Thesis (PhD(Agric) Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009. / The main goal of this project was to elucidate the underlying genetic factors responsible for the different fermentation phenotypes and physiological adaptations of industrial wine yeast strains. To address this problem an ‘omic’ approach was pursued: Five industrial wine yeast strains, namely VIN13, EC1118, BM45, 285 and DV10, were subjected to transcriptional, proteomic and exometabolomic profiling during alcoholic fermentation in simulated wine-making conditions. The aim was to evaluate and integrate the various layers of data in order to obtain a clearer picture of the genetic regulation and metabolism of wine yeast strains under anaerobic fermentative conditions. The five strains were also characterized in terms of their adhesion/flocculation phenotypes, tolerance to various stresses and survival under conditions of nutrient starvation. Transcriptional profiles for the entire yeast genome were obtained for three crucial stages during fermentation, namely the exponential growth phase (day 2), early stationary phase (day 5) and late stationary phase (day 14). Analysis of changes in gene expression profiles during the course of fermentation provided valuable insights into the genetic changes that occur as the yeast adapt to changing conditions during fermentation. Comparison of differentially expressed transcripts between strains also enabled the identification of genetic factors responsible for differences in the metabolism of these strains, and paved the way for genetic engineering of strains with directed modifications in key areas. In particular, the integration of exo-metabolite profiles and gene expression data for the strains enabled the construction of statistical models with a strong predictive capability which was validated experimentally. Proteomic analysis enabled correlations to be made between relative transcript abundance and protein levels for approximately 450 gene and protein pairs per analysis. The alignment of transcriptome and proteome data was very accurate for interstrain comparisons. For intrastrain comparisons, there was almost no correlation between trends in protein and transcript levels, except in certain functional categories such as metabolism. The data also provide interesting insights into molecular evolutionary mechanisms that underlie the phenotypic diversity of wine yeast strains. Overall, the systems biology approach to the study of yeast metabolism during alcoholic fermentation opened up new avenues for hypothesis-driven research and targeted engineering strategies for the genetic enhancement/ modification of wine yeast for commercial applications.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: In the present-day competitive global market, wine industries are constantly aiming to improve the wine-making process,including the role of yeast. The most commonly used wine yeast is Saccharomyces cerevisiae, which is able to produce high quality wines, but problem fermentations do sometimes arise. The occurrence of stuck and sluggish fermentations pose a serious problem leading to loss of productivity and quality. Although the precise mechanism leading to stuck fermentations is unknown, they are often correlated with high fructose to glucose ratios in the wine-must. S. cerevisiae is a glucophylic yeast, indicating its preference for consuming glucose over fructose. Both these hexose sugars are present in unfermented wine must, mostly in equal concentrations. As fermentation progresses, glucose is consumed at a faster rate than fructose, leading to an increase in the fructose to glucose ratio. Yeast are left with the undesirable fructose at the later stages of fermentation, when the environmental stresses on the yeast can lead to stuck or sluggish fermentation. This residual fructose can lead to undesirable sweetness, as fructose is about twice as sweet as glucose. Even with the extensive research into yeast metabolism, there is as yet no definitive explanation as to why yeasts ferment glucose faster than fructose. This study aimed to investigate the mechanism responsible for the faster consumption of glucose over fructose of a commercially used wine yeast strain S. cerevisiae VIN 13. The first two steps of sugar metabolism, uptake and phosphorylation, were investigated as the possible sites of discrepancy in fermentation rates. Enzyme rates and affinities for both glucose and fructose as substrates for the relevant enzymes were experimentally determined. These kinetic parameter values were used to improve an existing model of yeast glycolytic pathway to model wine fermentations. The feasibility of constructing and validating a kinetic model of wine fermentations were investigated, by comparing model predicted fluxes with experimentally determined fluxes. Another aspect of this study was an investigation into the effect of hexose sugar type on fermentation profiles. Wine fermentations were done with only one hexose sugar as carbon source to determine if it has an effect on the flux through metabolism. This work succeeded in the construction of a kinetic model that distinguished between glucose and fructose as carbon source. The glucose was consumed faster than fructose, with control lying in the hexose transport step. It was also established that fermentation prfiles of fermentations with only one sugar was the same for both one sugar type fermentations. Fermentation with either glucose or fructose as the sole carbohydrate source had the same specfic production and consumption rates as normal fermentations with both sugars. Construction of detailed kinetic models can aid in the metabolic and cellular engineering of novel yeast strains. By identifying the importance of hexose transport, and thus the glucophilic character of the yeast, in flux control, yeast transporters can be targeted for strain improvement. This may in turn lead to more effective fermentation practices for controlling problem fermentations, or to the development of novel strains that utilizes fructose in the same manner as glucose, and in so doing lower the risk of stuck or sluggish wine fermentation. / AFRIKAANSE OPSOMMING: In die hedendaagse kompeterende wynmark is wynmakers aanhoudend besig om die wynmaak proses te verbeter en dit sluit die verbetering van wyngis in. Die mees algemeenste gebruikte wyngis is Saccharomyces cerevisiae, omdat dit wyn van gehalte produseer, maar probleem fermentasies kom wel voor. Die verskynsel