The identified strains were screened in a synthetic wine medium for their ability to convert malic acid to lactic acid. Based on the LAB strain performance in the synthetic wine medium, seven strains of both O. oeni and Lb. plantarum were selected. These 14 strains were screened for the presence of genes encoding for enzymes responsible for biogenic amine production and were found to contain none of the genes associated with the formation of histamine, tyramine or putrescine. The LAB strains were genetically screened for enzymes associated with aroma modification by LAB during MLF. The enzymes of interest that were screened for included β-glucosidase, esterase, protease and phenolic acid decarboxylase (PAD). The Lb. plantarum strains were found to possess more diverse enzymatic profiles related to aroma than O. oeni. The biggest differences were observed for the presence of β-glucosidase and PAD. The second objective was to perform small-scale fermentations with the individual LAB isolates. The individual isolates were evaluated in Pinotage and based on these results; three strains of each O. oeni and Lb. plantarum were selected for evaluation in mixed culture fermentations. The mixed cultures were evaluated in Pinotage, Shiraz and Cabernet Sauvignon in the 2008 vintage. As a third objective, the wines were also analytically and sensorially evaluated to investigate the changes in the aroma profile that could be attributed to the presence of the mixed LAB isolates. Based on the fermentation data as well as data pertaining to the aroma modification, three mixed cultures were selected for evaluation in the 2009 vintage in Pinotage, Cabernet Sauvignon and Chardonnay. The mixed cultures were able to successfully complete MLF in fermentation periods comparable to that of a commercial culture used as control. The different LAB cultures had distinct and diverse effects on the wine aroma profile. The O. oeni strain played a larger role in the ester concentration present after MLF, while the Lb. plantarum strain had a larger effect on the higher alcohol and volatile fatty acid concentration upon completion of MLF. The results generated by this novel study clearly indicate the potential of a mixed LAB starter culture for conducting MLF. The mixed cultures successfully completed MLF and made a positive contribution to the wine aroma profile. / AFRIKAANSE OPSOMMING: Die kwaliteit van wyn word beïnvloed en bepaal deur verskeie faktore en wynbereidings prosesse, wat die proses van appelmelksuurgisting (AMG) insluit. AMG speel ’n integrale rol in die sensoriese profiel van meeste rooiwyne, sowel as sommige witwyne soos Chardonnay. AMG word gedefinieër as die omskakeling van L-appelsuur na L-melksuur en koolstofdioksied. Hierdie omskakeling kan toegeskryf word aan die teenwoordigheid van melksuurbakterieë (MSB), spesifiek spesies van die genera Oenococcus, Lactobacillus, Pediococcus en Leuconostoc. Vanuit hierdie wyn MSB, is Oenococcus oeni die spesies wat die beste aanpas en oorleef onder stresvolle wyn kondisies. Die omskakeling van appelsuur, ’n dikarboksielsuur, na melksuur, ’n monokarboksielsuur, lei tot ‘n vermindering in suurheid en ’n verhoging in pH. Hierdie vermindering in suurheid gee ’n sagter en meer geronde mondgevoel aan die wyn en dra by tot ‘n meer aangename geurprofiel. ’n Verdere rede vir AMG in wyn is om mikrobiese stabiliteit te verseker deurdat appelsuur verwyder word as ’n moontlike koolstof substraat vir mikroörganismes. Onlangs het navorsing begin fokus op AMG en die vermoë van MSB om die aroma profiel van wyn te beïnvloed deur die produksie/modifisering van sekere aroma komponente. Vir MSB om AMG te kan deurvoer, moet hulle kan oorleef in die stresvolle wynomgewing. Wyntoestande in Suid-Afrika is veral uitdagend vir die oorlewing van mikroörganismes as gevolg van lang, warm somers