Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The conversion of sugar into ethanol and carbon dioxide is a process that has been intertwined with human culture and long as civilized man has existed. This fermentation process has been dominated by the micro-organism Saccharomyces cerevisiae and from providing ancient seafaring explorers of a non perishable beverage to equipping bakers with a raising agent to turn flour into bread; this organism with its fermentative potential, has formed an essential part of most societies. In more recent times, many industries still rely on this basic principle. The complexities and efficiencies of the conversion of sugar into its various fermentative byproducts have been studied and optimised extensively to meet the specific demands of industries. Depending on the raw material used as starting point, the major beneficiaries of the useful characteristics have been alcoholic beverage producers (wine, beer, and whiskey amongst others), bakers (bread leavening) and biofuel producers. One of the obstacles in fermentation optimisation is the sugar consumption preferences displayed by the organism used. S. cerevisiae can consume a wide variety of sugars. Depending on the complexities of its structures, it shows a preference for the simpler saccharides. The fermentation of certain more complex sugars is delayed and runs the risk of being left residually after fermentation. Many of the crops utilised in fermentation-based products contain large amounts of starch. During the starch degradation process many different forms of sugars are made available for fermentation. Improved fermentation of starch and its dextrin products would benefit the brewing, whiskey, and biofuel industries. Most strains of Saccharomyces ferment glucose and maltose, and partially ferment maltotriose, but are unable to utilise the larger dextrin products of starch. This utilisation pattern is partly attributed to the ability of yeast cells to transport the aforementioned mono-, di- and trisaccharides into the cytosol. The inefficiency of maltotriose transport has been identified as the main cause for residual maltotriose. The maltotriose transporting efficiency also varies between different Saccharomyces strains. By advancing the understanding of maltotriose transport in yeast, efforts can be made to minimise incomplete fermentation. This aim can be reached by investigating the existing transporters in the yeast cell membrane that show affinity for maltotriose. This study focuses on optimising maltotriose transport through the comparison of the alpha glucoside transporter obtained from different strains of Saccharomyces. Through specific genetic manipulations the areas important for maltotriose transport could be identified and characterised. This study offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer, whiskey, and biofuel industries. / AFRIKAANSE OPSOMMING: Die transformasie van suiker na etanol en koolstof dioksied is so oud soos die beskawing self, en dit is van die vroegste tye af onlosmaaklik met die mens se kultuur verbind. Hierdie fermentasie-proses word gedomineer deur die Saccharomyces cerevisiae mikroorganisme. Hierdie organisme het antieke seevaarders voorsien van ‘n nie-bederfbare drankie en van ouds af aan bakkers ‘n rysmiddel verskaf waarmee meel in brood verander kon word. As gevolg van hierdie fermenteringspotensiaal het hierdie organisme ‘n onmisbare rol in meeste beskawings gespeel. Baie industrieë is steeds op hierdie basiese beginsel gebou. Die kompleksiteite en effektiwiteit van die transformasie van suiker na sy verskeie gefermeenteerde neweprodukte is breedvoerig bestudeer en geoptimiseer om aan die spesifieke behoeftes van verskeie industrieë te voeldoen. Afhangend van die grondstowwe wat as beginpunt gebruik is, is die primêre begunstigdes van die fermentasie proses die alkoholiese drankprodusente (onder andere die wyn-, bier- en whiskey produsente), bakkers en biobrandstofprodusente. Die suikerverbruik-voorkeur van die organisme wat die fermentering fasiliteer is een van die struikelblokke in die optimisering van die proses. S. cerevisiae kan ‘n wye spektrum van suikers verbruik maar dit toon ‘n voorkeur vir die eenvoudiger suikers. Die fermentasie van sekere van die meer komplekse suikers is vertraag en loop die risiko om agtergelaat te word na fermentasie. Vele van die gewasse wat in die gefermenteerde produkte gebruik word bevat groot hoeveelhede stysel. Vele soorte suikers word gedurende die afbreek van die stysel beskikbaar gestel vir fermentasie. Die brouers-, whiskey- en biobrandstof industrieë sal almal voordeel trek uit die verbeterde fermentasie van stysel en sy gepaardgaande dekstrin produkte. Meeste Saccharomyces gisrasse fermenteer glucose en maltose; maltotriose word gedeeltelik gefermenteer, maar die meer komplekse dekstrien produkte gevind in stysel word nie gefermenteer nie. Hierdie verbruikerspatroon kan gedeeltelik toegeskryf word aan die vermoë van gisselle om die bogenoemde mono-, di- and trisaccharides in die sitosol op te neem. Die oneffektiwiteit van maltotriose transport is identifiseer as die hoofoorsaak van post-fermentatiewe, oortollige maltotriose. Die effektiwiteit van maltotriose transport verskil ook tussen verskillende Saccharomyces rasse. Pogings om onvolledige fermentasie te veminder kan bevorder word deur die kennis rondom maltotriose transport in gis uit te bou. Hierdie oogmerk kan bereik word deur die bestaande transporters in die gissel se membraan wat ‘n affiniteit vir maltotriose toon te ondersoek. Hierdie studie fokus op die optimisering van maltotriose transport deur die vergelyking van die alpha glucoside transporter (AGT1) wat van verskillende Saccharomyces rasse afkomstig is. Die areas wat relevant is tot maltotriose transport kon deur spesifieke genetiese manipulasies identifiseer en gekarakteriseer word. Hierdie studie bevorder die vooruitsig op die ontwikkeling van gisrasse met verbeterde maltose en maltotriose transport vermoëns wat op sy beurt weer kan aanleiding gee tot die verbeterde fermentasie effektiwiteit in die bier, whiskey en biobrandstof industrieë.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Wine protein haze formation is a major challenge for wine makers, and several wine clarifying agents such as bentonite are used in the industry to protect wine from this occurrence. However, clarifying agents may have an undesirable impact on wine quality. Yeast mannoproteins have been shown to possess haze-protective properties, while also positively impacting on the sensorial properties of the product. However, while such mannoproteins are released into the wine during the wine making process, the amounts are low and therefore of limited oenological significance. However, and although commercial wine yeast strains display significant genotypic and phenotypic diversity, no broader assessment of haze protective activity and of mannoproteins release by different wine yeast strains has been undertaken. In this study, several yeast strains were screened for their impact on wine haze formation in Chardonnay must and in a grape juice model system. The data show that strains of the species Saccharomyces paradoxus possess better haze protective properties than the common Saccharomyces cerevisiae wine yeast strains. Differences in the nature of the proteins released by these two species were investigated, and indicated that several mannoproteins were released at significantly higher levels by S. paradoxus, and that some of these proteins might indeed contribute to the haze-protective activity. A further exploration of yeast cell wall properties indicated that the cell walls of haze-protective S. paradoxus strains contained higher levels of chitin than non-haze protective strains. Grape chitinases are likely to be primarily responsible for wine haze formation, and the data clearly demonstrate that these enzymes are able to bind to the yeast cell walls, and that strains with higher amounts of chitin in the cell wall will bind more chitinases. This finding suggests that the haze-protective nature of the strains is at least in part linked to the chitin levels of the strains. Furthermore, the impact of some genetic modifications in two wine strains (namely S. cerevisiae VIN13 and S. paradoxus RO88) suggests that several proteins contribute to wine haze protection. However, none of the mannoprotein-encoding flocculation genes, FLO1, FLO5, and FLO11 showed any impact on this property. Further studies are required to assess the full impact of the S. paradoxus strains on haze protection. In particular, the possible use of such strains as starter cultures or the use of S. paradoxus yeast hulls as clarifying agent needs to be further explored. / AFRIKAANSE OPSOMMING: Wyn proteïen-waas vorming is 'n groot uitdaging vir wynmakers en verskeie wyn verhelderings agente soos bentoniet word in die wynbedryf gebruik om wyn te beskerm teen die vorming van waas. Hierdie verheldering agente het egter 'n ongewenste impak op wynkwaliteit. Gis mannoproteïene is uitgewys as proteïene met moontlike waas-beskermende eienskappe wat ook 'n positiewe uitwerking op die sensoriese eienskappe van die produk het. Al word hierdie mannoproteïene egter vrygestel in die wyn tydens die wynmaak proses, is die hoeveelhede oor die algemeen te laag om van wynkundige belang te wees. Verder, ten spyte van die beduidende genotipiese en fenotipiese diversiteit van kommersiële wyngisrasse is daar nog geen breër assessering van die waas beskermende aktiwiteit van mannoproteïene, vrygestel deur verskillende rasse, tot dusver onderneem nie. In hierdie studie is verskeie gisrasse gekeur vir hul impak op wyn waas-vorming in Chardonnay mos en ook in 'n model druiwesap. Die data wys dat rasse van die spesie Saccharomyces paradoxus besit beter waas beskermende eienskappe as die algemene Saccharomyces cerevisiae wyngisrasse. Verskille in die aard van die proteïene wat vrygestel is deur hierdie twee spesies is ondersoek, en dit is aangedui aangedui dat verskeie mannoproteins vrygestel aan aansienlik hoër vlakke deur S. Paradoxus. Dit is ook aangedui dat sommige van hierdie proteïene wel bydra tot die waas-beskermende aktiwiteit. 