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

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The presence and growth of lactic acid bacteria (LAB) in wine and their influence on wine quality has received much attention in recent years. Lactic acid bacteria are responsible for conducting malolactic fermentation (MLF) in wine. The benefits associated with malolactic fermentation in terms of deacidification of wine and the contribution to wine flavour and complexity have also recently been the topic of research. It is impossible to describe malolactic fermentation as distinctly desirable or undesirable in terms of its influence on the final quality of wine. The benefits and disadvantages are dependent upon viticultural region, grape variety, wine composition, winemaking techniques and the style and objectives of the winemaker. Brandy production is a multi-stage process in which base wine production, distillation technique and wood maturation all have a large influence on the final chemical profile and organoleptic quality of the brandy. The volatile composition of the base wine, which basically undergoes a concentration process during the subsequent double distillation phase, is critical in determining the aroma and flavour quality of the final brandy product. Thus, the brandy is only as good as the base wine it is distilled from. The aims of this study were to determine the effect of lactic acid bacteria and spontaneous malolactic fermentation on the quality of brandy base wine and the resulting distillate, and to determine which LAB species had been responsible for the occurrence of spontaneous MLF. This study showed that LAB are present at high numbers and are able to conduct spontaneous MLF of brandy base wines. It was shown that the incidence of spontaneous MLF varied from year to year. In 1998, 50% of the commercially produced base wines had undergone partial MLF prior to distillation. In 1999 and 2000 respectively, 34% and 45% of the commercial base wines had undergone partial MLF prior to distillation. The occurrence of spontaneous MLF had an influence on the chemical composition and the sensory quality of the base wine and distillate. There was an increase in the concentrations of ethyl lactate, acetic acid and diethyl succinate in samples that had undergone MLF. There was also a decrease in the concentrations of esters, such as iso-amyl acetate, ethyl acetate, ethyl caproate, hexyl acetate and 2-phenethyl acetate in these same samples. Sensory evaluation of the base wines and distillates demonstrated that samples that had undergone MLF differed significantly from samples that had not undergone MLF. It was also shown that distillates that had not undergone MLF had a slightly better aroma profile than those that had. Sweet aromas, like chocolate and caramel, as well as negative aromas, like chemical or solvent, were more prominent in brandy distillates that had undergone MLF. Herbaceous and fruity aromas were more intense in distillates not having undergone MLF. Fifty-four strains, all Gram-positive and catalase negative, were isolated at different stages of brandy production. Seven strains were isolated from the grape juice, 15 strains were isolated from the base wine, 20 strains were isolated during MLF and 12 strains were isolated from the base wine after MLF had been completed. Based on C02 production from glucose and gluconate, 17 strains were classified as facultatively heterofermentative and 37 strains as obligately heterofermentative. Fifteen of the 37 obligately heterofermentative strains were rod-shaped and were regarded as lactobacilli. The remaining 22 strains were oval or cocci-bacilli shaped. The isolates were identified to species level by using numerical analysis of the total soluble cell protein patterns, 16S rRNAsequencing and polymerase chain reaction (PCR) with species-specific primers. The facultative heterofermentative lactobacilli were identified as Lactobacillus paracasei and Lactobacillus p/antarum. The fifteen obligately heterofermentative lactobacilli were identified as members of the species Lactobacillus brevis, Lactobacillus verrniforme, Lactobacillus buchneri and Lactobacillus hi/gardii. The 22 obligate heterofermentative isolates, with a coccoid morphology, could be grouped into two clusters and were identified as Oenococcus oeni. O. oeni was the species responsible for the occurrence of spontaneous MLF in most of the commercial base wines. Lb. brevis, Lb. hi/gardii and Lb. paracasei were also isolated from commercial base wines that had undergone spontaneous MLF. In nine out of 14 experimental base wine samples that had undergone spontaneous MLF, O. oeni was again the predominant species. Lb. brevis, Lb. hi/gardii and Lb. paracasei were identified in the remaining experimental base wine samples. This is the first report of the presence of Lb. perecese! and Lb. vermiforme in brandy base wine. It was shown that the occurrence of spontaneous MLF had a negative effect on the quality of brandy base wine, but that was shown to be due to the different species and strains performing MLF. In the non-preferred