Thesis (MScAgric (Viticulture and Oenology)--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: ‘Crimson Seedless’ is currently one of the most important and popular table grape cultivars produced in South Africa, and as such it is of great economic value for table grape producers. Major concerns with ‘Crimson Seedless’ is that it is prone to inadequate colouring, and with increased yields the berry size decreases. An additional difficulty is that methods used to increase berry size, further impede berry colouring. A plant growth regulator (PGR) commonly used in table grape production, to enhance colour formation, is ethephon (2-chloro-ethyl-phosphonic acid, 2-CEPA). In recent years significant research has been done on the effect of sunlight on anthocyanin production in grapes, although this has primarily been on wine grape cultivars. Currently, there is limited knowledge on the effect of sunlight on table grapes, and how this might influence their anthocyanin composition and content. The effect of ethephon on colour of grapes and other fruit have been extensively researched and well documented. However, the effect of ethephon on the anthocyanin composition of ‘Crimson Seedless’ is not well known. The current study aimed to explore the effect of sunlight (by matter of exclusion) and management practices, namely defoliation and ethephon application, on the anthocyanin profile and content of ‘Crimson Seedless’. Four different treatments were applied to two ‘Crimson Seedless’ vineyards, the first site located in Paarl, and the second in De Doorns. The treatments were: 1. Naturally exposed bunches, 2. Exposed bunches treated with ethephon, 3. Bunches kept in shade boxes, 4. Shaded bunches treated with ethephon. At the De Doorns site an additional defoliation treatment was superimposed over the above treatments. An HPLC technique was modified for the separation and detailed profiling of ‘Crimson Seedless’ anthocyanins and was used to analyse the effect of the reported treatments on the anthocyanin profile of berry skins. The predominant anthocyanin in ‘Crimson Seedless’ is peonidin-3-glucoside (Pn-gluc), and this was found to be significantly increased only by ethephon application, and was not altered by sunlight or leaf removal. The responses of the other anthocyanin types varied according to the respective treatments applied. However, a general observation was that ethephon application more consistently increased the concentration of anthocyanins in berry skins than did sunlight. Leaf removal had the least significant effect on anthocyanin concentration. / AFRIKAANSE OPSOMMIMG: ‘Crimson Seedless’ is tans een van die belangrikste en gewildste tafeldruif cultivars wat in Suid-Afrika verbou word en daarom is dit van groot ekonomiese waarde vir tafeldruifprodusente. ‘Crimson Seedless’ is egter daarvoor bekend dat dit te swak kleur (volgens uitvoer spesifikasies) en tweedens is die cultivar geneig om kleiner korrels te ontwikkel wanneer die oeslading vermeerder word. ‘n Addisionele probleem is dat die praktyke wat in die industrie gebruik word om korrels te vergroot ‘n verdere negatiewe impak op ‘Crimson Seedless’ se kleur ontwikkeling kan veroorsaak. Die plant-groei-reguleerder wat algemeen in tafeldruif verbouing gebruik word, ten einde beter gekleurde druiwe te produseer, is ethephon (2-chloro-ethyl-phosphonic acid, 2-CEPA). In die laaste paar jaar was daar baie navorsing gedoen oor die effek wat sonlig het op die antosianien produksie van druiwe, maar navorsing was gefokus op wyndruif cultivars. Huidiglik is daar beperkte tegniese kennis oor die effek wat sonlig op tafeldruiwe het, en hoe dit moontlik die antosianien samestelling en inhoud kan beïnvloed. Daar is ook reeds verskeie studies gedoen en data gepubliseer oor die invloed wat ethephon op die kleur het van druiwe en ander vrugte, maar die invloed wat ethephon op die antosianien samestelling van ‘Crimson Seedless’ het, is nie wel bekend nie. Die doel van hierdie studie was om die effek van sonlig (deur uitsluiting) en bestuurspraktyke (blaarverwydering en ethephon toediening) te bestudeer en hoe dit die antosianien samestelling van ‘Crimson Seedless’ beïnvloed. Vier verskillende behandelings is toegedien in twee ‘Crimson Seedless’ wingerde, die eerste proefperseel in die Paarl en die tweede proefperseel in De Doorns. Die behandelings was: 1. Natuurlik blootgestelde trosse, 2. blootgestelde trosse met ethephon, 3. Trosse met skadubokse omhul, 4. Skaduboks trosse met ethephon. By De Doorns is ‘n addisionele blaarverwydering proef bygebring. ‘n HPLC tegniek was aangepas om die antosianien samestelling en inhoud van ‘Crimson Seedless’ te bepaal, en om die effek van die behandelings te ondersoek. Die HPLC data het getoon dat peonodien-3-glukosied (Pn-gluc) die primêre antosianien in ‘Crimson Seedless’ is met die hoogste inhoud van al die antosianiene. Pn-gluc was betekenisvol beïnvloed deur ethephon toediening, terwyl die ander behandelings geen betekenisvolle effekte daarop gehad het nie. Die effekte wat die ander antosianiene gehad het, het gevarieer volgens die behandelings wat toegedien was. ‘n Algemene observasie was dat ethephon toediening die antosianien konsentrasie in ‘Crimson Seedless’ druiwe skille meer konsekwent vermeerder het as die sonlig blootstelling. Die blaarverwydering het die minste betekenisvolle effek op die antosianien inhoud van ‘Crimson Seedless’ gehad.