'n Verdere verkenning van gis selwand eienskappe het aangedui dat die selwande van waas-beskermende rasse van S. paradoxus hoër vlakke chitien as nie-waas beskermende stamme bevat. Druiwe chitinases is waarskynlik hoofsaaklik verantwoordelik vir wyn waas vorming, en die data toon duidelik dat hierdie ensieme in staat is om te bind aan die gis selwande, en dat die stamme met hoër vlakke chitien in die selwand meer chitinases sal bind. Hierdie bevinding dui daarop dat die waas-beskermende aard van die stamme ten minste gedeeltelik gekoppel is aan die chitien vlakke van die stamme. Die impak van sekere genetiese modifikasies in twee verskillende gisrasse, naamlik die S. cerevisiae ras VIN13 en die S. paradoxus ras RO88, dui verder daarop dat verskeie proteïene dra by tot die beskerming teen wyn waas. Geeneen van die mannoprotein-koderende flokkulasie gene, FLO1, FLO5 en FLO11 het egter 'n impak op hierdie eienskap nie. Verdere studies is nodig om die volle impak van die S. paradoxus rasse op waas beskerming te assesseer. In die besonder, die moontlike gebruik van sulke rasse as 'n inkolasie kultuur of die gebruik van S. paradoxus gis doppe as verheldering agent moet verder ondersoek word.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The yeast Brettanomyces was isolated from beer in 1904 and associated with wine thereafter. A sporulating form, Dekkera, was discovered later. Brettanomyces bruxellensis produces high levels of volatile phenol off-flavours in wine. Sulphur dioxide (SO2) is the most widely used chemical preservative in wine. Yeasts have several mechanisms to cope with the SO2, namely Ssu1p, a membrane bound SO2 transporter; sulphite reduction, sulphite oxidation and acetaldehyde production. In unfavourable environmental conditions, certain yeasts can enter a viable-but-non-culturable (VBNC) state which is characterised by reduced metabolic rate, inability to reproduce on solid media and a reduction of cell size. VBNC can be triggered by chemical stress such as high SO2 levels. The objectives of this study were to examine the SO2 tolerance of B. bruxellensis and Saccharomyces cerevisiae, to quantify their rates of SO2 accumulation and efflux, determine the effect of SO2 on their energy metabolism and investigate if B. bruxellensis possesses an orthologue to S. cerevisiae SSU1. In this study, the identity of a number of Brettanomyces/Dekkera strains was confirmed using 5.8S rDNA-ITS RFLP analysis and DNA sequencing. Sporulation assays were used to confirm whether these strains belonged to the Dekkera or Brettanomyces genus. A method to accurately quantify SO2 in laboratory conditions was optimised. Molecular SO2 tolerance was tested by spotting fresh yeast cultures on media with SO2 and/or ethanol. Tolerance to SO2 and/or ethanol showed highly strain dependent results with S. cerevisiae showing the highest tolerance levels while B. bruxellensis tolerated SO2 and ethanol poorly but certain strains grew well with only SO2. The SO2 accumulation and efflux rates of 3 S. cerevisiae strains and 3 B. bruxellensis strains were determined. It was shown that the S. cerevisiae strains followed the same trends as previously found in literature whereas B. bruxellensis strains showed similar trends but displayed highly variable strain-dependent results. B. bruxellensis CB63 and S. cerevisiae VIN13 were investigated for their response to SO2 in two different media, TA and SWM, over a 48-hour and 32-day period respectively. Acetic acid, acetaldehyde, D-glucose, D-fructose (only in SWM) and ethanol (only in TA) were regularly monitored over the time course of each experiment. SO2 had the greatest impact on B. bruxellensis with decreased rates of glucose consumption and ethanol production as well as increased acetic acid. Acetaldehyde peaked shortly after SO2 addition with the subsequent restarting of sugar consumption for certain samples. This suggests that sufficient acetaldehyde was produced to bind free SO2 to reduce SO2 stress. Volatile phenols were quantified for day 32 of the SWM experiment. An increase of 4-ethyl guaiacol was correlated to higher molecular SO2 levels. SO2 negatively affected both yeasts energy metabolism, forcing the yeasts metabolism to adapt to ensure survival. In general, SO2 was shown to have a negative impact on all aspects of a yeasts growth and metabolism and that SO2 tolerance is highly strain dependent and a far more complicated characteristic than currently understood. / AFRIKAANSE OPSOMMING: Die gis Brettanomyces is in 1904 uit bier geïsoleer en daarna met wyn geassosieer. 'n sporulerende vorm, Dekkera, is later ontdek. Brettanomyces bruxellensis produseer hoë vlakke van vlugtige fenol afgeure in wyn. Swaweldioksied (SO2) is die mees gebruikte chemiese preserveermiddel in wyn. Giste het verskeie meganismes om SO2 te hanteer, naamlik Ssu1p, 'n membraan-gebonde SO2 transporter, sulfietvermindering, sulfiet-oksidasie en asetaldehiedproduksie. In ongunstige omgewingstoestande kan sekere giste 'n lewensvatbare, maar nie-kultiveerbare (LMNK)-toestand aanneem wat gekenmerk word deur verlaagde metaboliese tempo, onvermoë om voort te plant op soliede media en 'n vermindering van die selgrootte. LMNK kan veroorsaak word deur chemiese stres, soos hoë SO2-vlak. Die doelwitte van hierdie studie was om die SO2 -bestandheid van B. bruxellensis en Saccharomyces cerevisiae te ondersoek, hul spoed van SO2 -opneming/akkumulasie en -uitskeiding te kwantifiseer, die invloed van SO2 op energiemetabolisme te bepaal en te ondersoek of B. bruxellensis oor ‘n soortgelyke geen as die S. cerevisiae SSU1 beskik. In hierdie studie is die identiteit van 'n aantal Brettanomyces/Dekkera-stamme bevestig deur 5.8S rDNA-ITS RFLP-analise en DNA-opeenvolging te gebruik. Sporulasietoetse is gebruik om te bevestig of hierdie stamme aan die genus Dekkera of Brettanomyces behoort. 'n Metode om SO2 onder laboratoriumtoestande akkuraat te kwantifiseer, is geoptimiseer. Molekulêre SO2- bestandheid is getoets deur vars giskulture op media met SO2 en/of etanol te groei. Bestandheid teen SO2 en/of etanol het stam-afhanklike resultate getoon, S. cerevisiae wat die hoogste toleransievlakke getoon het, terwyl B. bruxellensis SO2 en etanol swak tolereer, maar sekere stamme het goed gegroei met slegs SO2. Die SO2-akkumulasie en -uitskeidingtempo van 3 S. cerevisiae-rasse en 3 B. bruxellensis-stamme is bepaal. Daar is gevind dat die S. cerevisiae-rasse dieselfde tendens soos voorheen in die literatuur beskryf, gevolg het, terwyl B. bruxellensis-stamme soortgelyke tendense getoon het,maar hoogs veranderlike stamafhanklike resultate vertoon. B. bruxellensis CB63 en S. cerevisiae VIN13 is ondersoek vir hul reaksie tot SO2 in twee verskillende media, TA en SWM, oor 'n tydperk van 48-uur en 32-dae onderskeidelik. Asynsuur, asetaldehied, D-glukose, D-fruktose (slegs in SWM) en etanol (slegs in TA) is gereeld gemoniteer oor die verloop van elke eksperiment. SO2 het die grootste impak op B. bruxellensis met ‘n verlaagde tempo van glukoseverbruik en etanolproduksie, sowel as verhoogde asynsuur. ‘n Asetaldehiedhoogtepunt is bereik kort na die SO2-byvoeging met die daaropvolgende hervatting van suiker wat vir sekere monsters gebruik is. Dit dui daarop dat voldoende asetaldehied geproduseer is om vry SO2 te bind om SO2-stres te verminder. Vlugtige fenole is op dag 32 van die SWM-eksperiment gekwantifiseer. 'n Toename van 4-etiel-guajakol korreleer met hoër molekulêre SO2-vlakke. SO2 het beide giste se energiemetabolisme negatief beïnvloed, wat die gis dwing om sy metabolisme aan te pas om oorlewing te verseker. Oor die algemeen het SO2 'n negatiewe impak op alle aspekte van giste se groei en metabolisme, en SO2-bestandheid is hoogs stam–afhanklik. Dit is ook 'n baie meer ingewikkelde kenmerk as wat tans verstaan word.