distillate samples, Lactobacillus spp. had performed MLF or had developed after or during MLF. / AFRIKAANSE OPSOMMING: Die teenwoordigheid en die vermoë van melksuurbakterieë (MSB) om in wyn te groei, is 'n onderwerp wat al heelwat nagevors is. Melksuurbakterieë is verantwoordelik vir die uitvoering van appelmelksuurgisting (AMG) in wyn. Die voordele verbonde aan appelmelksuurgisting, ten opsigte van die verlaging van die totale suurinhoud en die bydrae tot die verbeterde geur en kompleksiteit van die wyn, is ook al goed bestudeer. Wat die invloed op die finale wynkwaliteit betref, is dit byna onmoontlik om AMG as uitsluitlik gewens óf ongewens te beskou. Die voordele en nadele van AMG is afhanklik van verskeie faktore, nl. wingerdkundige streek, druifkultivar, wynsamestelling, wynmaakpraktyke, asook die styl en doelwitte van die wynmaker. Die produksie van brandewyn is 'n multistapproses waarin die bereidingsmetode van die basiswyn, die distillasietegniek en houtveroudering 'n groot invloed op die finale kwaliteit en chemiese samestelling van die brandewyn het. Die vlugtige verbindings van die basiswyn, wat tydens die dubbele distillasieproses gekonsentreer word, is van wesenlike belang in die bepaling van die aroma en geur van die finale brandewynproduk. Brandewyn is dus inderdaad net so goed soos die basiswyn waarvan dit gestook is. Die doelwitte van hierdie studie was om te bepaal wat die invloed van MSB en die voorkoms van spontane AMG op die kwaliteit van die basiswyn en die distillaat is, asook om die MSB wat vir die voorkoms van spontane AMG verantwoordelik was, te identifiseer. Hierdie studie het bewys dat MSB in hoë getalle teenwoordig was en dat dit in staat is om die spontane AMG van basiswyne uit te voer. Daar is bewys dat die voorkoms van spontaneAMG moontlik van jaar tot jaar kan verskil. In 1998 het 50%, in 1999 het 34% en in 2000 45% van die kommersieel-geproduseerde basiswyn gedeeltelike AMG spontaan voor distillasie ondergaan. Daar is ook gevind dat spontane AMG 'n invloed op die chemiese samestelling en sensoriese kwaliteit van die basiswyn en die distillaat gehad het. Daar was 'n toename in die konsentrasies van etiellaktaat, asynsuur en diëtielsuksinaat in monsters wat spontane AMG ondergaan het. In dieselfde monsters was daar ook 'n afname in die konsentrasies van iso-amielasetaat, etielasetaat, etielkaproaat, heksielasetaat en 2-fenielasetaat. Sensoriese evaluering van die basiswyne en distillate het getoon dat daar betekenisvolle verskille was tussen die monsters wat AMG ondergaan het en dié wat nie AMG ondergaan het nie. Daar is bewys dat die distillate wat nie AMG ondergaan het nie, 'n beter aromaprofiel gehad het as dié wat AMG ondergaan het. Soet geure, soos sjokolade en karamel, en negatiewe geure, soos "chemies" en "oplosmiddel", was prominent in distillate wat AMG ondergaan het. Kruidagtige en vrugtige geure was meer intensief in distillate wat nie AMG ondergaan het nie. Vier-en-vyftig bakteriese rasse, almal Gram-positief en katalase-negatief, is gedurende die verskillende stadia van brandewynproduksie geïsoleer. Sewe rasse is uit druiwesap, 15 rasse gedurende die alkoholiese fermentasie, 20 rasse gedurende AMG en 12 rasse na voltooiing van AMG geïsoleer. Op die basis van koolstofdioksied (C02)-produksie vanaf glukose en glukonaat is 17 rasse as fakultatief heterofermentatief en 37 rasse as obligaat heterofermentatief geklassifiseer. Vyftien van die 37 obligaat-heterofermentatiewe rasse was staafvormig en is as lactobacilli geïdentifiseer. Die oorblywende 22 het ovaal of kokkus-bacillusvormige selmorfologie getoon. Identifikasie tot op spesievlak is gedoen deur van numeriese analise van die totale oplosbare selproteïenprofiele, 16S-rRNAvolgordebepalings en spesie-spesifieke inleiers vir die polimerasekettingreaksie (PKR) gebruik te maak. Die fakultatief-heterofermentatiewe rasse is as Lactobacillus paracasei en Lactobacillus p/antarum geklassifiseer. Die 15 obligaat heterofermentatiewe stafies is as Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus hi/gardii en Lactobacillus vermiforme geïdentifiseer. Die 22 ovaal, obligaat heterofermentatiewe isolate kon in twee groepe ingedeel word en is as Oenococcus oeni geïdentifiseer. Daar is bevind dat O. oeni-isolate vir die voorkoms van spontane AMG in die meeste van die kommersiêle basiswyne verantwoordelik was. Lb. brevis, Lb. hi/gardii en Lb. paracasei is ook uit kommersiêle basiswyne wat spontane AMG ondergaan het, geïsoleer. In nege uit 14 van die eksperimentele basiswyne wat spontane AMG ondergaan het, was O. oeni die dominante spesie. In die oorblywende eksperimentele wyne is Lb. brevis, Lb. hi/gardii en Lb. paracasei aangetref. Hierdie is die eerste vermelding van die teenwoordigheid van Lb. paracasei and Lb. vermiforrne in brandewynbasiswyn. Daar is gevind dat die voorkoms van spontane AMG "n negatiewe invloed op brandewynkwaliteit het, maar dit is as gevolg van die verskeidenheid van MSB-spesies en rasse wat voorkom. In die distillate wat deur die proepaneel afgekeur is, het Lactobacillus spesies die AMG deurgevoer, of het dit tydens of na AMG ontwikkel.