Thesis (PhD (Viticulture and Oenlogy))--University of Stellenbosch, 2008. / Includes bibliography and list of tables and figures. / ENGLISH ABSTRACT: Over the last decade research has shown the importance of small antimicrobial peptides in the innate immunity of plants. These peptides do not only play a critical role in the multilayered defense systems of plants, but have proven valuable in the engineering of disease resistant food crops towards the ultimate aim of reducing the dependency on chemical fungicides. As the lists of isolated and characterized peptides grew, it became clear that other biological activities, in addition to the antimicrobial capacity, could be linked to some of these peptides; these alternative activities could have important applications in the field of medicine. This has made the defensin encoding genes prime targets for the agricultural and medical biotechnology sectors. To this end we set out to evaluate South African flora for the presence of plant defensin sequences and to isolate plant defensin genes that might be useful in biotechnology applications. Moreover, by isolating and characterizing these novel peptides, also in an in planta environment and in interaction with fungal pathogens, important knowledge will be gained of the biological role and importance of the peptides in the plant body. The plant host targets were South Africa Brassicaceae species including indigenous species, as well as Vitis vinifera, as the most important fruit crop in the world and since no defensins have been isolated from this economically important crop plant. The Brassicaceae family has been shown to be abundant in defensin peptides and several of the best characterized peptides with potent activity have been isolated from this family. Based on initial activity screens conducted on selected South African Brassicaceae spp. we concluded that these spp. contain promising antifungal peptide activities, warranting further efforts to isolate the genes and encoding peptides and to characterize them further. The preliminary activity screens used a peptide-enrichment isolation strategy that favored the isolation of basic, heat-stable peptides; these properties are characteristic features of plant antimicrobial peptides. These peptide fractions showed strong antifungal activities against the test organisms. A PCR-amplification strategy was subsequently designed and implemented, leading to the isolation of 14 novel defensin peptide encoding genes from four South African Brassicaceae spp., including the indigenous South African species Heliophila coronopifolia. Amino acid sequence analysis of these peptides revealed that they are diverse in amino acid composition and share only 42% homology at amino acid level. This divergence in amino acid composition is important for the identification of new biological activities within closely related plant defensins. Single amino acid changes have been contributed with the divergent biological activities observed in closely related plant defensin peptides. Phylogenetic analysis conducted on the deduced amino acid sequences revealed that all the new defensins share a close relationship to other Brassicaceae members of the plant defensin superfamily and was furthest removed from the defensins isolated from the families Solanaceae and Poaceae. Classification analysis of these peptides showed that they belong to subgroup A3 of the defensin superfamily. A putative defensin sequence was also isolated from V. vinifera cultivar, Pinotage, and termed Vv-AMP1. Genetic characterization showed that only a single gene copy of this peptide is present within the V. vinifera genome, situated on chromosome 1. Genetic characterization of this peptide encoding gene within the Vitis genus showed that this gene has stayed conserved throughout the divergent evolution of the Vitis genus. Expression studies of Vv-AMP1 revealed that this gene is expressed in a tissue specific and developmentally regulated manner, being only expressed in grape berries and only at the onset of vèraison. Induction of Vv-AMP1 in grapevine leaf material could never be achieved through the external application of hormones, osmotic stress, wounding, or pathogen infection by Botrytis cinerea. Deduced amino acid analysis showed that Vv-AMP1 encoded for a 77 amino acid peptide consisting of a 30 amino acid signal peptide and a 47 amino acid mature peptide, with putative antifungal activity. The Vv-AMP1 peptide grouped with the subclass B type defensins, which have been documented to have both antifungal and antibacterial activities. The Vv-AMP1 signal peptide directed the green fluorescent protein (GFP) reporter gene to the apoplastic regions in cells with high levels of accumulation in the vascular tissue and the guard cells of the stomata. Recombinant Vv-AMP1 peptide was successfully purified from a bacterial host and shown to have a size of 5.495 kDa. Recombinant Vv-AMP1 showed strong antifungal activity at low concentrations against a broad