Thesis (MSc)--Stellenbosch University, 2012 / ENGLISH ABSTRACT: The aroma and flavour of wine are important aspects that form the basis for consumers’ organoleptic experience of wine. Therefore, an understanding of the chemical composition of wine aroma is of major importance, to establish possible links between wine chemistry, sensory attributes and consumer preference for a product. For this purpose analytical chemistry and multivariate techniques are indispensable tools for the metabolic profiling of wine. Chenin blanc is one of the most important South African export white wine varieties. However, despite its importance, very limited profiling of Chenin blanc aroma compounds has been done and information is restricted to isolated and dated reports on a few chemical compounds only. Therefore, the overall aim of this study was to obtain an in-depth view of the volatile chemical profile of this cultivar. The first task was to perform targeted volatile metabolic profiling of the three dry and offdry Chenin blanc styles, fresh and fruity, rich and ripe unwooded and rich and ripe wooded. To this end, a new, simple and robust liquid-liquid extraction technique using dichloromethane was developed and validated for extraction of analytes prior to gas chromatography flame ionization detection (GC-FID) analysis, to quantify 57 analytes in one rapid analytical procedure. This method was applied to profile 48 Chenin blanc wines. Very successful discrimination between the three styles, using the quantified volatile compounds, was obtained with two multivariate methods. These were partial least squares regression-discriminant analysis, (PLS-DA), as well linear-DA, using best subset selection for identifying the most important variables. According to the classification models, a higher content of maturation derived, malolactic fermentation derived and wood derived compounds were predominantly characteristic of the wooded wines. Higher content of some terpenes and ethyl esters were predominantly associated with the rich and ripe unwooded style Chenin blanc wines, while the fresh and fruity style were generally characterized by high levels of acetate esters. Secondly, untargeted analysis of 21 wines was done with gas chromatography mass spectrometry (GC-MS). Mathematical chromatography, using PARAllel FACtor analysis (PARAFAC and PARAFAC2), was applied to the GC-MS data for resolution of the complex chromatographic results by multi-way modeling, and to derive unbiased multivariate classification models of the three styles. This approach provided excellent style differentiation, without the arduous task of analysis of numerous standards and setting up of calibration curves, required by the targeted approach described above. Additionally, the data generated during this study will form part of the current South African wine aroma database, which does not contain any data regarding Chenin blanc at present. / AFRIKAANSE OPSOMMING: Die aroma en geur van wyn is belangrike aspekte aangesien dit die basis vorm van die wynverbruiker se organoleptiese ervaring van die produk. Derhalwe, is ‘n deeglike kennis van die chemiese samestelling van wynaroma baie belangrik, ten einde die korrelasies tussen wynchemie, sensoriese eienskappe en verbruikersvoorkeure te bepaal. Vir hierdie doel, is die insameling van analitiese chemiese data, tesame met multi-veranderlike tegnieke om relevante inligting uit die data te onttrek, onmisbaar vir die metaboliese profilering van wyn. Chenin blanc is huidiglik een van Suid-Afrika se belangrikste uitvoer wit wynvariëteite. Ten spyte hiervan, is daar tot hede egter baie min profilering van Chenin blanc se aromakomponente gedoen en die beskikbare inligting is beperk tot geïsoleerde en verouderde navorsingsbevindinge. In die lig van bogenoemde, is die oorkoepelende motivering vir hierdie studie dus om die vlugtige chemiese komponente se profiel in Chenin blanc wyn, in diepte te bepaal. Die eerste taak was die geteikende bepaling van die metaboliese profiel van drie droë of halfdroë Chenin blanc wynstyle, nl. vars en vrugtig, ryk en ryp ongehout, asook ryk en ryp gehout. Om dit te bereik, is ‘n nuwe eenvoudige en robuuste vloeistof-vloeistof ekstraksieprosedure met dichlorometaan ontwikkel, wat analise met gaschromatografie – vlamionisasie deteksie (GC-FID) voorafgaan, om die konsentrasies van 57 komponente in een vinnige analise te bepaal. Hierdie metode is gebruik om 48 Chenin blanc wyne te profileer. Deur gebruik te maak van multi-veranderlike data analitiese tegnieke, is die gekwantifiseerde vlugtige komponente data gebruik in diskriminant analise. Baie suksesvolle onderskeid tussen die drie style, is verkry deur gebruik te maak van twee multi-veranderlike metodes, naamlik: parsiële kleinste kwadrate regressie, diskriminant analise, asook liniêre diskriminant analise. Vir laasgenoemde analise, is die seleksie van die mees belangrike veranderlikes met beste sub-groep regressie bepaal. Volgens hierdie klassifikasie modelle is ‘n hoër inhoud van veroudering-, appelmelksuurgisting- en houtverwante aroma komponente baie kenmerkend in die houtbehandelde wyne. Hoër vlakke van sommige terpene en etiel esters was kenmerkend met betrekking tot ryk en ryp ongehoute Chenin blanc style, terwyl die vars en vrugtige style meer gekenmerk was met hoë vlakke van asetaat esters. Tweedens is ‘n seleksie van 21 wyne geanaliseer deur gebruik te maak van gaschromatografie – massa spektrometrie (GC-MS) in ‘n ongeteikende metaboliese profileringsbenadering. Wiskundige chromatografiese metodes, spesifiek, parallelle faktor analise (PARAllel FACtor analysis (PARAFAC and PARAFAC2)), was voorts gebruik om komplekse chromatogramme te prosesseer met wiskundige, multi-vlak modellering. Met hierdie nie-selektiewe benadering, is ook suksesvolle klassifikasiemodelle gegenereer vir die diskriminasie tussen die drie verskillende Chenin blanc style. Die voordeel van die ongeteikende GC-MS analise, gekoppel met die data hanterings- en prosesseringsprotokols in hierdie studie gebruik, is dat die arbeidsintensiewe taak om kalibrasiekurwes op te stel vir elke individuele komponent, soos wat vereis word in die geteikende benadering, nie nodig is nie. Die data wat ingewin is gedurende hierdie studie, sal ook bygevoeg word tot ‘n bestaande Suid-Afrikaanse aroma databasis, wat tans geen data aangaande Chenin blanc wyn bevat nie.

Thesis (MSc)--Stellenbosch University, 2007. / ENGLISH ABSTRACT: With ever increasing pressure on wine producers to lower the financial costs involved in winemaking to be able to compete in the market, all while maintaining a high level of wine quality, the focus on maintaining control over all aspects of the winemaking process are greatly emphasized. Malolactic fermentation (MLF) is one of the important processes in red wine production. The advantages of this process, when performed successfully, is widely known and accepted. One way to gain control over MLF is the use of MLF starter cultures. Starter cultures usually consist of Oenococcus oeni that has been isolated from grapes or wines and is in most cases available in a freeze-dried form ready for direct inoculation into the wine when MLF is desired. Starter cultures are induced into wine and usually ensure the immediate onset as well as a fast and clean execution of the process. Starter cultures used in South Africa are in most cases isolated from cooler viticultural regions in the Northern hemisphere. The constitution of wines from cooler viticultural regions, differ from those in South Africa, which has a warm climate. The most important difference is the acid content of the wines which is lower in South African must/wines and results into a higher pH. The three most important changes that develop in wine during MLF are a decrease in acidity due to the conversion of malic acid to the less harsh lactic acid, enhanced flavour and aroma of wine and an increase in the microbiological stability of wine. The decrease in acidity is very important for wines produced for grapes grown in cool viticulture regions. In South Africa though, the climate is warm and higher pH’s are present in the musts and wines and the de-acidification due to MLF is not the main aim but rather the microbiological stabilisation. One of the compounds that could be produced by lactic acid bacteria (LAB) is biogenic amines (BA’s). These compounds can be hazardous to human health. This thesis focussed on the performance of MLF starter cultures in high pH South African red wines. The first objective of the study was to stretch MLF starter cultures in high pH red wines of South Africa. Stretching means to use less than the prescribed dosage or the re-use of starter cultures. The difference in MLF rate, the influence of the natural occurring LAB and the levels of biogenic amines formed during MLF were determined for the different stretching treatments. The results showed that different rates in malic acid degradation were experienced between the treatments, but in all cases MLF fermentation was completed. Biogenic amines were formed at various levels and the influence of the natural occurring LAB also played a role. The second objective of the study was the evaluation of the effect of a wine isolated LAB (Lactobacillus) and an acetic acid bacteria (AAB), inoculated with a MLF starter culture had on MLF at different wine pH’s. It was found that especially in the case where the Lactobacillus was inoculated in combination with the MLF starter culture a possible stimulatory effect was experienced with regards to malic acid degradation rate. Biogenic amine concentration was measured