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

Thesis (MTech (Food Technology))--Cape Peninsula University of Technology, 2010. / Sulphiting is considered as the most reliable and understood preservation technique in the wine industry. Since sulphur dioxide (S02) has been associated with possible health risks, legislation as well as consumers, are becoming more reluctant about the general use of S02 in wine production. In order to avoid economic losses due to spoilage, the wine industry is seeking feasible techniques to possibly reduce the levels of S02 in wine. The purpose of this study was, therefore to determine the efficacy of ultraviolet radiation (UV)-C (254 nm) as an alternative technology to inactivate microorganisms in white and red grape juices and wines.

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

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

Thesis (Msc (Viticulture and Oenology))--University of Stellenbosch, 2007. / Among the factors contributing to wine complexity and quality, wine aroma is one of the most important factors. Wine aroma is the outcome of interaction among different compounds produced from the grapes, during fermentation as well as during the ageing process. Apart from its origin from grapes, fungi and yeasts, wine aroma can also be derived from the metabolic activity of wine lactic acid bacteria (LAB). These microorganisms are usually associated with malolactic fermentation (MLF) which normally occurs after alcoholic fermentation. MLF is beneficial to wine due to its contribution to deacidification, microbiological stabilisation and wine aroma formation, with the latter being the most important area of interest in our study. The production of volatile aromatic components in wine can, in part, be achieved through the hydrolytic action of enzymes produced by LAB associated with wine. These enzymes include β-glucosidase, protease, esterase, lipase and glucanase. Most of the work done on bacterial enzymes has been on LAB from food sources other than wine, in which these enzymes contribute to the flavour development of some cheeses, yoghurt and other fermented foods. The activity of these enzymes during wine fermentation has mostly been concerned with β-glucosidase from Oenococcus oeni. Only in recent years has there been a renewed interest in evaluating the activity of β-glucosidase in other genera of wine LAB. The overriding goal of this study was to screen and characterise wine-related enzymes produced by LAB associated with wine. All the LAB isolates tested in this study were obtained from IWBT culture collection and were previously isolated from five different wineries situated in the Western Cape region, South Africa. We first screened isolates using classical methods. The isolates were grown on agar medium supplemented with appropriate substrate analogues in order to evaluate the activity of enzymes (i.e. β- glucosidase, glucanase, lipase and esterase). The colonies exhibiting enzymatic activity ...