spectrum of fungal pathogens, which included Verticillium dahliae (IC50 of 1.8 μg mL-1) and the necrotrophic pathogen Botrytis cinerea (IC50 of 12-13 μg mL-1). Antifungal activity of Vv-AMP1 did not induce morphological changes in fungal hyphae, but its activity was associated with induced membrane permeabilization in treated hyphae. Vv-AMP1 was successfully introduced into Nicotiana tabacum as confirmed by Southern blot analysis and 20 individual lines were generated. Genetic characterization confirmed the integration and expression of the gene in the heterologous tobacco environment. The peptide was under control of its native signal sequence which has been shown to direct its product to the apoplastic regions of cells. The transgenic lines were analyzed to determine the presence and activity of the grapevine defensin peptide. Western blot analyses of partially purified plant extracts detected a signal of the expected size in both the untransformed control and the transgenic lines. Comprehensive analysis of EST databases identified three highly homologous sequences from tobacco that probably caused the background signal in the control. These crude protein extracts were able to inhibit the growth of V. dahliae in vitro when tested in a microtiter plate assay, but the inhibition could not be conclusively linked to the presence of the transgenic peptide, since non-expressing transgenic lines, included as controls, also showed inhibition. Similar results were obtained with infection studies, clearly showing that despite successful integration and expression of the transgene, the peptides was either not functional in the heterologous environment, or perhaps unstable under the particular regulatory conditions. This peptide belongs to a subclass of peptides known for associated activities that might activate tight control by plant hosts if threshold levels are reached. These aspects need further investigation, specifically since it is in stark contrast to previous results obtained with defensins from a different subclass. This study has also yielded significant other related resources that would be instrumental for further possible biotechnology exploitation of some of the novel peptides, but also to provide genetic constructs and plant material that would be invaluable to address fundamentally important questions such as the regulation and mode of action of defensin peptides, specifically in interaction with pathogen hosts. The novel peptides have been transformed to various hosts, including grapevine and these transgenic populations are available to facilitate the next rounds of research into this extremely promising group of antifungal peptides. / AFRIKAANSE OPSOMMING: In die laaste dekade het navorsing die belangrike rol van klein antimikrobiese peptiede in plantweerstandsmeganismes beklemtoon. Hierdie peptiede speel nie alleenlik 'n belangrike rol in die komplekse lae van plantweerstandstelsels nie, maar het ook hulle ekonomiese potensiaal getoon in die manipulering van siekteweerstandbiedendheid in voedselgewasse met die oorkoepelende doel om landbougewasse minder afhanklik van chemiese spuitstowwe te maak. Soos wat die hoeveelheid geïsoleerde en gekarakteriseerde peptiede toeneem, het dit duidelik geword dat ander biologiese aktiwiteite, bykomend tot die antimikrobiese kapasiteit, met sommige van dié peptiede verbind kan word; hierdie alternatiewe aktiwiteite het belangrike toepassing in veral die mediese veld. Dit het die defensin-koderende gene kernteikens vir die landbou- en mediese biotegnologiesektore gemaak. In die studie is daar begin om die Suid-Afrikaanse blommeryk te evalueer vir die teenwoordigheid van plantdefensingene en om dié gene te isoleer wat van ekonomiese belang vir die biotegnologiebedryf kan wees. Deur die in vitro- én in planta karakterisering van die unieke plantdefensinpeptiede word daar gemik daarna om belangrike inligting in te win oor die biologiese rol van die peptiede binne die plantligggaam. Die plantgashere wat geteiken is sluit in die Suid-Afrikaanse Brassicaceae-spesies, insluitende inheemse spesies, asook Vitis vinifera, wat as die belangrikste vrugtegewas ter wêreld beskou word. Die Brassicaceae-familie is welbekend daarvoor dat dit 'n ryk bron van plantdefensinpeptiede is en verskeie van die bes gekarakteriseerde antifungiese defensinpeptiede is van dié familie afkomstig. Aanvanklike aktiwiteitstoetse het getoon dat die Suid-Afrikaanse Brassicaceae-spesies belowende antifungiese aktiwiteit toon, wat die verdere isolering en karakterisering van dié gene en hul peptiedprodukte regverdig. Die aanvanklike aktiwiteitstoetse het 'n selektiewe peptiedverrykingstrategie gevolg wat die isolering van basiese, hittestabiele peptiede bevoordeel het; hierdie eienskappe is baie kenmerkend van plant-antimikrobiese peptiede. Die peptiedfraksies wat met hierdie metode geïsoleer is, het sterk antifungiese aktiwiteit teen die toetsorganismes getoon. Die resultate het gelei tot die ontwikkeling en toepassing van 'n