at the end of MLF and it was found that no histamine and tyramine were formed in any of the treatments, while the putrescine and cadaverine levels were found to be at approximately similar levels for the different treatments. The third objective was to evaluate the possible influence of commercial tannin additions and a pectolytic enzyme on rate of MLF and phenolic composition of high pH red wine. The commercial tannins had possible inhibitory as well as stimulatory effects on the rate of malic acid degradation especially during the initial stages of MLF, with the highest dosage having the significant effect. The BA results showed difference in the levels produced due to tannin additions as well as strain differences could exist. The phenolic content showed a decrease in colour density, total red pigments, total phenolics and anthocyanins between AF and MLF. The fourth objective was to evaluate inoculation time of MLF starter cultures. The results showed that the fastest AF/MLF time was with simultaneous inoculation of the yeast and MLF starter cultures. It was also for this treatment where no histamine or tyramine was detected at the end of MLF compared to the other inoculation strategies (before the end of AF and after AF). This study generated a large amount of novel data which made a valuable contribution with regards to MLF in high pH red wines of South Africa. / AFRIKAANSE OPSOMMING: Die druk om wyne van hoë gehalte teen lae insetkoste te lewer om deel te bly van ’n kompeterende mark, plaas die fokus weer sterk op onder andere die beheer van alle aspekte van die wynmaak proses. Appelmelksuurgisting (AMG) is een van die belangrikste prosesse van rooiwyn produksie. Die voordele van AMG, in die geval van die suksesvolle implementering daarvan is vandag bekend en word geredelik aanvaar. Een van die metodes om beheer te verkry oor the proses van AMG is deur die gebruik van AMG aanvangskulture. AMG aanvangskulture bestaan uit Oenococcus oeni wat geïsoleer word vanaf druiwe of mos/wyn en is in meeste gevalle beskikbaar in ’n gevries-droogte vorm wat direk in wyn geïnokuleer kan word. Aanvangskulture word in wyn geïnduseer om die onverpose aanvang van AMG te bewerkstellig asook om ’n vinnige en skoon deurvoering van die proses te verseker. Die aanvangskulture wat in Suid-Afrika vir hierdie doeleinde gebruik word is in meeste van die gevalle verkry uit koue wingerdbou gebiede in die Noordelike Halfrond. Die samestelling van druiwe van koue wingerdbou gebiede en dié van Suid-Afrikaanse warm wingerdbou gebiede verskil. Die belangrikste verskil word ervaar in die suur inhoud, wat laer is in Suid-Afrikaanse druiwe en dus lei tot ‘n hoër pH inhoud. Die drie mees belangrikste veranderinge wat gedurende AMG in wyn plaasvind is die vermindering van die suur, as gevolg van die omskakeling van appelsuur na melksuur, die verbetering van die aroma en geur van wyn en die verbeterde mikrobiologiese stabiliteit. Die afname in suur is veral belangrik in wyne van koue wingerbou gebiede omdat die suur-inhoud daarvan soveel hoër is. In Suid-Afrika kan hierdie verlaging in suur egter lei tot ’n verdere verhoging in die pH wat plat wyne en uiteindelik ’n verlaging in die kwaliteit van wyn tot gevolg kan hê. Biogene amiene (BA) is verbinding wat melksuurbakterieë (MSB) kan vorm gedurende AMG en kan ernstige implikasies hê vir die mens se gesondheid. Hierdie tesis fokus op die evaluering van AMG aanvangskulture in hoë pH rooi wyne van Suid-Afrika. Die eerste doelwit gedurende hierdie studie was om AMG kulture te rek en die invloed daarvan in hoë pH rooiwyn te evalueer ten opsigte van the tempo van AMG, die rol van die natuurlike MSB te bestudeer asook om die vlak van biogene amiene te bepaal vir die verskillende behandelings. Die resultate het aan die lig gebring dat die rek van kulture verskille in die tempo van appelsuur afbraak tot gevolg het, maar dat AMG in alle gevalle wel suksesvol deurgevoer kon word. Die BA’e wat gevorm is, was teenwoordig in verskillende hoeveelhede. Die tweede doelwit was om die effekt van die gesamentlike inokulasie van ’n wyn geisoleerde MSB (Lactobacillus) asook ’n asynsuurbakterie (ASB) met ’n kommersiële AMG aanvangskultuur op AMG te evalueer. Hierdie eksperiment is uitgevoer by verskillende pH’s. Daar is gevind dat veral in die kombinasie inokulasie met die Lactobacillus, die tempo van appelsuur afbraak moontlik gestimuleer was. Geen histamien of tiramien is tydens AMG gevorm in hierdie eksperiment gevorm nie, terwyl putresien en kadaverien teenwoordig was teen ongeveer gelyke vlakke vir die behandelings. Die derde doelwit was om die moontlike invloed van kommersiële tannien toevoegings en die toevoeging van ’n pektolitiese ensiem te evalueer ten opsigte van AMG tempo die fenoliese samestelling van rooiwyn te bestudeer. Verskillende kommersiële tanniene het ’n moontlike sowel as inhiberende uitwerking gehad, veral gedurende die aanvanklike stadium AMG. Die grootste verskille is waargeneem in die behandelings waar die hoogste dosisse tannien bygevoeg is. Die BA resultate toon dat verkillende vlakke geproduseer was en dat hierdie verskille onstaan het as gevolg van verskille in tannien dosisse sowel as aanvangskulture. Die fenoliese inhoud het ’n afname in kleur intensiteit, totale rooi pigmente, totale fenole en antosianiene getoon vir die periode vanaf AF tot die einde van AMG. Die vierde doelwit was om the tyd van inokulasie van AMG aanvangskulture te bestudeer. Die resultate het getoon dat die vinningste tydperk van AF/AMG was ondervind in die geval waar die gis aanvangskulture gelyktydig met die AMG aanvangskulture geïnokuleer was. Geen histamine en tyramine het ook in hierdie behandeling ontwikkel nie, terwyl daar wel vlakke teenwoordig was in die ander behandelings (inokulasie net voor die einde van AF en na afloop van AF). Tydens hierdie studie is ’n groot hoeveelheid nuwe data geskep wat ‘n groot bydrae ten opsigte van AMG in hoë pH rooi wyne vanaf Suid-Afrika kan lewer.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Producers of South African (SA) dry and semi-dry Chenin blanc table wines are currently experimenting with winemaking techniques to modulate the flavours of the predominantly fruity styles of this genre. An important stage during wine style development is sensory profiling paired with consumer acceptance testing, before wine is produced on industrial scale. With those aforementioned goals in mind, this study was conducted in partnership with two commercial SA wine cellars. The main focus of the study was an investigation into the treatment effects of two winemaking techniques, respectively grape skin contact and natural fermentation, on the sensory profiles of experimentally produced Chenin blanc wines. Results obtained with descriptive sensory analysis (DA) of the wines were compared to those obtained by two rapid sensory profiling methods, namely projective mapping (PM) and frequency of attribute citation (FC). A consumer preference study was also done on the wines. In order to understand the treatment effects better, the dominant non-Saccharomyces yeasts that were present during the natural fermentations were identified using polymerase chain reaction (PCR), while the major volatile chemical compounds were identified with chromatography and mass spectrometry. The sensory and chemical profiles of the naturally fermented wines were significantly different from those of the inoculated wines. PCR analysis identified some of the yeasts present during alcoholic fermentation. In comparison to the inoculated fermented wines, the naturally fermented wines were generally perceived to have more intense and riper tropical fruity aromas, with enhanced sweetness and reduced intensities of sourness, bitterness and astringency. The wines fermented on the skins (FoS) had lower ester and volatile thiol concentrations than the control wines (with no skin contact) and limited skin contact wines. Sensory attributes linked to the FoS wines included aromas of dried fruit, dried grass and a vegetative character, with an increased sour and bitter taste and astringent mouthfeel. In contrast, the wines that were produced with limited skin contact (12 hours) retained their tropical fruity aromas better than the FoS wines. Limited skin contact seemed to have had a less harsh effect on the taste and mouthfeel than the FoS wines. A consumer study was done to establish a Generation Y consumer group’s (18-35 years) preference for the different treated wines. Overall, the naturally fermented wines, which were described as having a strong tropical fruit character, were preferred. The FoS wines were generally disliked by the consumers. Results obtained from the three sensory analysis methods, respectively DA, PM and FC, were similar, thereby confirming the suitability of the rapid methods PM and FC, to extract qualitative information from the sensory profiling of white wine. The results of this study made a significant contribution towards validation of rapid sensory methods for wine evaluation, which are particularly valuable in the context of sustainability and technology transfer to research and industry alike. The knowledge gained on the chemical profiles of SA Chenin blanc is novel and this is one of the first reports on the volatile thiol content of SA Chenin blanc wine. / AFRIKAANSE OPSOMMING: Produsente van Suid-Afrikaanse (SA) droë en semi-droë Chenin blanc tafelwyne eksperimenteer tans met wynmaaktegnieke om die oorwegend vrugtige style van hierdie genre te varieer. ‘n Belangrike fase van wynstylontwikkeling is sensoriese profilering van die produkte met gepaardgaande verbruikersaanvaardingstoetse, voordat die wyne op industriële skaal geproduseer word. Met dié bogenoemde doelwitte in gedagte, was hierdie studie in vennootskap met twee kommersiële SA wynkelders gedoen. Die hooffokus van die studie was om die invloed van twee wynmaaktegnieke, onderskeidelik druifdopkontak en natuurlike fermentasie, op die sensoriese profiele van eksperimenteel geproduseerde Chenin blanc wyne te ondersoek. Resultate wat met beskrywende sensoriese analise (DA) van die wyne verkry is, is vergelyk met dié wat deur twee vinniger sensoriese profileringsmetodes metodes, naamlik projektiewe kartering (PM) en frekwensie van kenmerkaanhaling (FC) verkry is. 