van vasval of stadige fermentasies kan lei tot die verlies van produksie en kwaliteit. Die oorsaak van probleem fermentasies is gewoontlik veelvoudig, maar die verhouding van glukose tot fruktose in die wyn-mos kan ongunstig raak om fermentasies te onderhou. S. cerevisiae is 'n glukofiliese gis, wat sy voorkeur om glukose bo fruktose te gebruik beskryf. Albei hierdie heksose suikers is teenwoordig in ongefermenteerde wyn-mos, meestal in gelyke hoeveelhede. Soos fermentasies vorder word glukose vinniger verbruik as fruktose wat lei tot 'n toename in die fruktose tot glukose verhouding. Die gis moet dus die fruktose in die later stadium van fermentasie gebruik wanneer die omgewings druk op die gis kan lei tot probleem fermentasies. Die oorblywende fruktose kan lei tot ongewenste soetheid aangesien fruktose twee keer soeter is as glukose. Selfs met die ekstensiewe navorsing met betrekking tot gis metabolisme is daar nog nie 'n verduideliking hoekom gis glukose vinniger as fruktose gebruik nie. Hierdie studie het beoog om die meganisme wat lei tot die vinniger verbruik van glukose oor fruktose te ondersoek vir 'n kommersieël gebruikte gis S. cerevisiae VIN 13. Die eerste twee stappe van suiker metabolisme, suiker opname en fosforilasie, was ondersoek as die moontlike punt van die verskil in fermentasie tempo. Ensiem snelhede en affiniteite vir beide glukose en fruktose as substrate vir die ensieme van belang was eksperimenteel bepaal. Hierdie waardes is gebruik om 'n bestaande model van gis glikolise aan te pas vir wyn fermentasies. Die uitvoerbaarheid van saamstel en valideer van 'n kinetiese model van wyn fermentasies was ondersoek, deur model voorspelde fluksie waardes met eksperimentele fluksie waardes te vergelyk. 'n Ander aspek van die studie was die ondersoek van die effek van heksose suiker tipe op fermentasie profiel. Wyn fermentasies is gedoen met slegs een heksose suiker as koolstof bron om te bepaal of dit 'n invloed het op die fluksie deur metabolisme. Hierdie werk het daarin geslaag om 'n kinetiese model saamtestel wat onderskei tussen glukose en fruktose as koolstof bron. Die glukose is vinniger verbruik as fruktose, met beheer gesetel in die heksose opname stap. Dit was ook vasgestel dat fermentasie profiele van fermentasies met slegs een suiker nie verskil het vir fermentasies met slegs fruktose of glukose. Fermentasies met slegs een suiker het dieselfde spesifieke produksie en konsumpsie tempo gehad as die normale fermentasie met albei suikers. Die konstruksie van 'n gedetailleerde kinetiese model kan gebruik word in die metaboliese en sellulêre ontwikkeling van nuwe gisstamme. Deur die ontdekking van die belangrikheid van heksose opname in fluksie beheer, wat lei tot die glukofiliese karakter van gis, kan gis opname geteiken word vir gis ontwikkeling. Dit mag om die beurt lei tot meer effektiewe fermentasie praktyk in die beheer van probleem fermentasies, of die ontwikkeling van nuwe stamme wat fruktose in dieselfde manier as glukose benut, en sodoende die risiko van vasval of stadige wyn fermentasies verlaag. / National Research Foundation / Post-graduate Merit Bursary

Thesis (MSc)--Stellenbosch University, 1959. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (MSc (Chemistry and Polymer Science))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Wine quality is compromised by the presence of 3-hydroxypropionaldehyde (3-HPA) due to spontaneous conversion into acrolein under wine making conditions. Acrolein is highly toxic and is presence has been correlated with the development of bitterness in wine. Lactic acid bacterial strains isolated from South African red wine, Lactobacillus pentosus and Lactobacillus brevis, are implicated in accumulating 3- HPA during anaerobic glycerol fermentation. The environmental conditions leading to its accumulation are elucidated. In aqueous solution 3-HPA undergoes reversible dimerization and hydration, resulting in an equilibrium state between different derivatives. Interconversion between 3-HPA derivatives and acrolein is a complex and highly dynamic process driven by hydration and dehydration reactions. Acrolein is furthermore highly reactive and its steady-state concentration in complex systems very low. As a result analytical detection and quantification in solution is problematic. This study highlights the roles played by natural chemical derivatives and shows that the acrolein dimer can be used as a marker for indicating the presence of acrolein in wines. Solid-phase microextraction (SPME) coupled to gas chromatograph mass spectrometry (GC-MS) was validated as a technique for direct detection of the acrolein dimer in wine. The potential of a recently introduced sorptive extractive technique with a sample enrichment probe (SEP) was also investigated. The SPME technique simplifies the detection process and allows for rapid sampling of the acrolein marker, while SEP is more sensitive. / AFRIKAANSE OPSOMMING: Die teenwoodigheid van 3-hidroksiepropioonaldehied (3-HPA) in wyn het ‘n negatiewe invloed op kwaliteit as gevolg van die moontlike omskakeling na akroleien tydens die wynmaak prosses. Akroleien is hoog toksies en is moontlik betrokke by die ontwikkeling van ‘n bitter komponent in wyn. Hierdie studie wys dat stamme van die melksuurbakteriëe Lactobacillus pentosus en Lactobacillus brevis, geisoleer uit Suid-Afrikaanse wyn, 3-HPA tydens anaerobiese alkoholiese fermentasie kan opbou. Kondisies wat ontwikkeling beinvloed is bestudeer. 3-HPA ondergaan omkeerbare dimerisasie en hidrasie in oplossing en het ‘n ewewig tussen veskillende derivate tot gevolg. Omkakeling tussen 3-HPA derivate en akroleien is ‘n komplekse en hoogs dinamiese prosses wat gedryf word deur hidrasie en dehidrasie reaksies. Akroleien is verder hoogs reaktief en die ewewigskonsentrasie van hierdie aldehied in komplekse omgewings is laag. Analitiese waarneming en kwantifisering is gevolglik problematies. Hierdie studie lig die rol wat natuurlike chemise derivate speel duidelik uit en wys dat die akroleien dimeer as ‘n merker gebruik kan word om die teenwoodigeid van akoleien in wyn te staaf. Soliede-fase mikro-ekstraksie (SPME) gekoppel aan gas chromatografie massa spektroskopie (GC-MS) is gevalideer as ‘n tegniek vir die direkte waarneming van die akroleien dimeer in wyn. Die potensiaal van ‘n nuwe ekstraksie tegniek, gebasseer op ‘n peiler wat vir die monster verreik (SEP), was ook ondersoek. Die SPME tegniek is vinnig en vergemaklik analiese, terwyl SEP meer sensitief is.