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2005. / The quality of wine is influenced by a variety of factors, most noticeably the quality of the grapes, winemaking practices and the yeast strains used for alcoholic fermentation. Although several yeast strains are present in the must at the beginning of fermentation, strains of S. cerevisiae quickly dominate and survive alcoholic fermentations. This dominance of S. cerevisiae prompted research that led to the development of a multitude of industrial yeast starter cultures. Starter cultures are usually capable of quick and complete fermentations, with minimal production of deleterious substances such as volatile acidity, H2S, SO2 and ethyl carbamate. Yeast strains should be able to survive the stressful environment created during alcoholic fermentation, whilst possibly offering novel oenological benefits such as pectinolytic activity, killer activity and malic acid degradation. The increased production of volatile esters and higher alcohols may also be desirable, as this will allow the production of wines that are more aromatic. In this study, VIN13 was crossed with S. paradoxus strain RO88 and WE14 by using a micomanipulator. VIN13 was chosen for its fast and complete fermentation ability and moderate aroma production potential. Other factors such as the presence of killer activity and low production of volatile sulphur compounds also favoured the selection of VIN13. S. paradoxus strain RO88 was selected for its ability to degrade malic acid and the favourable impact on aroma production during fermentation. Hybrids between these yeasts may have the potential to produce more aromatic wines, with the added bonus of pectinolytic activity and a strong fermentation capacity. The first crossing yielded 5 hybrids between VIN13 and S. paradoxus strain RO88. Two of these hybrids stood out in the sense that they were able to degrade more malic acid than VIN13 and they also possessed killer and pectinolytic activity. Cinsaut wine was made and the 2 hybrids were shown to have higher aroma compound capacity than the parental yeasts. This was also confirmed during sensory evaluation. The second crossing between VIN13 and WE14 yielded 10 hybrids with low H2S production potential and killer activity. WE14 was selected for its ability to produce very aromatic wines and also the slower fermentation capacity. Hybrids between these yeast may have the potential to produce wines with an increased aromatic content and the fermentation rate might be slower, thereby improving the aroma profile of the wine. After microvinification, 5 hybrids were selected on the basis of fermentation rate differing from that of the parental yeasts and favourable oenological traits, such as fast and complete fermentation, high production of glycerol and low production of volatile acidity. Pinotage wine was made and it was shown that some of the hybrids produced more esters and higher alcohols than the parental yeasts. Sensory evaluation also showed the aroma production potential of the hybrids, as some of the hybrids were shown to score higher for banana, cherry and tobacco characteristics.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Fermentation is a complex process in which raw materials are transformed into high-value products, in this case, grape juice into wine. In this modern and economically competitive society, it is increasingly important to consistently produce wine to definable specifications and styles. Process management throughout the production stage is therefore crucial to achieve effective control over the process and consistent wine quality. Problematic wine fermentations directly impact on cellar productivity and the quality of wine. Anticipating stuck or sluggish fermentations, or simply being able to foresee the progress of a given fermentation, would be extremely useful for an enologist or winemaker, who could then take suitable corrective steps where necessary, and ensure that vinifications conclude successfully. Conventional methods of fermentation monitoring are time consuming, sometimes unreliable, and the information limited to a few parameters only. The current effectiveness of fermentation monitoring in industrial wine production can be much improved. Winemakers currently lack the tools to identify early signs of undesirable fermentation behaviour and to take preventive actions. This study investigated the application of Fourier transform mid infrared (FT-IR) spectroscopy in transmission mode, for the quantitative and qualitative monitoring of alcoholic fermentation during industrial wine production. The major research objectives were firstly to establish a portfolio of quantitative calibration models suitable for quantification