polimerasekettingreaksie-strategie, wat daartoe gelei het dat 14 nuwe defensingene van vier Suid-Afrikaanse Brassicaceae-genera, insluitend die inheemse spesie Heliophila coronopifolia, geïsoleer kon word. Afgeleide aminosuurvolgorde-analises van die nuwe defensinpeptiede het gewys dat hulle slegs 42% homologie het. Hierdie diversiteit in aminosuurvolgorde is belangrik vir die identifisering van nuwe biologiese aktiwitiete binne die groep van verwante peptiede. Navorsing het verder getoon dat enkel-aminosuurverskille bydra tot die diverse spektrum van biologiese aktiwiteite binne 'n groep van verwante defensinpeptiede. Filogenetiese analise van die aminosuurvolgordes het getoon dat al die nuwe defensinpeptiede 'n sterk verwantskap met plantdefensinpeptiede, wat van ander Brassicaceae-spesies geïsoleer is, toon. Daarteenoor het dit die kleinste verwantskap getoon met plantdefensinpeptiede wat van die Solanaceae- en Poaceae-families geïsoleer is. Klassifikasiestudies het bewys dat die nuwe peptiede saam met subgroep A3 van die plantdefensin-superfamilie groepeer. 'n Moontlike plantdefensingeen, genaamd Vv-AMP1, is ook van die V vinifera-kultivar, Pinotage, geïsoleer. Genetiese karakterisering het aangedui dat slegs 'n enkele kopie van die geen in die V. vinifera-genoom teenwoordig en op chromosoom 1 geleë is. Genetiese karakterisering van Vv-AMP1 binne die Vitus-genus het gewys dat die geen binne die genus evolusionêr gekonserveerd is. Uitdrukkingstudies van Vv-AMP1 het verder bewys dat die geen uitgedruk word op 'n weefselspesifieke, ontwikkelingsgekoppelde wyse, naamlik slegs in druiwekorrels en slegs tydens rypwording. Vv-AMP1-uitdrukking kon nooit geïnduseer word in wingerdblare deur die uitwendige toediening van hormone, osmotiese stres, wonding of patogeeninfeksie deur Botrtys cinerea nie. Ontleding van die afgeleide aminosuurvolgorde het gewys dat Vv-AMP1 kodeer vir 'n 77-aminosuurpeptied, wat uit 'n 30-aminosuurseinpeptied en 'n 47-aminosuur-aktiewe peptied met voorspelde antifungiese aktiwiteit bestaan. Die Vv-AMP1-peptied is gegroepeer met subgroep B van die plantdefensin-superfamilie, 'n subgroep wat vir beide antifungiese en antibakteriese aktiwiteit gedokumenteer is. Die Vv-AMP1-seinpeptied het die groen fluoressensie-indikatorproteïen (GFP) na die apoplastiese areas van die plantselle gelei, met hoë vlakke van lokalisering in die vaatbundelweefsel en sluitselle van die huidmondjies. Die rekombinante Vv-AMP1-peptied is suksesvol geproduseer en uit 'n bakteriese produksieras gesuiwer, en het 'n molekulêre massa van 5.495 kDa gehad. Die gesuiwerde peptide het by lae konsentrasies 'n sterk aktiwiteit getoon teen 'n breë spektrum van fungiese patogene, wat Verticllium dahliae (IC50 van 1.8 μg mL-1) en die nekrotrofiese patogeen, B. cinerea (IC50 van 12-13 μg mL-1), ingesluit het. Vv-AMP1-aktiwiteit het geen ooglopende morfologiese veranderinge in die fungi-hifes veroorsaak nie, maar hulle aktiwiteit is verbind met 'n verhoogde membraandeurdringbaarheid in behandelde fungi-hifes. Suksesvolle intergrasie van Vv-AMP1 in die Nicotiana tabacum-genoom is deur Southern-kladontledings bevestig en 20 individuele transgeniese lyne is ontwikkel. Genetiese karakterisering van die transgeniese lyne het gewys dat Vv-AMP1 suksesvol geïntegreer is en ook in die transgeniese tabakomgewing uitgedruk word. Die peptied is uitgedruk onder beheer van sy eie seinpeptied, wat die aktiewe produk na die apoplastiese areas van die plantselle teiken. Die transgeniese tabaklyne is ook ontleed om te bepaal of die wingerdpeptied suksesvol geproduseer word en sy aktiwiteit in die transgeniese omgewing behou. Western-kladanalise van semi-gesuiwerde plantproteïenekstrakte het 'n positiewe sein gelewer in beide die kontroleplante en die transgeniese plantlyne. Bestudering van tabakgeenuitdrukkings-databasisse het drie nukleotiedvolgordes opgelewer wat homologie met Vv-AMP1 toon en moontlik verantwoordelik kan wees vir die positiewe sein in die ongetransformeerde kontroleplante. Kru proteïenekstrakte van die transgeniese tabaklyne het in vitro-aktiwiteit teen V. dahliae getoon. Geen oortuigende ooreenkoms kon egter gevind word tussen V. dahliae-inhibisie en die teenwoordigheid van die transgeniese Vv-AMP1-peptied nie, aangesien kontroleplante wat Southern-klad-positief is, maar nie geenuitdrukking toon nie, ook inhibisie van V. dahliae veroorsaak het. Soortgelyke resultate is met infeksiestudies verkry. Alle resultate dui daarop dat, al is daar suksesvolle integrasie en uitdrukking van die geen in tabak verkry, dat die Vv-AMP1 peptied óf onaktief óf onstabiel in die transgeniese tabakomgewing is. Die peptied behoort aan 'n subgroep peptiede met aktiwiteite wat, sodra sekere vlakke van peptied oorskry word, die moontlik streng kontrole op proteïenvlak in die gasheerplant kan uitlok. Sekere aspekte van die studie sal verder bestudeer moet word, aangesien die data teenstrydig is met data wat verkry is met soortgelyke plantdefensinpeptiede wat aan 'n ander subgroep behoort. Die studie het baie hulpbronne gegenereer wat vir die biotegnologiesektor belangrik kan wees, veral op ekonomiese gebied. Verder is die geenkonstrukte en plantlyne wat ontwikkel is waardevol om fundamentele vrae rondom die regulering en meganisme van aksie van defensinpeptiede, spesifiek plantpatogeeninteraksie, te beantwoord. Die nuwe plantdefensingene is na verskeie gasheerplante, insluitende wingerd, getransformeer waar die transgeniese lyne die volgende rondte van navorsing oor die bestudering oor die belangrike groep van antifungiese peptiede, sal aanvul.