Thesis (MSc)--Stellenbosch University, 2002. / Full text to be digitised and attached to bibliographic record. / ENGLISH ABSTRACT: The winemaking process constitutes a unique ecological niche that involves the interaction of yeasts, lactic acid bacteria and acetic acid bacteria. Saccharomyces cerevisiae has established its importance as a wine yeast and also proven itself as a reliable starter culture organism. Its primary role is to convert the grape sugar into alcohol and, secondly, its metabolic activities result in the production of higher alcohols, fatty acids and esters, which are important flavour and aroma compounds that are essential for consistent and predictable wine quality. There is a growing consumer demand for wine containing lower levels of alcohol and chemical preservatives. Glucose oxidase (GOX) has received considerable research interest regarding its potential application in the wine industry to reduce alcohol levels and as a biocontrol agent. Several physical processes are used for the removal or reduction of alcohol in wine and some of them are sometimes used in combination. These processes tend to involve expensive equipment and can be intensive from a processing point of view. An alternative approach was introduced with the concept of treating grape must with GOX to reduce the glucose content of the must, and therefore produce a wine with a reduced alcohol content after fermentation. Due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing quest for specialised wine yeast strains possessing a wide range of optimised, improved or novel oenological properties. The first and main objective of this study was to genetically engineer wine yeasts to produce wine with a reduced alcohol content. In order to do this, the structural glucose oxidase (gox) gene of Aspergillus niger was cloned into an integration vector (Ylp5) containing the yeast mating pheromone a-factor secretion signal (MFa1 s) and the phosphoglycerate kinase 1 gene promoter and terminator (PGK1PT). This PGK1p-MFa1sgox- PGKh gene cassette (designated GOX1) was introduced into a laboratory strain of S. cerevisiae (~1278). Results obtained indicated the production of biologically active glucose oxidase and showed that it is secreted into the culture medium. This would mean that the enzyme will convert the glucose to gluconic acid in the medium before the yeast cells are able to metabolise the glucose to ethanol. Microvinifications performed with Chardonnay grapes showed that the laboratory yeast starter cultures transformed with GOX1 were indeed able to reduce the total amount of alcohol in the finished product. The second objective of this study involved the potential application of GOX as a biocontrol agent. Screening was performed for wine spoilage microorganisms, such as acetic acid bacteria and lactic acid bacteria, using plate assays. The wine spoilage microorganisms tested formed different sized inhibition zones, indicating varying degrees of inhibition. The inhibition of some of the wine spoilage microorganisms was confirmed under a scanning electron microscope. The total collapse of the bacterial cell wáll could be seen and might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide (H202). The produced H202 leads to hyperbaric oxygen toxicity, a result of the peroxidation of the membrane lipid, and a strong oxidising effect on the bacterial cell, which is the cause of the destruction of basic molecular structures, such as nucleic acids and cell proteins. In this exciting age of molecular yeast genetics and modern biotechnology, this study could pave the way for the development of wine yeast starter culture strains for the production of wine with a lower alcohol content and reduced levels of chemical preservatives, such as sulphur dioxide. The use of genetically modified organisms (GMOs) within the wine industry is a limiting factor at present and credible means must be found to effectively address the concerns of traditionalists within the wine industry and the negative overreaction by some consumer groups. There is a vast potential benefit to the wine consumer and industry alike and the first recombinant wine products therefore should unmistakably demonstrate safe products free of potentially harmful compounds, and have organoleptic, hygienic and economic advantages for both the wine producer and consumer. / AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n ekologiese interaksie tussen gis, asynsuurbakterieë en melksuurbakterieë. Saccharomyces cerevisiae het homself alreeds bewys as 'n belangrike en betroubare inisiëringsgis in wyn. Die hoofdoel van die gis is om druifsuikers na etanol om te skakel. Tweedens lei die gis se metaboliese aktiwiteite tot die produksie van hoër alkohole, vetsure en esters, wat tot die konsekwente voorspelbare smaak en aromaverbindings in herhaalbare kwaliteit wyn bydra. Daar is 'n toenemende aanvraag na wyne met 'n laer alkoholinhoud en minder preserveermiddels. Glukoseoksidase (GOX) het heelwat navorsing in die wynindustrie uitgelok omdat dit gebruik kan word om die alkoholinhoud in wyn te verlaag, asook as 'n biologiese beheermiddel kan funksioneer. Daar is reeds sekere fisiese prosesse wat gebruik kan word om die alkohol in wyn te verwyder of te verminder. Sommige van hierdie prosesse word soms in kombinasie gebruik. Die nadeel is egter dat hierdie prosesse baie duur en intensief is, veral ten opsigte van prosessering. 