of the major quality determining parameters in fermenting must. The second major research objective focused on a pilot study aimed at exploring the use of off-line batch multivariate statistical process control (MSPC) charts for actively fermenting must. This approach used FT-IR spectra only, for the purpose of qualitative monitoring of alcoholic fermentation in industrial wine production. Towards these objectives, a total of 284 industrial-scale, individual, actively fermenting tanks of the seven major white cultivars and blends, and nine major red cultivars, of Namaqua Wines, Vredendal, South Africa, were sampled and analysed with FT-IR spectroscopy and appropriate reference methods during vintages 2007 to 2009. For the quantitative strategy, partial least squares regression (PLS1) calibration models for determination of the classic wine parameters ethanol, pH, volatile acidity (VA), titratable acidity (TA) and the total content of glucose plus fructose, were redeveloped to provide a better fit to local South African samples. New PLS1 models were developed for the must components glucose, fructose and yeast assimilable nitrogen (YAN), all of which are frequently implicated in problem fermentations. The regression statistics, that included the standard error of prediction (SEP), coefficient of determination (R2) and bias, were used to evaluate the performance of the redeveloped calibration models on local South African samples. Ethanol (SEP = 0.15 %v/v, R2 = 0.999, bias = 0.04 %v/v) showed very good prediction and with a residual predictive deviation (RPD) of 30, rendered an excellent model for quantitative purposes in fermenting must. The models for pH (SEP = 0.04, R2 = 0.923, bias = -0.01) and VA (SEP = 0.07 g/L, R2 = 0.894, bias = -0.01 g/L) with RPD values of 4 and 3 respectively, showed that the models were suitable for screening purposes. The calibration model for TA (SEP = 0.35 g/L, R2 = 0.797, bias = -0.004 g/L) with a RPD of 2, proved unsatisfactory for quantification purposes, but reasonable for screening purposes. The calibration model for the total content of glucose plus fructose (SEP = 0.6.19 g/L, R2 = 0.993, bias = 0.02 g/L) with a RPD of 13, showed very good prediction and can be used to quantify total glucose plus fructose content in fermenting must. The newly developed calibration models for glucose (SEP = 4.88 g/L, R2 = 0.985, bias = -0.31 g/L) and fructose (SEP = 4.14 g/L, R2 = 0.989, bias = 0.64 g/L) with RPD values of 8 and 10 respectively, also proved fit for quantification of these important parameters. The new calibration models of ethanol, total glucose plus fructose; and glucose and fructose individually, showed an excellent relation to local South African samples and can be easily implemented by the wider wine industry. Two calibration models were developed to determine YAN in fermenting must by using different reference methods, namely the enzyme-linked spectrophotometric assay and Formol titration method, respectively. The results showed that enzyme-linked assays provided a good quantitative model for white fermenting must (SEP = 14.10 mg/L, R2 = 0.909, bias = -2.55 mg/L, RPD = 6), but the regression statistics for predicting YAN in red fermenting must, were less satisfactory (data not shown). The Formol titration method could be used successfully in both red- and white fermenting must (SEP = 16.37 mg/L, R2 = 0.912, bias = -1.01 mg/L, RPD = 4). A minor, but very important finding was made with respect to the storage of must samples that were taken from tanks, but that could not immediately be analysed with FT-IR spectroscopy or reference values. Principal component analysis (PCA) of frozen samples showed that must samples could be stored frozen for up to 3 months and still be used to expand the calibration sample sets when needed. Therefore, samples can be kept frozen to a later stage if immediate analyses are not possible. For the purpose of the pilot study that focused on the use of FT-IR spectroscopy for qualitative off-line monitoring of alcoholic fermentation, a total of 21 industrial-scale fermentation tanks were monitored at 8- or 12-hourly intervals, from the onset of fermentation to complete consumption of the grape sugars. This part of the work excluded quantitative data, and only used FT-IR spectra. MSPC charts were constructed on the PLS scores of all the FT-IR spectra taken at the various time intervals of the different batches, using time as the y-variable. The primary aim of this research objective was to evaluate if the PLS batch models could be used to discriminate between normal and problem alcoholic fermentations. The models that were constructed clearly showed the variations in patterns over time, between red- and white wine alcoholic