wat lei tot ’n matriks met ’n hoë suikerkonsentrasie en wyn met ’n hoë etanolkonsentrasie. ‘n Omgewing met ‘n hoë pH en lae appelsuur konsentrasie, kan ook bydrae tot stresvolle kondisies vir MSB. Hierdie parameters, tesame met die gebruik van swaweldioksied, maak dit moeilik vir MSB om AMG te inisieer en te voltooi. Sommige van die kommersiële aanvangskulture wat tans beskikbaar is, bevat nie MSB wat onder Suid-Afrikaanse wyntoestande geïsoleer is nie en daarom is dit nie altyd optimaal vir gebruik nie. Oenococcus oeni is ook die enkele MSB kultuur wat in alle kommersiële kulture gebruik word. Die hoofdoelwit van hierdie studie was om ’n potensiële kommersiële aanvangskultuur te ontwikkel wat ‘n mengsel van MSB bevat. Hierdie aanvangskultuur moet AMG suksesvol kan voltooi, mikrobiologiese stabiliteit bevorder en steeds die wynaroma positief kan beïnvloed. Bakterierasse van O. oeni en Lb. plantarum is geselekteer vir gebruik in hierdie studie. Lactobacillus plantarum het reeds in vorige studies potensiaal getoon as ‘n moontlike aanvangskultuur. Die MSB isolate vir hierdie studie is geselekteer uit die Instituut vir Wynbiotegnologie se kultuurversameling en geïsoleer uit spontane AMG fermentasies. Die eerste doelwit was om hierdie MSB isolate te karakteriseer vir belangrike eienskappe en die moontlike gebruik as ’n kommersiële AMG aanvangskultuur. ‘n Totaal van 23 O. oeni en 19 Lb. plantarum isolate is geïdentifiseer. Hierdie isolate is in ’n sintetiese wynmedium geëvalueer vir hul vermoë om appelsuur na melksuur om te skakel. Op grond van hul reaksie in die sintetiese wynmedium, is sewe isolate van elk van die O. oeni en Lb. plantarum geselekteer. Hierdie 14 isolate is ondersoek vir die teenwoordigheid van die gene wat kodeer vir biogeenamien produksie en daar is gevind dat geen van die isolate enige van die biogeenamien gene wat ondersoek is, naamlik histamien, tiramien en putresien besit nie. Die MSB isolate is geneties ondersoek vir die teenwoordigheid van dié gene wat kodeer vir ensieme wat die aromaprofiel tydens AMG beïnvloed. Dié ensieme sluit β-glukosidase, esterase, protease, fenoliese suurdekarboksilase en sitraatliase in. Daar is gevind dat die Lb. plantarum isolate meer diverse ensiemprofiele as O. oeni besit. Die grootste verskille in die ensiemprofiele kan toegeskryf word aan die teenwoordigheid van β-glukosidase en fenoliese suurdekarboksilase. Die tweede doelwit was om kleinskaalse AMG fermentasies met die individuele MSB isolate uit te voer. Die individuele isolate is in Pinotage geëvalueer. Volgens hierdie resultate is drie isolate van elk van die O. oeni en Lb. plantarum geselekteer om in gemengde kulture getoets te word. Die gemengde kulture is in Pinotage, Shiraz en Cabernet Sauvignon in 2008 geëvalueer. As ’n derde doelwit is hierdie wyne ook analities en sensories geëvalueer om die veranderinge in die aromaprofiele as gevolg van die teenwoordigheid van die MSB te ondersoek. Op grond van die fermentasiedata, sowel as die data oor die aromaveranderinge, is drie gemengde kulture geselekteer vir evaluering in Pinotage, Cabernet Sauvignon en Chardonnay in 2009. Die gemengde kulture kon AMG suksesvol voltooi met fermentasietempo’s wat vergelykbaar was met dié van ‘n kommersiële AMG kultuur wat as kontrole gebruik is. Die verskillende MSB kulture het spesifieke en uiteenlopende uitwerkings op die wynaroma gehad. Die O. oeni isolaat in die gemengde kultuur blyk ‘n belangriker rol te speel in die esterkonsentrasie na AMG, terwyl die Lb. plantarum isolaat ’n groter effek het op die hoër alkohol en vlugtige vetsuurinhoud na AMG. Die resultate wat deur hierdie unieke studie gegenereer is, gee ’n aanduiding van die potensiaal van ’n gemengde MSB aanvangskultuur vir AMG. Die gemengde kulture kon AMG suksesvol voltooi en ‘n positiewe bydrae tot die aromaprofiel van die wyn lewer.