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2007. / The South African and international table grape industries are growing rapidly, which necessitates the production of high quality export fruit at competitive production costs. For this reason, alternative irrigation methods are required to utilise water optimally while still attaining good quality table grapes. An increase in agricultural productivity may be dependent on either the availability of more water for irrigation or an increase in the efficiency of water use. The first aim of this study was to evaluate the effectiveness of the Partial Rootzone Drying (PRD) irrigation strategy in Crimson Seedless and Dauphine table grape production. This irrigation system is based on the drying of half of the vine roots, thereby allowing the plant to produce hormones like abscisic acid (ABA) in reaction to water stress. The hormone production in turn results in stomatal closure and the reduction of water loss via transpiration. The drying cycle is then repeated after 10 to 15 days on the other side of the vine, irrigating the previously dried roots. PRD will encourage a consistent production of the stress hormone abscisic acid (ABA), without actual water stress. This strategy reduces the amount of water used for irrigation, without an accompanying loss in fruit yield, as compared to conventional techniques. In this study, conventionally treated vines were irrigated according to historical block data and PRD-treated vines were irrigated at the same times. The second aim of this study was to monitor the efficacy of a foliar nutrient, Croplife. This foliar nutrient allegedly improves the uptake of foliar applied nutrients, assists with transport of all minerals through the leaves and enables the plant to attain higher pest and disease resistance thresholds. Conventionally treated vines that did not receive foliar nutrient treatment were compared to vines that received foliar nutrients as prescribed by the manufacturer. Vine cultivars Crimson Seedless and Dauphine, were grown under open hydroponic principles with drip and drip irrigation respectively in this experiment. For the hydroponic vines (Crimson Seedless), all vines were situated in the same row and 72 vines were divided into mini-plots of three vines. Treatments were then assigned to an equal number of plots at random. The same procedure was followed for the drip irrigated vines (Dauphine) but the vines were situated in two rows of equal length. Treatment effects were followed from budburst until harvest, where after post-harvest analyses were conducted. The first aim, namely to show that PRD is an effective irrigation strategy for table grape production in Crimson Seedless and Dauphine cultivars , has shown that vines did not exhibit signs of stress even though they received only half the conventional amount of water. This study was conducted over only one growth season and therefore no definite conclusions could be drawn about the long term effectiveness of PRD on table grapes. It did, however, confirm numerous results obtained from different studies on the use of PRD in wine grape production. The results obtained in the second part of the study were inconclusive and could not show that Croplife is effective in improving the uptake and transport of applied foliar nutrients. Because Crimson Seedless is cultivated under open hydroponic principles, nutrients can be absorbed by the roots via the soil and micronutrients are also available from chemical sprays during the season. There was no evidence to indicate that the use of Croplife increased nutrient absorption and transport, neither did it supplement or detract form the observed effect of PRD. Despite the limitations experienced during this study, it has shown that the use of PRD for table grape production may be a useful tool for improving water utilisation efficiency in future. The strategy will have to be developed systematically through experimentation to fully unlock the potential of the PRD management system for table grape production. This study provides a good starting point for future research required to elucidate numerous aspects of the PRD system and has clearly shown that established vineyards can be switched to a PRD system without a loss in table grape quality. It is envisaged that the advantages of this system could have a positive effect on the production of high quality fruit for the international market.