fermentations. One Colombar tank that was fermented at very low temperature in order to achieve a specific wine style, was characterised by a fermentation pattern that clearly differed form the rest of the Colombar fermentations. This atypical fermentation was identified by the batch models constructed in this study. PLS batch models over all the Colombar fermentations clearly identified the normal and problem fermentations. The results obtained in this study showed that FT-IR spectroscopy showed great potential for effective quantitative and qualitative monitoring of alcoholic fermentation during industrial wine production. The work done in this project resulted in the development of a portfolio of calibration models for the most important quality determining parameters in fermenting must. The quantitative models were subjected to extensive independent test set validation, and have subsequently been implemented for industrial use at Namaqua Wines. Multivariate batch monitoring models were established that show good discriminatory power to detect problem fermentations. This is a very useful diagnostic tool that can be further developed by monitoring more normal and problem fermentations. Future work in this regard, will focus on further optimisation and expansion of the quantitative and qualitative calibration models and implementation of these in the respective wineries of Namaqua Wines. / AFRIKAANSE OPSOMMING: Fermentasie is ‘n komplekse proses waartydens rou material getransformeer word na produkte van hoë waarde, in hierdie geval, druiwesap na wyn. In die huidige ekonomies-kompeterende samelewing, is dit al hoe meer belangrik om volhoubaar wyn te produseer wat voldoen aan definieerbare spesifikasies en style. Goeie prosesbestuur tydens die wynproduksie stadium is baie belangrik om herhaalbaarheid en gehaltebeheer te verseker. Problematiese wynfermentasies het ’n direkte impak op beide kelderproduktiwiteit en wynkwaliteit. Die voorkoming van slepende- of steekfermentasies, of selfs net om probleme te voorsien, sou uiters bruikbaar wees vir ‘n wynkundige of wynmaker, wat dan die toepaslike regstellende stappe kan neem waar nodig, om te verseker dat die wynbereiding suksesvol voltooi word. Konvensionele metodes van monitering van alkoholiese fermentasie is tydrowend, soms onbetroubaar en die inligting beperk tot ‘n paar parameters. Die huidige effektiwiteit van fermentasie monitering in industriële wynproduksie kan heelwat verbeter word. Wynmakers ervaar tans ’n behoete aan tegnologië wat die vroeë tekens van ongunstige fermentasiepatrone kan identifiseer, en hul doeltreffendheid om moontlike regstellende aksies te neem, is dus beperk. Hierdie studie het die toepassing van Fourier transformasie mid-infrarooi (FT-IR) spektroskopie in transmissie, ondersoek met die oog op kwantitatiewe en kwalitatiewe monitering van alkoholiese gisting tydens industriële wynproduksie. Die vernaamste navorsingsdoelwitte was eerstens om ’n portefeulje van kwantitatiewe kalibrasiemodelle te vestig, wat geskik is om die belangrikste kwaliteitsbepalende parameters in gistende mos te kwantifiseer. Die tweede hoofnavorsingsdoelwit was ’n loodsstudie wat ondersoek ingestel het na die opstel van multiveranderlike statistiese proseskontrole grafieke van aktief-gistende mos, met die oog op aflyn-kwalitatiewe monitering van alkoholiese gisting in industriële wynproduksie. Hiervoor is slegs FT-IR spektra gebruik. Vir die doel van hierdie studie is monsters van ’n totaal van 284 individuele, aktief-gistende tenke van die sewe hoof wit kultivars en hul versnydings en nege hoof rooi kultivars van Namaqua Wyne, Vredendal, Suid Afrika, geneem. Al die monsters is met toepaslike chemiese metodes en FT-IR spektroskopie analiseer tydens die parsseisoene van 2007 tot 2009. Vir die kwantitatiewe strategie is parsiële kleinste kwadraat (PKK1) kalibrasiemodelle vir die bepaling van die klassieke wynparameters etanol, pH, vlugtige suur (VS), titreerbare suur (TS) en die totale konsentrasie van glukose plus fruktose herontwikkel, om beter te pas op plaaslike Suid-Afrikaanse monsters. Nuwe PKK1 kalibrasiemodelle is ontwikkel vir die komponente glukose, fruktose en gis-assimileerbare stikstof, aangesien hierdie komponente gereelde aanduidings van probleemgisting is. Die regressiestatistieke het die standaardvoorspellingsfout (SVF), bepalingskoëffisiënt (R2) en sydigheid ingesluit en was gebruik om die prestasie van die herontwikkelde kalibrasiemodelle vir plaaslike Suid-Afrikaanse monsters te evalueer. Etanol (SVF = 0.15 %v/v, R2 = 0.999, sydigheid = 0.04 %v/v) het baie goeie regressiestatistiek getoon en met ‘n relatiewe voorspellingsafwyking (RVA) van 30, was dit ‘n uitstekende model vir kwantifisering in gistende mos. Die