'n Alternatief om die alkoholinhoud van wyn te verlaag, het egter na vore gekom toe daar voorgestel is om die mos met GOX te behandel. As gevolg van die veeleisende aard van moderne wynmaakpraktyke en gesofistikeerde wynmarkte, is daar 'n nimmereindigende soektog na meer gespesialiseerde wyngisrasse wat 'n wye reeks van geoptimiseerde en verbeterde, en selfs unieke, wynkundige einskappe bevat. Die hoofdoelwit van hierdie navorsingsprojek behels die genetiese manipulasie van 'n gisras sodat dit in staat is om wyn met 'n laer alkoholinhoud te produseer. Om hierdie doel te verwesentlik, is die strukturele glukoseoksidasegeen (gox) van Aspergillus niger in 'n integreringsvektor gekloneer. Transkripsie-inisiëring en -terminering is deur fosfogliseraatkinase-1-promotor en -termineerder (PGK1PT) bewerkstellig. Die a-spesifieke gisferomoon-a-faktor (MFa1 s) is gebruik om die uitskeiding van GOX uit die gis te bewerkstellig. Saam vorm bogenoemde die PGK1p-MFals-gox-PGKh-geenkasset, wat as GOX1 bekend is. GOX1 is na 'n labaratoriumras van S. cerevisiae (:E1278) getransformeer. Resultate dui aan dat biologies aktiewe GOX geproduseer en uitgeskei word. Dit beteken dat van die glukose in die medium reeds na glukoonsuur omgesit sal word voordat die gis dit kan begin benut en alkohol produseer. Kleinskaalse wynmaakprosesse wat met Chardonnay-druiwe en GOX-produserende labaratoriumgis uitgevoer is, het inderdaad tot laer alkoholpersentasies gelei. Die tweede doelwit van die navorsingsprojek was om te bepaal of GOX die potensiaal as biologiese beheermiddel het. Daar is ondersoek ingestel na sekere wynbederfsorganismes soos asynsuur- en melksuurbakterieë en die inhibisie van die organismes is op agarplate gemonitor. Verskillende grade van inhibisie, soos die grootte van die inhibisiesone, was sigbaar vir die verskillende wynbederfsorganismes wat getoets is. Die inhibiese van sekere wynbederfsorganismes is ook met behulp van 'n skandeerelektronmikroskoop bevestig. Die totale ineenstorting van die bakteriële selwand was sigbaar en kan verklaar word deur die teenwoordigheid van waterstofperoksied (H202). Laasgenoemde is 'n byproduk van die laaste metaboliese reaksie en staan as 'n antimikrobiese middel bekend. Die byproduk (H202) gee aanleiding tot hiperbariese suurstoftoksisiteit, 'n gevolg van die peroksidasie van membraanlipiede en 'n sterk oksiderende effek t.o.v. die bakteriële selwand. Dit lei tot die vernietiging van die basiese molekulêre strukture, soos die nukleïensure en selproteïene. Tydens hierdie opwindende era van molekulêre gisgenetika en biotegnologie kan hierdie navorsing die fondament lê vir die ontwikkeling van 'n wyngiskultuur wat in staat is om wyn met 'n laer alkoholinhoud te produseer. Die gebruik van geneties gemanupileerde organismes (GMO's) in die wynbedryf is egter nog 'n beperkende faktor. 'n Geloofwaardige manier moet dus gevind word om die bekommernisse van tradisionaliste, asook die negatiewe oorreaksies van sommige verbruikers, aan te spreek en hok te slaan. Daar is groot potensiaal en voordele vir beide die verbruiker en industrie. Dit is dus belangrik dat die eerste rekombinante wynprodukte wat die mark betree, veilig en vry van potensieel skadelike verbindings is, asook organoleptiese, higiëniese en ekonomiese voordele toon te opsigte van beide die wynprodusent en gebruiker.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Spoilage, be it due to microbial actions, chemical reactions or both, poses a serious threat to the food and beverage industries. Not only can spoilage lead to great economic losses, but it can also cause industries to lose their competitive edge in the economic and consumer market. Considering all the modern technologies and the range of preservation techniques that are available, it is surprising that spoilage is still an economic problem. Wine spoilage due to unpalatable bitterness, and the role of lactic acid bacteria (LAB) in causing this bitterness, have received much attention over the years, but no definite understanding has yet emerged. The first objective of this study was to isolate, enumerate and identify the LAB from three red grape varieties, viz. Pinotage, Merlot and Cabernet Sauvignon. The LAB populations on the grapes of all three varieties ranged from 102 to 104 cfu/ml during the 2001 and 2002 harvest seasons. The Cabernet Sauvignon grapes had slightly higher numbers than the Pinotage and Merlot. The LAB population in the Cabernet Sauvignon, Pinotage and Merlot wines after completion of the alcoholic fermentation ranged from 102 to 105 cfu/ml, while during 2002 the numbers in wine undergoing malolactic fermentation (MLF) ranged from 104 to 108 cfu/ml. The isolated LAB were divided