Thesis (MscAgric (Viticulture and Oenology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Tannins and colour components in red wine are important quality parameters. These factors can be manipulated in the vineyard by grape growing techniques or in the cellar by different winemaking practices. Grape seeds make a significant contribution to tannin concentration in wine when compared to those from the skins and pulp. Tannins contribute to the ageing potential, organoleptic properties and stabilisation of red wine colour. The colour of a red wine is also influenced by malolactic fermentation, the biological process that transforms malic acid into lactic acid which normally leads to an increase in pH. The subsequent change in pH alters the anthocyanin equilibrium, the primary colour components in red wine. Oxygen contributes to the polymerisation of anthocyanins over time to form more stable pigments that are less sensitive to pH fluctuations and sulphur dioxide bleaching. Limited research has been done on the use of oxygen after alcoholic fermentation and the impact it has on the phenolic composition of red wines. Similarly, only a few studies have examined the impact of either the addition or removal of seeds to the phenolic composition of a red wine in combination with oxygen addition. Additionally, little published data seems to exist on the effect of different pHs on red wine’s phenolic and colour development after oxygen addition. In our results we have shown that it is possible to stabilise wine colour by adding supplementary seeds before alcoholic fermentation. This led to an increase in colour intensity in certain cases. Some red wines produced without seeds had significantly lower colour intensities. This clearly suggests that catechins and proanthocyanidins are extracted from seeds and contribute to wine colour as they combine with other pigments to stabilise wine colour. Spectrophotometric and HPLC analyses have shown that the total phenolic content increased with seed concentration. However, we have observed that a wine may possibly become saturated with phenols when supplementary seeds are added. Anthocyanin concentrations often decreased when oxygen was added, while polymeric phenols and polymeric pigments sometimes increased. When applying different oxygen dosages to a red wine on commercial scale with microoxygenation, it was found that monomeric anthocyanins decreased as more oxygen was added and this decrease in anthocyanins led to the formation of stable polymeric pigments. This was reflected in the significant increase in colour intensity for the wines receiving oxygen. Small differences were detected in the total phenol and tannin concentration for the control and oxygenated wines. However, some of these phenolic and colour differences disappeared during subsequent ageing of the wine. When making wines of different initial pHs, we observed that the colour density decreased as the pH increased. The application of oxygen reduced the decrease in colour during MLF, especially at a lower pH. The addition of oxygen did not result in significantly different polymeric pigment concentrations in the various pH treatments, although the results could have been different if the wines were aged. However, pH differences in the range between 3.4 and 4.0 did not significantly influence the phenol composition of the wines under our conditions. This study led to a better understanding on the effect of oxygen additions under different conditions on red wine's phenolic and colour composition. / AFRIKAANSE OPSOMMING: Tanniene en kleur komponente teenwoordig in rooiwyn is belangrike kwaliteit parameters. Hierdie faktore wat wynkwaliteit beïnvloed kan gemanipuleer word in die wingerd deur verskeie verbouingstegnieke toe te pas en in die kelder deur die toepassing van verskillende wynbereidingsmetodes. Die bydrae van sade tot die finale tannienkonsentrasie in rooiwyn is groot in vergelyking met dié van die pulp en doppe. Tanniene dra by tot die verouderingspotensiaal, organoleptiese eienskappe en die stabilisasie van die wynkleur. Die kleur van ‘n rooi wyn word ook beïnvloed deur appelmelksuurgisting (AMG), die biologiese proses wat appelsuur omskakel na melksuur en ‘n gevolglike toename in die pH van die wyn veroorsaak. Hierdie verandering in die pH van die wyn beïnvloed die antosianien ewewig, die primêre kleur komponente teenwoordig in rooiwyn. Suurstof dra by tot die polimerisasie van antosianiene oor tyd om meer stabiele kleur pigmente te vorm met ‘n hoër kleurintensiteit wat minder sensitief is teenoor pH veranderinge en die bleikingseffek van swaweldioksied. Beperkte navorsing is gedoen op die gebruik van suurstof na alkoholiese gisting en die impak daarvan op die fenoliese samestelling van ‘n rooiwyn. Slegs ‘n paar studies het die invloed van die verwydering of byvoeging van sade in kombinasie met suurstoftoediening op ‘n rooiwyn se fenoliese samestelling ondersoek. Dit wil voorkom of beperkte gepubliseerde data beskikbaar is oor die effek wat verskillende pH’s het op rooi wyn se fenoliese en kleurontwikkeling na suurstof byvoeging. Ons resultate het aangedui dat dit wel moontlik is om in sekere gevalle die kleur van ‘n rooiwyn te stabiliseer deur addisionele sade by te voeg voor alkoholiese fermentasie. Hierdie byvoeging het ‘n toename in kleurintensiteit tot gevolg gehad. Sekere wyne wat gemaak is sonder sade het ‘n kenmerkend laer kleur intensiteit gehad. Hierdie bevinding is ‘n duidelike bewys dat katesjiene en prosianidiene geëkstraheer word vanuit die sade en bydra tot wynkleur deurdat hulle met ander pigmente verbind om die kleur sodoende te stabiliseer. Spektrofotometriese en hoë druk vloeistof chromatografie (HDVC) analises het gewys dat die totale fenoliese konsentrasie neem toe met ‘n toename in saad konsentrasie. Daar is egter waargeneem dat ‘n wyn moontlik versadig kan raak met fenole wanneer addisionele sade bygevoeg word. Antosianien konsentrasies het meestal afgeneem wanneer suurstof bygevoeg is, maar polimeriese fenole en polimeriese pigmente het partykeer toegeneem. Met die toediening van verskillende suurstof dosisse tot ‘n rooiwyn op kommersiële skaal het ons bevind dat monomeriese antosianiene afneem wanneer meer suurstof bygevoeg word. Hierdie afname in antosianiene het egter gelei tot die vorming van stabiele polimeriese pigmente. Dié bevinding was gereflekteer in die toename in kleurintensiteit van wyne wat met suurstof behandel is. Klein verskille was waargeneem vir die totale fenol en tannien konsentrasies tussen die kontrole en wyne behandel met suurstof. Sekere van hierdie fenoliese kleur verskille het egter afgeneem tydens die daaropvolgende veroudering van die wyne. Wyne wat gemaak is met verskillende aanvanklike pH’s se kleurintensiteit neem af soos die pH toeneem. Die toediening van suurstof het die kleurverlies tydens AMG verminder, veral by ‘n laer pH. Die toediening van suurstof het nie verskillende polimeriese pigment konsentrasies by verskillende pH’s veroorsaak nie, maar ‘n verskil kon moontlik waargeneem word indien die wyne verouder was. pH verskille tussen 3.4 en 4.0 het egter nie die fenoliese samestelling van die wyne onder ons omstandighede beduidend beïnvloed nie. Hierdie studie het gelei tot meer kennis oor die effek van suurstoftoedienings onder verskillende kondisies op rooiwyn se fenoliese en kleursamestelling.