modelle vir pH en VS met RVA waardes van 4 en 3 onderskeidelik, is geskik vir semi-kwantitatiewe toepassings. Die kalibrasiemodel vir TS met ‘n RVA waarde van 2, was nie geskik vir akkurate kwantifisering nie, maar wel vir semikwantitatiewe analises. Die kalibrasiemodel vir die totale glukose plus fruktose inhoud in gistende mos, met ‘n RVA waarde van 13, het uitstekende regressiestatistiek gegee en is geskik vir akkurate kwantifiseringsdoeleindes. Die nuut-ontwikkelde kalibrasiemodelle vir glukose en fruktose, met RVA waardes van onderskeidelik 8 en 10, is geskik vir akkurate kwantifisering van hierdie belangrike parameters. Die kalibrasiemodelle vir etanol, totale glukose plus fruktose, en glukose en fruktose afsonderlik, het uitstekende korrelasies getoon met plaaslike Suid-Afrikaanse monsters en is gereed om toepassing te vind in die wyer wynindustrie. Twee kalibrasiemodelle is ontwikkel om gis-assimileerbare stikstof in gistende mos te bepaal, deur gebruik te maak van verskillende verwysingsmetodes van analise; hierdie metodes was ‘n ensiem-gekoppelde spektrofotometriese toets en die Formoltitrasie metode. Resultate het getoon dat goeie regressiestatistiek vir FT-IR spektroskopie-gebaseerde kalibrasiemodelle waar data wat met die ensiem-gekoppelde toetse verkry is, as verwysingwaardes gebruik is, in wit gistende mos (SVP = 14.10 mg/L, R2 = 0.909, sydigheid = -2.55 mg/L, RVA = 6), maar nie in rooi gistende mos nie. Die Formoltitrasie metode as verwysingsmetode, was geskik vir die ontwikkeling van goeie kalibrasiemodelle in beide rooi- en wit gistende mos (SVP = 16.37 mg/L, R2 = 0.912, sydigheid = -1.01 mg/L, RVA = 4). ’n Sekondêre, maar baie belangrike bevinding is gemaak met betrekking tot die stoor van mosmonsters wat geneem is van tenke, maar wat nie dadelik met die verwysingsmetodes en FT-IR spektroskopie analiseer kon word nie. Multiveranderlike hoofkomponentanalise op vars en gevriesde sapmonsters het getoon dat gevriesde monsters gebruik kan word om die kalibrasie datastel uit te brei, wanneer benodig. Dus, sapmonsters kan gevries word tot ’n later stadium as onmiddelike analises nie moontlik is nie. Vir die doel van die tweede navorsingsdoelwit van die studie, naamlik kwalitatiewe af-lyn monitering van alkoholiese fermentasie met FT-IR spektroskopie, is ‘n totaal van 21 industriëlegrootte fermentasietenks ge-monitor deur sapmonsters met 8- tot 12-uurlikse intervalle te trek, vanaf die begin van fermentasie, totdat al die druifsuiker gemetaboliseer is. Vir hierdie deel van die werk is die kwantitatiewe data nie gebruik nie; slegs die FT-IR spektra. Multiveranderlike statistiese proseskontrole grafieke is opgestel op grond van die PKK tellings wat bereken is op al die FT-IR spektra wat gemeet is by die verskillende tydsintervalle. Vir hierdie analise is tyd as y-veranderlike gebruik. Die vernaamste doel van hierdie ondersoek was om te evalueer of die PKK-gebaseerde modelle kon onderskei tussen normale en slepende gistings. Die modelle wat verkry is, het die variasie oor tyd in die fermentasiepatrone tussen wit- en rooiwyn fermentasies tydens alkoholiese gisting, duidelik uitgewys. Een Colombar tenk wat teen baie lae temperatuur gefermenteer is om ‘n spesifieke wynstyl te verkry, se fermentasiepatroon het aansienlik verskil van die ander Colombar tenks wat gemonitor is, en hierdie atipiese patroon is ook deur die kwalitatiewe modelle identifiseer. ‘n PKK model oor al die Colombar fermentasies kon duidelik tussen normale en slepende gistings onderskei. Die resultate wat in hierdie studie verkry is, het getoon dat FT-IR spektroskopie baie goeie potensiaal toon vir die aanwending van kwantitatiewe en kwalitatiewe monitering van alkoholiese fermentasie tydens industriële wynproduksie. Die werk wat in hierdie projek gedoen is, het gelei tot die vestiging van ‘n portefeulje van kalibrasiemodelle vir die belangrikste kwaliteitsbepalende parameters in fermenterende mos. Die kwantitatiewe modelle is baie deeglik getoets met onafhanlike toets datastelle, en daarna is die kalibrasiemodelle geimplementeer vir industriële gebruik by Namaqua Wyne. Multiveranderlike statistiese proseskontrole grafieke wat baseer is op data wat vanaf 21 verskillende fermentasietenks verkry is, het baie goeie potensiaal getoon om probleemfermentasies vroeg te identifiseer. Dié grafieke is ‘n baie nuttige diagnostiese hulpmiddel wat verder ontwikkel kan word om verskillende tipes probleemfermentasies te monitor. Toekomstige navorsing in hierdie konteks, sal toegespits word op die optimisering en uitbreiding van die kwantitatiewe en kwalitatiewe modelle, sowel as toepassing van die tegnieke in die onderskeie kelders van Namaqua Wyne.

Thesis (MScAgric)--Stellenbosch University, 1948. / NO ABSTRACT AVAILABLE