into the three metabolic groups, with 59% belonging to the facultatively heterofermentative group, 26% to the obligately heterofermentative group and 15% to the obligately homofermentative group. The isolates were identified by means of species-specific primers as Leuconostoc mesenteroides (4), Oenococcus oeni (28), Lactobacillus brevis (15), Lb. hilgardii (15), Lb. plantarum (98), Lb. pentosus (12), Lb. paraplantarum (3), Lb. paracasei (28), Pediococcus acidilactici (2) and Pediococcus spp. (35). The most predominant species isolated was Lb. plantarum, followed by Pediococcus spp. The results suggest that Pinotage carries a more diverse LAB population in comparison to Merlot and Cabernet Sauvignon. The second objective of this study was to determine the presence of the glycerol dehydratase gene in the LAB strains by using the G01 and G02 primers. Twenty-six strains tested positive, namely Lb. plantarum (15), Lb. pentosus (1), Lb. hilgardii (5), Lb. paracasei (2), Lb. brevis (2) and a Pediococcus spp. (1). Interestingly, 62% of these strains were isolated from Pinotage. The strains all had the ability to degrade glycerol by more than 90%, and no significant differences were observed between the species. The GO-possessing strains exhibited varying degrees of inhibition towards Gram-positive and Gram-negative bacteria, and the results suggest that this inhibition activity may be similar to that of reuterin, which is produced by Lb. reuteri. This study can form the foundation for unravelling the causes of bitterness in red wines. Combining the results of this study with analytical, sensory and molecular data may very well provide the industry with valuable tools with which to combat the occurrence of bitterness. / AFRIKAANSE OPSOMMING: Bederf as gevolg van mikrobiese aksies, chemiese reaksies of beide, hou 'n groot bedreiging vir die voedsel- en drankbedrywe in. Nie net kan bederf lei tot groot ekonomiese verliese nie, maar dit kan ook veroorsaak dat bedrywe hul kompeterende voordeel in die ekonomiese en verbruikersmarkte verloor. As die moderne tegnologie en die reeks preserveringstegnieke wat beskikbaar is, in ag geneem word, is dit verbasend dat bederf steeds 'n ekonomiese probleem is. Wynbederf as gevolg van oormatige bitterheid en die rol van melksuurbakterieë (MSB) in die ontwikkeling van hierdie bitterheid het oor die jare heen baie aandag geniet, maar geen definitiewe verklaring is nog daarvoor gevind nie. Die eerste doelwit van hierdie studie was om MSB vanaf drie rooidruifvariëteite, nl. Pinotage, Merlot en Cabernet Sauvignon, te isoleer, te kwantifiseer en te identifiseer. Die MSB-populasies op die druiwe van al drie variëteite het gedurende die 2001- en 2002-parsseisoene tussen 102 en 104 kvu/ml gevarieer. Die Cabernet Sauvignon-druiwe het effens hoër getalle as die Pinotage- en Merlot-druiwe gehad. Die MSB-populasies in die Cabernet Sauvignon-, Pinotage- en Merlot-wyne aan die einde van die alkoholiese fermentasie het tussen 102 en 1055 kvu/ml gevarieer. Gedurende 2002 het die MSB-getalle in die wyne waarin appelmelksuurgisting (AMG) aan die gang was tussen 104 en 108 kvu/ml gevarieer. Die geïsoleerde MSB was onderverdeel in die drie metaboliese groepe, met 59% wat behoort aan die fakultatiewe, heterofermentatiewe groep, 26% aan die obligate, heterofermentatiewe groep en 15% aan die obligate, homofermentatiewe groep. Die isolate is geïdentifiseer as Leuconostoc mesenteroides (4), Oenococcus oeni (28), Lactobacillus brevis (15), Lactobacillus hi/gardii (15, Lactobacillus p/antarum (98), Lactobacillus pentosus (12), Lactobacillus parap/antarum (3), Lactobacillus paracasei (28), Pediococcus acidi/actici (2) en Pediococcus spp. (35) deur middel van spes iespesifieke inleiers. Die mees algemeen geïsoleerde spesies was Lb. p/antarum, gevolg deur Pediococcus spp. Die resultate impliseer dat Pinotage 'n meer uiteenlopende MSB-populasie in vergelyking met Merlot en Cabernet Sauvignon dra. Die tweede doelwit van hierdie studie was om die teenwoordigheid van die gliseroldehidratase-geen in die MSB-isolate deur middel van die GD1- en GD2- inleiers te bepaal. Ses-en-twintig isolate was positief, nl. Lb. p/antarum (15), Lb. pentosus (1), Lb. hi/gard;; (5), Lb. paracasei (2), Lb. brevis (2) en 'n Pediococcus spp. (1). 'n Interessante resultaat was dat 62% van hierdie isolate vanaf Pinotage geïsoleer is. Die isolate was almal in staat om meer as 90% van die gliserol te gebruik en geen noemenswaardige verskille is tussen die isolate waargeneem nie. Die GD-bevattende isolate het verskillende grade van inhibisie teenoor Grampositiewe en Gram-negatiewe bakterieë getoon, en die resultate impliseer dat hierdie inhiberende aktiwiteit dieselfde is as dié van reuterin wat deur Lb. reuteri geproduseer word. Hierdie studie kan dus die basis vorm vir die ontrafeling van die oorsake van bitterheid in rooiwyne. Deur die resultate van hierdie studie met analitiese, sensoriese en molekulêre data te kombineer, kan die wynbedryf voorsien word van waardevolle metodes om die voorkoms van bitterheid mee te bekamp.