'n Verbruikervoorkeurstudie is ook op die wyne gedoen. Ten einde die behandelingseffekte beter te verstaan, is die dominante nie-Saccharomyces giste wat teenwoordig was tydens die natuurlike fermentasie geïdentifiseer met behulp van die polimerasekettingreaksie (PCR), terwyl die vernaamste vlugtige chemiese verbindings geïdentifiseer is met chromatografie en massaspektrometrie. Die sensoriese en chemiese profiele van die natuurlike gefermenteerde wyne was beduidend anders as dié van die geiinokuleerde wyne. PCR analise het sommige giste wat teenwoordig was tydens alkoholies fermentasie geïdentifiseer. In vergelyking met die geiinokuleerde wyne, het die natuurlik gefermenteerde wyne intenser en ryper tropiese vrugtige aromas getoon, met ‘n verhoogde perspesie van soetheid en laer intensiteite van suurheid, bitterheid en vrankheid. Die wyne wat berei is deur fermentasie op die druifdoppe (FoS) het laer konsentrasies van esters en vlugtige swawelverbinding gehad, as die kontrole wyne (geen dopkontak) en beperkte dopkontak wyne. Sensoriese eienskappe gekoppel aan die FoS wyne was geasssosieer met gedroogde vrugte, gedroogde gras en 'n vegetatiewe aroma, met gepaardgaande verhoogde suur en bitter smaakpersepsies en ‘n frank mondgevoel. In teenstelling, het die wyne wat geproduseer is met 'n beperkte dopkontak (12 uur) hul tropiese vrugtige aroma beter behou as die FoS wyne. Beperkte dopkontak het ook ‘n minder negatiewe uitwerking op die smaakpersepies van bitterheid en vrankheid gehad. ‘n Verbruikerstudie is gedoen om 'n groep jong Generasie Y verbruikers (18-35 jaar) se voorkeure vir die verskillende behandelings te bepaal. Oor die algemeen is die natuurlike gefermenteerde wyne, wat beskryf is met 'n sterk tropiese vrugtekarakter verkies, terwyl die groep nie van die FoS wyne gehou het nie. Resultate wat verkry is met drie sensoriese analise metodes, onderskeidelik DA, PM en FC, was soortgelyk en die geskiktheid van die vinniger metodes, PM en FC, om kwalitatiewe inligting van witwyn se sensoriese eienskappe te verkry, is bevestig. Die resultate verkry met hierdie studie maak ‘n beduidende bydrae tot die validering van vinniger sensoriese metodes vir wynevaluering, wat veral waardevol is in die konteks van volhoubaarheid en tegnologieoordrag na navorsing en die industrie. Die nuwe kennis wat in hierdie studie gegenereer is met die chemiese profilering van SA Chenin blanc wyn is baie waardevol en hierdie is een van die eerste narvorsingsprojekte oor die inhoud van vlugtige swawelverbings in SA Chenin blanc wyn.

Dissertation (PhD(Agric)) -- Stellenbosch University, 2005. / ENGLISH ABSTRACT: Due to increased consumer demand for products labelled by origin, and the requirement that these labels are a guarantee of both quality and product character, there is an increasing global focus on delimitation of denominations of origin. The integrity of denominations of origin and their defensibility can be ensured through the use of terroirs as a basis for delimitation. The aims of this study were to establish the dominant environmental criteria that affect the viticultural behaviour and wine character of two important cultivars (Cabernet Sauvignon and Sauvignon blanc) in the Stellenbosch Wine of Origin District, to use an appropriate methodology to identify viticultural terroirs in this district based on these criteria and with the use of a geographic information system, and finally to use these viticultural terroirs to identify denominations of origin within the same area. A terroir can be defined as a grouping of homogenous environmental units, or natural terroir units, based on the typicality of the products obtained. Identification and characterisation of terroirs depends on knowledge of environmental parameters, the functioning of the grapevine and the characteristics of the final product. Field studies, resulting in point data, are necessary to investigate the functioning of the grapevine but in order for this information to be of use within zoning studies it must be placed in a spatial context. As a first phase in data acquisition, the Stellenbosch Wine of Origin District was characterised and natural terroir units were identified using existing digital data and a geographic information system. A natural terroir unit (NTU) can be defined as a unit of land that is characterised by relatively homogenous topography, climate, geological substrate and soil. A total of 1389 NTUs were identified in the Stellenbosch Wine of Origin District (84 537 ha). The identified NTUs were homogenous with respect to terrain morphological unit, altitude, aspect and soil type. Each of the identified units was further described with respect to the extent of the expected sea breeze effect and, for certain of the soil types, the associated parent material. As a second phase of data acquisition, a network of plots of Sauvignon blanc and Cabernet Sauvignon were delimited in commercial vineyards in proximity to weather stations and their viticultural and oenological response monitored for a period of seven years. Regression tree analyses were performed on the complete data set and the relative importance of the environmental and management related variables determined for each dependent variable. Excepting for scion clone, which had a high relative importance for bunch mass of Sauvignon blanc and yield to pruning mass index of Cabernet Sauvignon, no other non-environmental variable included in the analyses appeared to have a strong effect on grapevine performance and wine character. The performance of Cabernet Sauvignon was affected by the potassium content of the subsoil and the climate of the season. The performance of Sauvignon blanc appeared to be related to soil texture, wind exposure and temperature, both during the green berry growth stage and the month prior to ripening. From the results presented, it appears that environmental parameters have an overriding effect on the performance of both Cabernet Sauvignon and Sauvignon blanc, but that these two cultivars react differently to environmental stimuli. A knowledge-driven model used the rules generated in the regression tree analyses to directly classify natural terroir units with respect to expected response of Cabernet Sauvignon and Sauvignon blanc in the Stellenbosch Wine of Origin District. The natural terroir units were thus grouped into terroir units that were homogenous with respect to predicted viticultural and oenological response for each cultivar. The use of representative sites to determine the response of the grapevine to its environment is time consuming and costly and limits terroir studies to research related investigations. Vineyard managers were therefore surveyed with respect to the functioning of established Sauvignon blanc vineyards in the Stellenbosch Wine of Origin District in an attempt to obtain the necessary data. Comparison of the data generated with these questionnaires to measured data in commercial vineyards suggested that the vineyard managers were able to characterise the performance of vineyards with respect to vigour, signs of drought stress and yield. Each vineyard was mapped and the responses were linked to modelled environmental variables. Classification and regression trees were used to construct decision trees, which could be applied to environmental data in a geographic information system to determine viticultural terroirs for production of Sauvignon blanc. These terroirs, although fewer, were comparable to those generated using field data. Data gathered during terroir studies, and the identified viticultural terroirs for Cabernet Sauvignon and Sauvignon blanc, were used to revisit the boundaries of the Stellenbosch Wine of Origin District and the Simonsberg-Stellenbosch ward. Modifications were proposed based on expected wine characteristics. Boundaries for two new wards in the Helderberg basin were proposed. It was also possible to identify vineyards within a ward for the production of terroir specific wines. / AFRIKAANSE OPSOMMING: Toenemende verbruikersaanvraag vereis produkte waarvan die etikette nie net die oorsprong aandui nie, maar ook kan dien as ‘n waarborg vir die produk se kwaliteit en kenmerkendheid. Hierdie tendens verklaar die toenemende wereldwye fokus op afgebakende areas van oorsprong. Die integriteit van die afgebakende areas van oorsprong sowel as hul verdedigbaarheid kan gewaarborg word deur terroirs as basis vir afbakening te gebruik. Die doelstellings van die studie was om die oorheersende omgewingseienskappe wat wingerdprestasie en wynkarakter van twee belangrike cultivars, naamlik Cabernet Sauvignon en Sauvignon blanc, in die Stellenbosch Wyn van Oorsprongsdistrik bemvloed, te bepaal; tweedens om ‘n toepaslike metodologie te gebruik om terroirs in die die distrik te bepaal wat gebaseer is op die geidentifiseerde omgewingseienskappe met die gebruik van ‘n geografiese inligtingstelsel; en, ten slotte, om hierdie terroirs vir wingerbou te