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))--University of Stellenbosch, 2009. / South Africa is the eighth largest wine producing country in the world and face stiff competition on the world market. Cabernet Sauvignon is the most planted red cultivar in the world as well as in South Africa and can be seen as the wine by which countries are judged. The aim of this study was to investigate suitable, practical maturity parameters or combinations thereof to determine the optimal time to harvest Cabernet Sauvignon grapes under South African conditions. The following parameters were investigated during this study: seed lignification, maturity indexes, anthocyanin concentration per berry, sensory criteria (grape skins tasting and wine) and phenolic content. Berry development in four Cabernet Sauvignon vineyards in different South African winegrowing areas were investigated over the 2003, 2004 and 2005 seasons. The first parameter to be investigated was seed lignification percentages. Seasonal differences at commercial harvest were observed with values of 2004 varying between 73% and 91% compared to 59% and 80% for the 2003 and 2005 seasons but commercial harvest was two weeks later during the 2004 season. During this study it was found that seeds never reached 100% lignification for Cabernet Sauvignon as was found in previous work to indicate grape maturity. The development of anthocyanins also peaked well before the maximum seed lignification was reached. It therefore appears that seed lignification is not suitable for the determination of grape maturity for Cabernet Sauvignon grapes under South African conditions. The second parameter to be investigated was maturity indexes (Balling / Titratable Acidity (TA), Balling × pH, Balling × pH2). The best wine values were used to determine the optimal maturity index values. Morgenster was the only vineyard to consistently give values that corresponded to previously reported data (index values). Anhöhe and Plaisir de Merle reported higher maturity values than that reported in literature and seasonal variation was observed. Maturity index values for the best wines varied between 88 and 101 (Balling × pH) for Anhöhe during 2003 and 2005 seasons, but increased too between 97 and 107 (Balling × pH) for 2004. The maturity index values were found to be vineyard and season dependant, with warmer areas reaching higher values. From this study it appears that maturity index values as a singular maturity parameter does not give a good indication of berry maturity in all seasons or vineyards. Thirdly, the berry anthocyanin concentration (mg / berry and mg / g berry) were investigated and comparable trends were found between the four vineyards. However vineyards in warmer, drier regions (Anhöhe) tended to have higher anthocyanin concentrations per gram berry. The more vigorous vineyard of Morgenster consistently exhibited a higher anthocyanin concentration per berry. This can be explained by the ratio of skin to pulp between small berries (Anhöhe, 0.95 g - 2004) and larger berries South Africa is the eighth largest wine producing country in the world and face stiff competition on the world market. Cabernet Sauvignon is the most planted red cultivar in the world as well as in South Africa and can be seen as the wine by which countries are judged. The aim of this study was to investigate suitable, practical maturity parameters or combinations thereof to determine the optimal time to harvest Cabernet Sauvignon grapes under South African conditions. The following parameters were investigated during this study: seed lignification, maturity indexes, anthocyanin concentration per berry, sensory criteria (grape skins tasting and wine) and phenolic content. Berry development in four Cabernet Sauvignon vineyards in different South African winegrowing areas were investigated over the 2003, 2004 and 2005 seasons. The first parameter to be investigated was seed lignification percentages. Seasonal differences at commercial harvest were observed with values of 2004 varying between 73% and 91% compared to 59% and 80% for the 2003 and 2005 seasons but commercial harvest was two weeks later during the 2004 season. During this study it was found that seeds never reached 100% lignification for Cabernet Sauvignon as was found in previous work to indicate grape maturity. The development of anthocyanins also peaked well before the maximum seed lignification was reached. It therefore appears that seed lignification is not suitable for the determination of grape maturity for Cabernet Sauvignon grapes under South African conditions. The second parameter to be investigated was maturity indexes (Balling / Titratable Acidity (TA), Balling × pH, Balling × pH2). The best wine values were used to determine the optimal maturity index values. Morgenster was the only vineyard to consistently give values that corresponded to previously reported data (index values). Anhöhe and Plaisir de Merle reported higher maturity values than that reported in literature and seasonal variation was observed. Maturity index values for the best wines varied between 88 and 101 (Balling × pH) for Anhöhe during 2003 and 2005 seasons, but increased too between 97 and 107 (Balling × pH) for 2004. The maturity index values were found to be vineyard and season dependant, with warmer areas reaching higher values. From this study it appears that maturity index values as a singular maturity parameter does not give a good indication of berry maturity in all seasons