Thesis (MSc)--Stellenbosch University, 2003. / Full text to be digitised and attached to bibliographic record. / ENGLISH ABSTRACT: Traditionally, wine has been produced by the spontaneous fermentation of grape juice by yeast that originate from the grapes and winery equipment. Research has shown that the population composition and dynamics of these yeasts and other microorganisms are very complex. Kloeckera and its anamorph, Hanseniaspora, dominate the yeast population found on the surfaces of grapes, although prevailing Saccharomyces cerevisiae strains complete the fermentation process. The yeast S. cerevisiae is an important factor contributing to the quality of wines and, therefore, the improvement of wine yeasts receives considerable attention worldwide. Apart from classical yeast breeding studies, genetic engineering and recombinant DNA techniques are increasingly being used in strain development research programmes. These techniques might enable the wine yeasts to produce heterologous enzymes that degrade polysaccharides, convert malic acid to lactic acid, increase glycerol production, release roam and flavour compounds, secrete antimicrobial peptides, etc. The release of recombinant yeast strains (genetically modified organisms, GMOs) is subject to statutory approval. Therefore, it is important to answer several questions prior to the use of such genetically improved yeast in the commercial production of wine. For example, will recombinant yeast strains be able to multiply and spread in nature, and will this GMO be able to out-compete the natural microflora because of its newly acquired genetic traits. Since existing commercial wine yeasts are used in the abovementioned strain development research, it is essential to determine already at this early stage to what extent these wine yeast strains survive and spread in nature and to what extent they influence the fermentations of the following vintages. This study is divided into two sections. The aim of the first section is to sample a representative number of yeast strains from various vineyards in different climatological areas, mainly in the Western Cape, South Africa. These yeast strains were identified mainly by electrophoretic karyotyping (contour-clamped homogenous electric field electrophoresis; CHEF). The second part of the study summarises the results obtained when Fourier transform infrared (FT-NIR) spectroscopy was used to differentiate commercial wine yeast strains. Sets of data, containing the spectra of the mostly used commercial wine yeast strains, were constructed and used as a reference library. The spectra of the isolated yeast strains were then compared to the reference dataset with specific FT-NIR computer software using mathematical calculations. In conclusion, the two methods used in conjunction with one another proved that the commercial wine yeast strains do not easily disperse from the cellar into the vineyard. The commercial wine yeast strains are also more likely to be found near the cellar and the places where the grape skins are dumped. Therefore, should a recombinant yeast strain be used in winemaking, it would not be dispersed into the vineyard. It therefore appears that the commercial use of genetically improved yeast does not pose a high risk in terms of dominance of the indigenous microbial population in the environment / AFRIKAANSE OPSOMMING: Wyn is tradisioneel gemaak deur die natuurlike gisting van druiwesap deur giste wat op die druiwe en keldertoerusting voorkom. Navorsing het getoon dat die samestelling en dinamika van die gispopulasie en ander mikro-organismes baie kompleks is. Kloeckera en sy anamorf, Hanseniaspora, domineer die inheemse gispopulasie op druiwedoppe, terwyl Saccharomyces cerevisiae in baie klein getalle op die druiwedoppe voorkom, maar later die fermentasie oorheers en uiteindelik voltooi. Die gis S. cerevisiae speel 'n baie belangrike rol in die kwaliteit van wyn en daarom geniet die verbetering van wyngiste wêreldwyd besondere aandag. Benewens die klassieke gistelingstudies, word genetiese manipuleringstegnieke toenemnd in navorsingsprojekte gebruik wat daarop gefokus is om wyngisrasse te verbeter. Hierdie tegnieke mag die giste in staat stelom heteroloë ensieme te produseer wat polisakkariedes afbreek, appelmelksuur afbreek, gliserolproduksie verhoog, smaak- en geurkomponente vrystel, antimikrobiese peptiede afskei, ens. Voordat sulke geneties gemanipuleerde giste het egter in kommersiële wynproduksie gebruik sal kan word, is daar heelwat wetlike vereistes waaraan voldoen sal moet word en vrae wat vooraf beantwoord sal moet word. Byvoorbeeld, sal die rekombinante giste in staat wees om vinniger te vermeerder as gevolg van die nuwe genetiese eienskappe en sodoende die natuurlike populasies onderdruk? Omdat kommersiële wyngiste in bogenoemde gisverbeteringprogramme gebruik word, is dit noodsaaklik om nou reeds die verspreiding van die kommersiële giste te monitor en te bepaal hoe geredelik hulle in die natuur kan versprei en oorleef, en hoe hulle wynfermentasies van die daaropvolgende jare beïnvloed. Die studie is in twee gedeeltes verdeel. Die doel van die eerste gedeelte was