gebruik om afgebakende areas van oorsprong in dieselfde omgewing te identifiseer. ‘n Terroir kan gedefinieer word as ‘n samestelling van homogene omgewingseenhede, of natuurlike terroir-eenhede (NTE), wat gebaseer word op die kenmerkende eienskappe van die produkte wat daaruit verkry word. Identifisering en karakterisering van terroirs sal afhang van kennis van die omgewingsparameters, die funksionering van die wingerdstok en die eienskappe van die finale produk. Veldstudies waaruit puntdata verkry word, is noodsaaklik om die funksionering van die wingerdstok te ondersoek. Dit is egter noodsaaklik om eers hierdie inligting in ruimtelike konteks te plaas alvorens die inligting vir soneringstudies gebruik kan word. As 'n eerste fase van datagenerering, was die Stellenbosch Wyn van Oorsprongsdistrik gekarakteriseer en NTE’s geidentifiseer deur gebruik te maak van bestaande digitale data en ‘n geografiese inligtingstelsel. ‘n NTE kan gedefinieer word as ‘n landseenheid wat gekarakteriseer word deur ‘n relatiewe homogene topografie, klimaat, geologiese substraat en grondtipe. ‘n Totaal van 1389 NTE’s is geidentifiseer in die Stellenbosch Wyn van Oorsprongsdistrik (84 537 ha). Die geidentifiseerde NTE’s was homogeen met betrekking tot die terrein morfologiese eenheid, hoogte bo seespieSI, hellingsaspek en grondtipe. Elk van die geidentifiseerde eenhede was verder beskryf volgens die omvang van die seewindinvloed en, vir toepaslike grond tipes, die geassosieerde moedermateriaal. As ‘n tweede fase van datagenerering is ‘n netwerk van persele van Cabernet Sauvignon en Sauvignon blanc afgebaken binne bestaande kommersiele wingerde in die nabyheid van weerstasies. Hul wingerd- en wynkundige respons is vir ‘n periode van sewe jaar gemonitor. Regressieboomanalises is gebruik om die volledige stel data te analiseer en om die relatiewe belang van omgewings- en bestuurspraktykverbonde veranderlikes te bepaal. Die bostokkloon (wat 'n hoe relatiewe belang vir die trosmassa van Sauvignon blanc en die oes- tot snoeimassa verhouding van Cabernet Sauvignon het) is die enigste van die nie-omgewingsparameter wat ‘n sterk invloed op wingerdprestasie of wynkarakter blyk te he. Die prestasie van Cabernet Sauvignon is beTnvloed deur die kaliuminhoud van die ondergrond sowel as die seisoensklimaat. By Sauvignon blanc het dit voorgekom of die prestasie verband hou met grondtekstuur, windblootstelling en temperatuur tydens die groen fase van korrelgroei sowel as die maand voor rypwording. Alhoewel dit blyk uit die resultate dat omgewingsparameters 'n oorheersende invloed op die prestasie van beide Cabernet Sauvignon en Sauvignon blanc uitoefen, reageer die twee cultivars verskillend op omgewingsprikkels. ‘n Kennisgedrewe model waarvan die riglyne uit resultate van die regressieboomanalise saamgestel is, word gebruik om NTE direk te klassifiseer ten opsigte van die verwagte respons van Cabernet Sauvignon en Sauvignon blanc in die Stellenbosch Wyn van Oorsprongsdistrik. Die NTE is dus gegroepeer om terroir eenhede te vorm wat homogeen was ten opsigte van die verwagte wingerd- en wynkundige respons vir elke cultivar. Die gebruik van verwysingspersele om die respons van die wingerdstok teenoor sy onmiddelike omgewing te bepaal, is tydrowend en duur en beperk sodoende terroir studies tot navorsing. Gevolglik is ‘n opname onder wingerdbestuurders gemaak om inligting oor die prestasie van Sauvignon blanc in die Stellenbosch Wyn van Oorsprongsdistrik in te win en sodoende die nodige data te verkry. Na vergelyking van die ingewinde data, wat uit die opname verkry is, met gemete data vanaf kommersiele wingerde, kon afgelei word dat wingerdbestuurders by magte is om die prestasie van wingerde ten opsigte van groeikrag, tekens van droogtestres en opbrengs te karakteriseer. Elke wingerd is gekarteer en die respons is gekoppel aan die gemodeleerde omgewingsparamters. Klassifikasie en regressiebome is gebruik om besluitnemingsmodelle saam te stel wat toegepas kon word op omgewingsdata in ‘n geografiese inligtingstelsel om terroirs vir die produksie van Sauvignon blanc te bepaal. Hierdie terroirs, alhoewel minder, was vergelykbaar met die wat gegenereer was met behulp van veldstudies. Data verkry met terroirstudies, sowel as gei'dentifiseerde terroir-eenhede, was gebruik om die grense van die Stellenbosch Wyn van Oorsrongsdistrik en die Simonsberg-Stellenbosch wyk te herbepaal. Voorgestelde modifiserings was gebaseer op verwagte wyneienskappe. Grense vir twee nuwe wyke in die Helderbergkom is voorgestel. Dit was ook moontlik om wingerde binne ‘n wyk te identifiseer vir die produksie van terroir-spesifieke wyne.

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: During winemaking, a number of biochemical changes occur as a result of the metabolic activity of wine lactic acid bacteria (LAB) associated with malolactic fermentation (MLF). The latter process, which occurs mostly after alcoholic fermentation by wine yeasts, involves the conversion of L-malate to L-lactate and CO2, thus resulting to wine acidity reduction, microbiological stabilization and alterations of wine organoleptic quality. Although Oenococcus oeni is predominantly the most preferred species suitable for carrying out MLF in wine owing to its desirable oenological properties, Lactobacillus plantarum has also been considered as a potential candidate for MLF induction. Other species in the genera of Lactobacillus and Pediococcus are often associated with wine spoilage. These microorganisms induce wine spoilage by producing off-flavours derived from their metabolic activity. It is therefore of paramount importance to understand the mechanism by which wine microbiota cause spoilage. The purpose of this study was to investigate the presence of genes encoding enzymes of oenological relevance in wine-associated LAB strains. In order to achieve this, different sets of specific primers were designed and employed for a wide-scale genetic screening of wine LAB isolates for the presence of genes encoding enzymes involved in various metabolic pathways, such as citrate metabolism, amino acid metabolism, hydrolysis of glycosides, degradation of phenolic acids as well as proteolysis and peptidolysis. PCR detection results showed that the majority of the tested strains possessed most of the genes tested for. It was also noted that, among the O. oeni strains tested for the presence of the pad gene encoding a phenolic acid decarboxylase, only two strains possessed this gene. None of the O. oeni strains has previously been shown to possess the pad gene, and this study was the first to report on the presence of this gene in O. oeni strains. In an attempt to genetically characterize this putative gene, DNA fragments from the two positive O. oeni strains were sequenced. The newly determined sequences were compared to other closely related species. Surprisingly, no match was found when these sequences were compared to the published genomes of three O. oeni strains (PSU-1, ATCC BAA-1163 and AWRI B429). This reinforced a speculation that the pad gene in these two strains might have been acquired via the horizontal gene transfer. In addition, it remains to be further determined if the presence of this gene translates to volatile phenol production in wine. In this study, a novel strain isolated from South African grape and wine samples was also identified and characterized. The identification of this strain was performed through the 16S rDNA sequence analysis, which indicated that this strain belongs to Lactobacillus florum (99.9% sequence identity). A novel PCR assay using a species-specific primer for the rapid detection and identification of Lb. florum strains was also established. For further characterization, this strain was also investigated for the presence of genes encoding enzymes of oenological relevance. PCR detection results indicated that the Lb. florum strain also possess some of the genes tested for. In addition to genetic screening of wine LAB isolates for the presence of different genes, this study was also aimed at evaluating the regulation of the mleA gene encoding malate decarboxylase in three oenological strains of O. oeni. The regulation of this gene was tested in a synthetic wine medium under various conditions of pH and ethanol. From the expression analysis, it was observed that the mleA gene expression was negatively affected by high ethanol content in the medium. On the other hand, low pH of the medium seemed to favour the expression of this gene as the mleA gene expression was more pronounced at pH 3.2 than at pH 3.8. The findings from this study have shed more light on the distribution of a wide array of enzyme-encoding genes in LAB strains associated with winemaking. However, it remains unknown if the enzymes encoded by these genes are functional under oenological conditions, given that wine is such a hostile environment encompassing a multitude of unfavourable conditions for the enzymes to work on. Evaluating the expression of these genes will also help give more insights on the regulation of the genes under winemaking conditions. / AFRIKAANSE OPSOMMING: Gedurende wynmaak, sal 'n aantal biochemiese veranderinge plaasvind as gevolg van die metaboliese aktiwiteit van wyn melksuurbakterieë (MSB) wat betrokke is by appelmelksuurgisting (AMG). Die laasgenoemde proses, wat meestal na alkoholiese fermentasie deur wyngiste plaasvind, behels die omskepping van L-malaat na L-laktaat en CO2, om sodoende die wyn se suur te verminder, mikrobiologiese stabiliteit en verandering van wyn organoleptiese kwaliteit. Alhoewel Oenococcus oeni hoofsaaklik die mees gewenste spesies