or vineyards. Thirdly, the berry anthocyanin concentration (mg / berry and mg / g berry) were investigated and comparable trends were found between the four vineyards. However vineyards in warmer, drier regions (Anhöhe) tended to have higher anthocyanin concentrations per gram berry. The more vigorous vineyard of Morgenster consistently exhibited a higher anthocyanin concentration per berry. This can be explained by the ratio of skin to pulp between small berries (Anhöhe, 0.95 g - 2004) and larger berries (Morgenster, 1.82 g – 2004). Wine colour density (A420+A520) followed the same trend as the anthocyanin concentrations of the homogenate. Grape skins (G) were used to make an artificial wine that was evaluated by an expert panel to determine the development of the grapes. Wines (W) made from sampled batches were also evaluated by an expert panel for: colour intensity, vegetative, red berry, black berry with spice, acidity, astringency and general quality. Vegetative aromas and acidity decreased and red and black berry with spice increased during ripening for both berries and wine. Colour intensity also increased, corresponding to an increase in perceived general quality score. Correlations between general quality of both the grape skins tasting and wines were investigated. Balling showed a strong correlation with general quality of the grape skins tasting (r = 0.76; p = 0.00) but not as strongly with subsequent wines (r = 0.57; p = 0.00). Anthocyanin concentration (mg / g berry) of the berries (r = 0.36; p = 0.00), perceived colour intensity of grapes (r = 0.69; p = 0.00) and wine (r = 0.84; p = 0.00) correlated with general wine quality. The tasting panel identified wines that were statically better than the rest for each season and vineyard. Maximum berry anthocyanin concentration coincided with wines rated as the best by the tasting panel. More than one wine was identified during the maximum anthocyanin peak that did not differ statistically from the best wine. It appears from this study that a window period exists at the maximum anthocyanin peak, where wines of comparable quality, but different style, can be produced. Principal component analysis (PCA) was used to determine the least number of suitable parameters that could distinguish between unripe and ripe grapes in order to establish a grape maturity model. These differences were successfully described by Balling, TA, pH, potassium (K+), tartaric and malic acid. Anthocyanin concentration could further distinguish between ripe and overripe grapes in the model. From these parameters the minimum and maximum values were used to construct a universal ripeness model containing data from all four vineyards. Variation between the four vineyards caused too much overlapping in the universal model data as the vineyards were situated in different climatic regions according to the Winkler temperature model. On a per vineyard basis this did not occur to the same extend. The best rated Cabernet Sauvignon wines correlated strongly with soluble solid content; colour and quality perceptions of grapes, but large seasonal differences resulted in larger grape compositional variances than that of the individual vineyards in the different climatic zones. This illustrated the difficulty of pinpointing a specific parameter to indicate optimal ripeness. From this study it is clear that a universal maturity model for Cabernet Sauvignon berries is not attainable at present, but individual vineyard models shows the most potential. A preliminary study into the differences of the phenolic composition was done using reverse phase high performance liquid chromatography (RP-HPLC) on the homogenate and wine. Malvidin-3-glucoside and total anthocyanins followed comparable trends to that found for the Iland method. Strong correlations (r > 0.9) were found between the malvidin- 3-glucoside and malvidin-3-glucoside-acetate and p-coumarate; this was also true for the total anthocyanins in both homogenate and wine. Wines identified by a tasting panel to be the best quality, corresponded with the maximum anthocyanin concentration (mg / L) peak in the homogenate. Dense canopies at the Morgenster vineyard over the three seasons lead to lower total anthocyanin and quercetin-3-glucuronide concentrations compared to the Anhöhe and Plaisir de Merle vineyards. The shading of bunches by the dense canopy most likely contributed to this. Catechin, epicatechin, proanthocyanidin and polymeric phenol concentrations decreased significantly from veraison until harvest. Seasonal differences were noted in the four vineyards. No correlations could be found between the general wine quality and the phenolic compounds, but a weak trend was observed for total anthocyanins in the homogenate. A trend was found with the total flavan-3-ol to anthocyanin ratio determined by RP-HPLC analysis of the grape homogenates (r = 0.40, p = 0.00). This ratio varied between 1 and 3 for the wines rated as being the best quality. Phenols by themselves do not give a clear indication of optimal harvest time. From this study it appears that no single parameter could consistently indicate optimal ripeness over the seasons or per vineyard, but the maximum berry colour (anthocyanin concentration) did give an indication of optimal harvesting time. It is clear that a combination of parameters could predict the optimal time more precisely as with the above mentioned model but more research is needed to this end.

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.