om 'n verteenwoordigende aantal gisrasse uit die wingerde van 'n aantal wynplase in verskillende klimaatstreke te isoleer, spesifiek in die Wes-Kaap, Suid-Afrika. Die gisrasse was grotendeels deur elektroforetiese kariotipering (kontoer-geklampte homogene elektriese veld; CHEF) geïdentifiseer. Die tweede deel van die navorsing was gefokus op die onderskeiding tussen die mees gebruikte kommersiële wyngiste met 'Fourier-Transform Near Infrared' (FTNIR) spektroskopie. Eerstens is 'n stel data, bestaande uit die spektrum data oor die kommersiële wyngiste opgestel om as 'n verwysingsbiblioteek te dien. Tweedens is die spektrum van data oor die geïsoleerde giste onder presies dieselfde toestande met die verwysingsbiblioteek vergelyk. Dié tegniek maak dit moontlik om tussen die kommersiële wyngiste te onderskei. As die twee metodes saam gebruik word vir identifikasie, kan die afleiding gemaak word dat kommersiële wyngiste nie maklik vanaf die kelder na die wingerd versprei nie. Die kommersiële wyngiste is ook meestal naby die kelder en die dopstortingsterreine gevind. Sou 'n rekombinante gisras dus gebruik word om wyn te maak, sal dit nie maklik versprei nie. Die kommersiële gebruik van geneties gemanipuleerde wyngiste behoort dus nie In groot omgewingsrisiko in te hou nie.

Thesis (MSc)--Stellenbosch University, 2003. / Full text to be digitised and attached to bibliographic record. / ENGLISH ABSTRACT: Winemaking is an age-old tradition that dates back to as early as 6000 BC. In our modern era there are several insects and microorganisms that pose a threat to the grapevine, the environment and the final wine product. Farmers and winemakers are becoming aware of the threat and the fight against disease, spoilage and/or pathogenic microorganisms is on the rise. Currently, the natural environment is being altered through rural developments, pollution and disaster, which in turn is responsible for altering the natural micro flora. The result is a harsh battle between man and microorganism. The weapon used often against microorganisms is chemical preservatives, such as sulphur dioxide. These chemical preservatives change the nutritional value, quality and wholesomeness of the wine. Chemical preservatives suppress the quality of the wine with a reduction in wine consumption by the consumers. Until the 18th century, wine was regarded as a safe drink and prescribed by doctors. In the zo" century alcohol consumption became the focus point of some health campaigners. Medical science restored the good name of wine in the 1990s when it came to light that moderate red wine consumption may aid in preventing heart disease and assist in stress management. The only drawback that lowers consumption levels is the use of chemical preservatives. It is of utmost importance to place the focus on health issues and the development of natural preservation methods that are environmentally friendly and contributes to the overall wholesomeness of the wine. Due to these demands, the scientific community placed the focus of research projects on the development and enhancement of biopreservation methods, in order to minimise chemical preservation use. One of the most promising biocontrol agents is bacteriocins. These proteinaceous molecules produced by various lactic acid bacteria exert antimicrobial activity towards closely related organism. Research has shown that bacteriocins may aid in the prevention of wine-spoilage and enhance natural preservation techniques. Most of the research on biopreservation in food and beverages has been performed on the bacteriocins of LAB. No evidence could be found that indicated bacteriocin production by wine isolated LAB in South Africa. This study is therefore, of utmost importance and is considered to be novel pioneering work for the South African wine industry. The main objective of this study was to screen wine isolated LAB for the production of antimicrobial and/or antifungal compounds. This was followed by the isolation and characterisation of the produced bacteriocins. This study forms part of a greater project that focuses on wine preservation, under the auspices of the Institute for Wine Biotechnology.The research results in this study indicated the production of bacteriocins by wine isolated LAB of South African origin. It was found that numerous isolates exerted antimicrobial activity towards other wine associated LAB. The most predominant species that gave the highest activity was Lactobacillus brevis and Lactobacillus paracasei. Experimental results indicated that the bacteriocins produced by these two species were thermo-stable and active over a wide pH range, including the temperatures and pH values that reign in the South African wine environment. The antimicrobial activity was lost after treatment with proteolytic enzymes, such as proteinase K and lysozyme. The size, production and growth kinetic curves of the bacteriocins under investigation showed similar results that are comparable to other findings in the literature. Antifungal activity was detected against Botryfis cinerea that indicated limited inhibitory activity towards spore germination, but had no effect on hyphal