is wat geskik is vir die uitvoering van AMG in wyn weens sy geskikte wynkundige eienskappe, Lactobacillus plantarum word ook beskou as 'n potensiële kandidaat vir AMG induksie. Ander spesies in die genera Lactobacillus en Pediococcus word dikwels geassosieer met wynbederf. Hierdie mikro-organismes veroorsaak wynbederf deur die produksie van wangeure as gevolg van hul metaboliese aktiwiteite. Dit is dus van kardinale belang dat die meganisme van die wynbederf verstaan word. Die doel van hierdie studie was om die teenwoordigheid van koderend ensieme gene van wynkundige belang in wynverwante MSB stamme te ondersoek. Ten einde dit te bereik, was verskillende stelle van spesifieke peilers ontwerp en toegepas vir 'n groot skaal se genetiese toetsing van wyn MSB isolate vir die teenwoordigheid van ensiemkoderende gene betrokke by verskeie metaboliese paaie, soos sitraat metabolisme, aminosuur metabolisme, hidrolise van glikosiede, agteruitgang van fenoliese sure sowel as proteolise en peptidolise. PKR opsporings resultate het getoon dat die meerderheid van die stamme getoets, die meeste van die gene getoets voor besit. Dit is ook opgemerk dat, onder die O. oeni stamme getoets vir die teenwoordigheid van die pad geen, slegs twee stamme hierdie geen besit. Geen O. oeni stamme het voorheen gewys dat hul die pad geen besit, en hierdie studie was die eerste bewys oor die teenwoordigheid van hierdie geen in O. oeni stamme. In 'n poging om die geen geneties te karakteriseer, is DNA-fragmente van die twee positiewe O. oeni stamme se sekwens volgorde bepaal. Die DNA volgorde is vergelyk met ander nouverwante spesies. Verrassend, was geen passende DNA volgorde gevind met die gepubliseerde genome van drie O. oeni stamme (PSU-1, ATCC BAA-1163 en AWRI B429) nie. Dit versterk die spekulasie dat die pad geen in hierdie twee stamme via die horisontale geen-oordrag verkry is. Verder moet dit nog bepaal word of die teenwoordigheid van hierdie geen lei na vlugtige fenol produksie in wyn. In hierdie studie, is ongeïdentifiseerde stam geïsoleerd van Suid-Afrikaanse druiwe en wyn monsters ook geïdentifiseer en karakteriseer. Die identifisering van hierdie stam is uitgevoer deur middel van die 16S rDNA volgorde analise, wat aangedui het dat hierdie stam behoort aan Lactobacillus florum (99.9% volgorde identiteit). PKR toetse met behulp van die spesie-spesifieke peiler vir die vinnige opsporing en identifikasie van Lb. florum stamme is ook ontwikkel. Vir verdere karakterisering, was hierdie stam ook ondersoek vir die teenwoordigheid van koderende ensiem gene van wynkundige belang. PKR opsporings resultate het aangedui dat die Lb. florum stam ook oor 'n paar van die gene getoets voor besit. Bykomend tot genetiese toetsing van wyn MSB isolate vir die teenwoordigheid van verskillende gene, het die studie ook die evaluering van die regulering van die mleA geen, kodering malaatdekarboksilase in drie wyn stamme van O. oeni. Die regulering van hierdie geen was getoets in die sintetiese wynmedium onder verskillende pH en etanol kondisies. Van die uitdrukkingsresultate, is daar waargeneem dat die mleA geenuitdrukking is negatief geraak deur hoë etanol-inhoud in die medium. Aan die ander kant, in die lae pH medium was die uitdrukking van hierdie geen bevoordeel by pH 3.2 as by pH 3.8. Die bevindinge van hierdie studie het meer lig gewerp op die verspreiding van die wye verskeidenheid van ensiem-koderende gene in MSB stamme wat verband hou met wynmaak. Dit bly egter steeds onbekend of die ensieme gekodeer deur hierdie gene funksioneel is onder wynkondisies, gegewe dat wyn so 'n vyandige omgewing is menigte ongunstige toestande vir die werking van ensieme. Evaluering van die uitdrukking van hierdie gene sal ook help om meer insigte gee oor die regulering van die gene onder wynmaak toestande.

Thesis (PhD (Wine Biotechnology))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Alcoholic fermentation, and especially wine fermentation, is one of the most ancient microbiological processes utilized by man. Yeast of the species Saccharomyces cerevisiae are usually responsible for most of the fermentative activity, and many data sets clearly demonstrate the important impact of this species on the quality and character of the final product. However, many aspects of the genetic and metabolic processes that take place during alcoholic fermentation remain poorly understood, including the metabolic processes that impact on aroma and flavour of the fermentation product. To contribute to our understanding of these processes, this study took two approaches: In a first part, the initial aim had been to compare two techniques of transcriptome analysis, DNA oligo-microarrays and Serial Analysis of Gene Expression (SAGE), for their suitability to assess wine fermentation gene expression changes, and in particular to assess their potential to, in combination, provide combined quantitative and qualitative data for mRNA levels. The SAGE methodology however failed to produce conclusive data, and only the results of the microarray data are shown in this dissertation. These results provide a comprehensive overview of the transcriptomic changes during model wine fermentation, and serve as a reference database for the following experiments and for future studies using different fermentation conditions or genetically modified yeast. In a second part of the study, a screen to identify genes that impact on the formation of various important volatile aroma compounds including esters, fatty acids and higher alcohols is presented. Indeed, while the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the genetic regulation of the network and the physiological roles of individual pathways within the network. Various factors that directly or indirectly affect and regulate the network have been proposed in the past, but little conclusive evidence has been provided. To gain a better understanding of the regulations and physiological role of this network, we took a functional genomics approach by screening a subset of the EUROSCARF strain deletion library, and in particular genes encoding decarboxylases, dehydrogenases and reductases. Thus, ten genes whose deletion impacted most significantly on the aroma production network and higher alcohol formation were selected. Over-expression and single and multiple deletions of the selected genes were used to genetically assess their contribution to aroma production and to the Ehrlich pathway. The results demonstrate the sensitivity of the pathway to cellular redox homeostasis, strongly suggest direct roles for Thi3p, Aad6p and Hom2p, and highlight the important role of Bat2p in controlling the flux through the pathway. / AFRIKAANSE OPSOMMING: Alkoholiese fermentasie, en veral die maak van wyn, is een van die vroegste mikrobiologiese prosesse wat deur die mensdom ingespan is. Die gisspesie Saccharomyces cerevisiae is gewoonlik grotendeels verantwoordelik vir die fermentasie and verskeie vorige studies het gedemonstreer dat hierdie spesie ‘n baie belangrike rol speel in die uiteindelike kwaliteit en karakter van die voltooide produk. Nieteenstaande die feit is daar steeds baie aspekte van beide die genetiese en metaboliese prosesse wat plaasvind tydens alkoholiese fermentatsie wat nog swak verstaan word, insluitende metaboliese padweë wat ‘n impak het op die smaak en aroma van die fermentasie produk. Om ons kennis van die veld uit te brei het die studie twee aanslae geneem: In die eerste geval is gepoog om twee tegnieke van transkriptoom analiese, nl. DNA oligomikro- arrays en Serial Analysis of Gene Expression (SAGE) te bestudeer vir hul vermoë om geen ekspressie veranderinge tydens wynfermentasie te ondersoek en meer spesifiek om hul potensiaal om ‘n kombinasie van kwantitatiewe sowel as kwalitatiewe data met betreking to mRNA vlakke te produseer. Die SAGE metode kon egter geen betroubare resultate produseer nie en dus word slegs die resultate van die mikro-array eksperimente in die tesis bespreek. Die resultaat is ‘n geheeloorsig oor die geenekspressie veranderinge wat so ‘n wyngis tydens alkoholiese fermentasie ondergaan en dien as ‘n verwysingsraamwerk vir toekomstige studies met geneties gemodifiseerde gis of selfs verskillende fermentasieparameters. Die tweede deel van die studie het gefokus op die identifikasie van gene wat ‘n impak het op die vorming van belangrike, vlugtige aroma komponente, o. a. Esters vetsure en hoër alkohole d.m.v. ‘n siftingseksperiment. Alhoewel daar redelik baie inligting is oor die onderligende metaboliese netwerke wat lei tot die vorming van die verbindings, is daar min kennis van die genetiese regulasie van die netwerk en die fisiologiese rol van individuele padweë wat die netwerk vorm. Verskeie faktore – wat of die netwerk direk of indirek affekteer – is al voorgestel, meer met min konkrete bewyse. Dus het ons gepoog om meer lig op die onderwerp te laat m.b.v. ‘n funksionele genoom aanslag deur ‘n siftingseksperiment te doen op ‘n subgroep (spesifiek gene wat kodeer vir dekarboksilase, dehidrogenase en reduktase ensieme) van die EUROSCARF delesiebiblioteek. Dus is tien gene geïdentifiseer – die delesie waarvan ‘n merkbare effek het op die aroma produksie netwerk en spesifiek die van hoër alkohole. Ooruitdrukkings en enkel en meervoudige delesie rasse van die tien gene is gemaak om d.mv. genetiese analiese, hulle rol in aroma produksie en die Ehrlich padweh uit te pluis. Die resultate toon dat hierdie padweg sensitief is teenoor die sellulêre redoks balans en dui op direkte rolle vir Thi3p, Aad6p en Hom2p, asook dat Bat2p ‘n baie belangrike rol speel in die werking van die padweg.

Thesis (PhD(Agric))--Stellenbosch University, 2014. / ENGLISH ABSTRACT: This study focussed on the effect of oxygen on the chemical and sensory evolution of Sauvignon blanc wine under controlled oxidation conditions. The sensorial interactive effects between Sauvignon blanc varietal aroma compounds and compounds that typically arise during oxidation of white wines were also investigated. In the first research chapter the sensorial interactive effects of Sauvignon blanc impact compounds with aldehydes typically originating from oxidation of white wines were investigated. Four compounds, 3‐ mercaptohexan‐1‐ol (3MH), 3‐isobutyl‐2‐methoxypyrazine (IBMP), 3‐(methylthio)‐propionaldehyde (methional) and phenylacetaldehyde were added together in a model wine medium at varying concentrations. The concentrations chosen were according to those reported in literature to occur in Sauvignon blanc wines. The sensory effects of these compounds were profiled using a trained sensory panel. Compounds were first profiled individually and results showed that the change in comcpound concentration not only led to a change in intensity ratings but also in some cases a change in the descriptor. All four compounds in the same sample showed complicated interactive effects. Data were statistically analysed using relatively novel techniques such as statistical networks that allowed deeper insights into the interactions involved. Various observations were made such as the contribution of 3MH to the ‘green’ character of the wine, the potent suppressing abilities of methional on 3MH and IBMP and the additive effect of methional and IBMP contributing to the ‘cooked’ character of the sample. In the second research chapter the effect of repetitive oxidation on a fresh and fruity style Sauvignon blanc wine was investigated. Results showed the progress and evolution of aromatic and non‐aromatic compounds during an oxidative aging period. A large range of chemical analyses were conducted together with extensive sensory profiling. Results showed a decrease in volatile thiols responsible for the fruity nuances and an increase in oxidation‐related compounds, such as acetaldehyde, during the course of the oxidation. Sensory profiling showed the evolution of the wine aroma during oxidation. The wine evolved from a fresh and fruity wine to slight oxidation and then developed extreme oxidative characteristics. The Control samples (no oxygen added) developed a ‘cooked’ character which could indicate the formation of reductive compounds in these wines. Conversely, the wines that received a single dose of oxygen did not develop this flavour and were perceived to be more fresh and fruity than the Control samples. The evolution of the wine colour was also monitored using a spectrophotometer as well as a sensory panel and results suggest that the colour of the wine evolved before the disappearance of the pleasant aroma. The advantages and disadvantages of oxygen exposure to this type of wine style are discussed as well as the complexity of the wine matrix and sensory interactions occurring in the specific wine. The aim of the third research chapter was again to investigate further sensory interactions between Sauvignon blanc varietal aroma compounds and an oxidation‐related compound. After the observations reported in the second research chapter, the inclusion of acetaldehyde in an interaction study seemed to be of critical importance. Acetaldehyde can reach significant concentrations during oxidation and can have a detrimental effect on wine aroma. This interaction study included three compounds, 3MH, IBMP and acetaldehyde. Acetaldehyde was able to effectively mask the ‘green’ character of the sample, while it also enhanced certain fruity nuances when present at specific concentrations. 3MH was able to suppress the oxidising character of acetaldehyde when present at sufficient concentrations. The results from this study clearly showed the complexity of the wine medium and the interactions involved. It also highlighted the importance of performing these types of sensory studies in a simple medium opposed to a complex matrix such as wine. The effect of oxygen on various aspects of the wine was investigated and the combination of chemical and sensory data delivered some interesting conclusions also involving interactions that occurred. This study paved the way for future investigations on the sensory relationships of Sauvignon blanc aroma compounds and the role of proper oxygen management in the production of quality wines. / AFRIKAANSE OPSOMMING: Die doelwit van die studie was om die effek van gekontroleerde oksidasie op die ontwikkeling en verloop van verskeie chemiese komponente asook die ontwikkeling van die sensoriese profiel van ‘n Sauvignon blanc wyn te ondersoek. Bykomende studies ondersoek die interaksie/wisselwerking tussen aromakomponente in ‘n eenvoudige matriks. Interaksies tussen spesifieke Sauvignon blanc kultivargeassosieerde aromakomponente en komponente wat normaalweg tydens die oksidasie van witwyne ontwikkel, word ondersoek. Die eerste navorsingshoofstuk het ten doel om die interaksie tussen tipiese Sauvignon blanc aromakomponente (of impakkomponente) met aldehiede wat normaalweg tydens oksidasie van witwyne ontwikkel, na te vors. Vier komponente is in ‘n modelwyn gevoeg teen verskeie konsentrasies, wat oor die algemeen in die literatuur gerapporteer is om voor the kom in Sauvignon blanc wyne. Die komponente wat ondersoek is, is: 3‐merkaptoheksanol (3MH), 3‐isobutiel‐2‐metoksiepirasien (IBMP), 3‐ (metieltio)‐propionaldehied (methional) en fenielasetaldehied. Die sensoriese effekte van die komponente is deur ‘n opgeleide sensoriese paneel geëvalueer. Komponente is eers individueel geanaliseer en die resultate het getoon dat dat die intensiteit van die spesifieke aroma verander namate die konsentrasie verander. In sommige gevalle het die beskrywende woord vir die aroma ook verander. Deur al vier komponente in dieselfde monster te voeg word die ondersoek gekompliseer. Die data is statisties geanaliseer deur gebruik te maak van relatiewe nuwe tegnieke soos statistiese netwerke wat dieper insig in die betrokke interaksies bewerkstellig. Verskeie waarnemings word gerapporteer onder andere die bydrae van 3MH tot die ‘groen’ karakter van die wyn, die kragtige onderdrukkingsvermoë van methional op 3MH en IBMP asook die opbouende effek van methional en IBMP wat bydra tot die ‘gekookte’ karakter van die monster. Die tweede navorsingshoofstuk is daarop gemik om die effek van herhalende oksidasie op 'n vars en vrugtige styl Sauvignon blanc wyn te ondersoek en om die vordering en ontwikkeling van aromatiese en nie‐aromatiese komponente gedurende hierdie tydperk te analiseer. ‘n Wye reeks chemiese komponente is geanaliseer tesame met omvangryke sensoriese analise. ‘n Afname in die vlugtige tiole, wat verantwoordelik is vir die vrugtige geure, is gevind tesame met ‘n toename in oksidasie‐verwante komponente (soos asetaldehied). Sensoriese ondersoeke toon ook die evolusie van die wynaroma tydens oksidasie. Die wyn het ontwikkel van ‘n vars en vrugtige styl na effense oksidasiegeure waarna ekstreme oksidasiekarakters waargeneem is. Die Kontrole monsters het ‘n ‘gekookte’ karakter ontwikkel wat ‘n aanduiding van die ontwikkeling van ‘reduktiewe’ komponente in hierdie wyn kan wees. Aan die ander kant het wyne wat een suurstofdosering ontvang het, geen van hierdie geure ontwikkel nie en die wyn is as varser en vrugtiger beskryf in vergelyking met die Kontrole monsters. Die ontwikkeling van die wynkleur is ook gemonitor deur gebruik te maak van ‘n spektrofotometer asook 'n sensoriese paneel. Resultate stel voor dat die kleur van die wyn ontwikkel voor die aangename geure begin verdwyn. Die voor‐ en nadele van suurstofblootstelling aan hierdie tipe wynstyl word bespreek asook die kompleksiteit van die wynmatriks en sensoriese interaksies wat in hierdie spesifieke wyn voorkom. Die derde navorsingshoofstuk is weereens daarop gemik om die sensoriese interaksie tussen tipiese Sauvignon blanc kultivar‐geassosieerde aromakomponente en nog ‘n oksidasie‐geassosierde aromakomponent te ondersoek. Die resultate vanuit die tweede navorsingshoofstuk het die insluiting van asetaldehied in die interaksiestudie genoodsaak. Asetaldehied kan betekenisvolle konsentrasies tydens oksidasie behaal en kan ook nadelige effekte op wynaroma hê. Hierdie interaksiestudie het die volgende drie komponente ingesluit: 3MH, IBMP en asetaldehied. Asetaldehied het die ‘groen’ karakter van IBMP effektief gemaskeer terwyl dit die waarneming van die vrugtige aroma ondersteun en selfs verhoog het wanneer dit teen sekere konsentrasies teenwoordig was. 3‐Merkaptoheksanol het die oksidasiekarakter van asetaldehied onderdruk wanneer dit teen genoegsame konsentrasies teenwoordig was. Die kompleksiteit van wyn as ‘n navorsingsmedium is duidelik vanuit die studie veral in die ondersoeking van interaksie‐effekte tussen komponente. Die belangrikheid van die gebruik van 'n eenvoudige medium teenoor ‘n komplekse medium vir soortgelyke studies is dus duidelik. Die effek van suurstof op verskeie aspekte van witwyn is ondersoek en die kombinasie van chemiese en sensoriese data het interessante gevolgtrekkings gelewer. Die studie het die pad vir toekomstige studies gebaan in terme van sensoriese interaksies met betrekking tot Sauvignon blanc aroma. Die belangrikheid van oordeelkundige suurstofbestuur tydens die produksie van kwaliteit wyne is ook uitgelig.