growth. This study provides novel information regarding bacteriocin production by LAB isolated from the South African wine industry. The results indicate the suitability of these bacteriocins as possible biopreservatives in the wine environment. The proposed results obtained in this study will aid in the development of bacteriocinproducing, tailored made wine yeast or LAB that may in future, play vital roles in the winemaking process. / AFRIKAANSE OPSOMMING: Wynmaak is 'n eeu oue tradisie wat terugdateer tot so vroeg soos 6000 jaar v.C. In ons moderne eeu is daar verskeie insekte en mikro-organismes wat In bedreiging vir die wingerdstok, asook die omgewing en die finale wynproduk inhou. Boere en wynmakers word al hoe meer bewus van hierdie bedreiging, terwyl die stryd teen siektes, bederf en/of patogene mikro-organismes ook aan die toeneem is. Tans word die natuurlike omgewing deur landelike ontwikkeling, besoedeling en natuurlike rampe verander, wat op sy beurt weer verantwoordelik is vir die verandering van mikroflora. Die gevolg is 'n harde stryd tussen die mens en mikro-organismes. Die wapen wat gereeld ingespan word in die stryd teen mikro-organismes, is chemiese preserveermiddels, soos swaweidioksied. Hierdie chemiese preserveermiddels verander die voedingswaarde, kwaliteit en die voedsaamheid van die wyn. Dit onderdruk ook die gehalte van wyn, wat meebring dat minder wyn deur die verbruiker gedrink word. Tot en met die agtiende eeu is wyn deur dokters as 'n veilige drankie voorgeskryf. In die twintigste eeu het alkoholverbruik die fokuspunt van gesondheidskamvegters geword. In die 1990's het die mediese wetenskap wyn se goeie naam in ere herstel toe dit aan die lig gekom het dat In matige verbruik van rooiwyn moontlik hartsiektes kan voorkom en help om stres te beheer. Die enigste nadelige faktor wat verbruikersvlakke verlaag, is die gebruik van chemiese preserveermiddels. Dit is uiters noodsaaklik om die fokus op gesondheidskwessies te plaas en die ontwikkeling van natuurlike preserveermetodes wat omgewingsvriendelik is en tot die algehele voedsaamheid van wyn bydra. As gevolg van hierdie eise het wetenskaplikes die fokus geplaas op navorsingsprojekte vir die ontwikkeling en verbetering van biopreserveringsmetodes met die doelom die gebruik van chemiese preserveermiddels te verminder. Een van die belowendste biokontrolemiddels is bakteriosiene. Hierdie proteïenagtige molekule word deur verskeie melksuurbakterieë vervaardig en oefen anti-mikrobiese aktiwiteit teenoor nabyverwante organismes uit. Navorsing het getoon dat bakteriosiene moontlik kan help in die voorkoming van wynbederf en natuurlike preserveertegnieke kan verbeter. Die meeste van die navorsing op biopreservering in voedsel en drank is op die bakteriosiene van melksuurbakterieë uitgevoer. Geen bewys kon gevind word in Suid Afrika wat bakteriosienproduksie deur wyn-geïsoleerde melksuurbakterieë aangedui het nie. Hierdie studie is daarom baie belangrik en word as baanbreker werk vir die Suid Afrikaanse wynbedryf beskou. Die hoofdoel van hierdie studie was om wyn-geïsoleerde melksuurbakterieë vir die produksie van anti-mikrobiese en/of anti-fungiese substanse te toets. Dit is gevolg deur die isolasie en karakterisering van die geproduseerde bakteriosiene. Hierdie studie maak deel uit van 'n groter projek wat fokus op wynpreservering en wat onder leiding van die Instituut van Wynbiotegnologie uitgevoer word. Navorsingsresultate van hierdie studie dui op die produksie van bakteriosiene deur wyn-geïsoleerde melksuurbakterieë van Suid Afrikaanse oorsrong. Daar is gevind dat verskeie isolate anti-mikrobiese aktiwiteit teenoor ander wynverwante malksuurbakterieë uitgeoefen het. Die oorheersende spesie wat die hoogste aktiwiteit getoon het, was Lactobacillus brevis en Lactobacillus paracasei. Eksperimentele uitslae dui daarop dat die bakteriosiene wat deur hierdie twee spesies geproduseer word, termostabiel en aktief is oor 'n wye pH reeks, insluitende die temperature en pH-waardes wat in die Suid Afrikaanse wynomgewing voorkom. Die anti-mikrobiese aktiwiteit het verlore gegaan na behandeling met proteolitiese ensieme soos proteïnase K. Die groote, produksie en groeikinetika kurwes van die bakteriosiene wat ondersoek is, toon vergelykbare resultate met ander bevindings in die literatuur. Anti-fungiese aktiwiteit is opgemerk teen Botrytis cinerea, wat beperkte inhiberende aktiwiteit ten opsigte van spoorontkieming aangedui het, maar geen effek op hifegroei gehad nie. Hierdie studie verskaf nuwe inligting aangaande bakteriosienproduksie deur melksuurbakterieë wat van die Suid Afrikaanse wynomgewing geïsoleer is. Die resultate dui op die geskiktheid van hierdie bakteriosiene as moontlike biopreserveermiddels in die wynbedryf. Die voorgestelde resultate deur hierdie studie verkry sal help in die ontwikkeling van bakteriosien produserende, spesifiek vervaardigse wyngis of melksuurbakterieë, wat in die toekoms 'n baie belangrike rol in die wynmaakproses sal speel.

Thesis (MScAgric)--Stellenbosch University, 1954. / No Abstract Available

Thesis (MScAgric)--Stellenbosch University, 1978. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming