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, 2014. / ENGLISH ABSTRACT: Previous studies have indicated that fermentation with yeast strains whose cell walls contain higher chitin levels may lead to reduced wine haze formation. In order to adjust cell wall chitin levels, more information on the regulation of chitin synthesis in wine-relevant yeast is required. Yeast cells are known to increase chitin levels when subjected to certain environmental changes such as an increase in temperature. The main aim of this project was to investigate chitin accumulation and synthesis in wine yeast strains when exposed to environmental change. This was achieved by subjecting the strains to various environmental conditions and comparing chitin levels. The information gained may aid future selection and/or manipulation of yeast strains for the production of higher chitin levels. Three Saccharomyces cerevisiae strains and two Saccharomyces paradoxus strains were subjected to conditions that had been linked to a change in chitin synthesis in past studies in laboratory yeast strains. Of the conditions used in this study, the addition of calcium to a rich media led to the highest cell wall chitin levels. The data also show that chitin synthesis is largely strain dependant. Two conditions which resulted in increased chitin deposition were chosen for gene expression analyses, using strains with strongly diverging average chitin levels. Results showed that an increase in chitin levels correlates with an increase in expression of GFA1, the gene encoding for the first enzyme of the chitin synthesis pathway. Overall, this study provides novel insights into chitin synthesis in Saccharomyces cerevisiae wine yeast strains as well as Saccharomyces paradoxus strains, with possible future implications on haze prevention studies. / AFRIKAANSE OPSOMMING: Vorige studies het aangetoon dat fermentasie met gisrasse waarvan die selwande hoë chitienvlakke bevat, kan lei tot verminderde wynwaasvorming. Om selwandchitienvlakke aan te pas, word daar meer inligting rakende die regulering van chitienvlakke in wyn gisrasse verlang. Dit is bekend dat gisselle chitienvlakke verhoog wanneer die selle onderwerp word aan sekere veranderinge in die omgewing soos ’n verhoging in temperatuur. Die hoofdoel van hierdie projek was om die chitienopbou en -sintese in wyngisrasse te ondersoek waar gis blootgestel word aan omgewingsveranderinge. Dit is bereik deur die selle aan verskeie omgewingstoestande bloot te stel en chitienvlakke met mekaar te vergelyk. Die inligting hieruit verkry kan toekomstige gisraskeuses asook die manipulering van gisrasse met die oog op hoër vlakke van chitienproduksie vergemaklik. Drie Saccharomyces cerevisiae rasse en twee Saccharomyces paradoxus rasse is onderwerp aan toestande wat in vorige studies gekoppel is aan ’n verandering in chitienvorming in laboratorium-gisrasse. Van die toestande toegepas in hierdie studie, het die toevoeging van kalsium tot ’n nutrientryke medium gelei tot die hoogste chitienvlakke in selwande. Die data toon ook aan dat chitiensintese hoofsaaklik rasverwant is. Twee toestande wat gelei het tot verhoogde chitienafsetting is gekies vir geen-uitdrukkingsanalise, terwyl rasse gebruik is met gemiddelde chitienvlakke wat wyd uiteenlopend is. Die resultate het getoon dat ’n verhoging in chitienvlakke ooreenstem met ’n verhoging in die uitdrukkingsvlakke van GFA1, die geen wat kodeer vir die eerste ensiem in die chitiensintesebaan. Oor die algemeen verskaf hierdie studie nuwe insigte oor chitiensintese in Saccharomyces cerevisiae wyngisrasse en Saccharomyces paradoxus rasse en verskaf dit belangrike inligting vir moontlike toekomstige studies oor waasvoorkoming.

Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Wine is a product of the fermentation of grape juice. Alcoholic fermentation is mainly conducted by the yeast Saccharomyces cerevisiae which metabolises grape sugars to mainly ethanol, CO2 and glycerol. Aside from these primary fermentation compounds, the yeast also produces many secondary metabolic by-products that are important to wine quality and style. Malolactic fermentation (MLF) is a secondary fermentation that normally occurs after alcoholic fermentation. Lactic acid bacteria (LAB) are responsible for the conversion of malic acid to lactic acid and CO2 during MLF, which is important for wine deacidification and also contributes to microbial stability. Malolactic fermentation and LAB strains can also influence the aroma profile of wines. The main genera associated with this process are Oenococcus, Lactobacillus, Pediococcus and Leuconostoc. Oenococcus oeni is the main species associated with MLF because it is able to survive the harsh physiochemical environment of winemaking. Recently L. plantarum has also been introduced as a commercial MLF starter culture. Research has started to focus on the potential of wine yeast and LAB interactions or combinations to alter the wine aroma profile via the production and/or degradation of aroma compounds. The overriding goal of this study is to unravel the interactions between wine yeast and different LAB strains and their impact on wine aroma and flavour. The first aim was to assess LAB growth during co- and sequential inoculation strategies, the ability to complete MLF and the impact on the production of aroma compounds in combination with two different yeast strains in a medium containing full complement of nitrogen supplementation. Malolactic fermentation was successful in the different inoculation strategies and the bacterial combination (L. plantarum and O. oeni) completed MLF in the shortest time. The impact of the bacterial strains on the modification of aroma compounds was bigger in co- than sequential inoculation. A general increase in total esters (contributing to the fruity character of wines) especially ethyl lactate and ethyl acetate was observed. The production of esters, volatile fatty acids and higher alcohols proved to be dependent on either the yeast strain used and/or the LAB strains used. The second aim of the research was to assess the effect of NH4Cl (ammonium) and amino acids supplementation on yeast and LAB strains (both in co- and sequential inoculation strategies) and the impact on the aroma profile of the fermented must. Fermentations supplemented with ammonia as sole nitrogen source showed the highest total bacterial growth in terms of cell numbers. Malolactic fermentation was completed in the shortest time with O. oeni and the bacterial combination inoculums. The co-inoculated strategies in combination with amino acids supplementation showed the biggest impact on the aroma compound profiles of the different fermentation strategies and bacterial treatments. A general increase in total esters was observed for NH4Cl additions with ethyl lactate and ethyl acetate showing the highest concentrations. The concentration of esters, volatile fatty acids and higher alcohols were strongly influenced by the yeast and the single LAB strains used. The results generated from this study showed that the chemical composition of the fermentation medium and the selection of yeast and LAB strains are important because these factors have an influence on the aroma and flavour profiles of wines. / AFRIKAANSE OPSOMMING: Wyn is die produk van gefermenteerde druiwe. Die gis, Saccharomyces cerevisiae is verantwoordelik vir alkoholiese fermentasies waar druiwe suikers na hoofsaaklik etanol, CO2 en gliserol gemetaboliseer word. Die gis produseer ook sekondêre metaboliete wat ‘n belangrike bydrae lewer tot wynstyl en kwaliteit. Appelmelksuurgisting (AMG) is ‘n sekondêre fermentasie wat gewoonlik na alkoholiese fermentasie plaasvind. Melksuurbakterieë (MSB) speel ‘n sleutel rol in die omskakeling van appelsuur na melksuur en CO2 gedurende AMG. Hierdie fermentasie lei tot ‘n afname in die suurheidsgraad en verbeter die mikrobiese stabiliteit van die wyn. Appelmelksuurgisting en MSB rasse kan die aroma- en geurprofiel van wyne beïnvloed. Die belangrikste genera wat met AMG geassosieer word is Oenococcus, Lactobacillus, Pediococcus en Leuconostoc. Oenococcus oeni is die mees algemene ras wat vir AMG gebruik word omdat dit in uiterste wyn toestande kan oorleef. Mees onlangs is Lactobacillus plantarum as kommersiële aanvangskultuur vir AMG geïdentifiseer. Navorsing het onlangs meer begin fokus op gis en MSB interaksie of kombinasies as ‘n strategie om die aroma profiele van wyne te verander. Die hoofdoel van die studie is om die interaksie tussen wyngiste en verskillende MSB rasse en die effek op die aroma profile van wyne te bestudeer. Die eerste doelwit was om die impak van die twee giste op die groei en AMG vermoeë van MSB gedurende ko- en sekwensiële inokulasie praktyke en die impak op die produksie van aroma komponente, in ‘n medium wat die volledige stikstof aanvullings bevat, te bestudeer. Appelmelksuurgisting was suksesvol in die verskillende inokulasie praktyke en die bakteriese kombinasie (L. plantarum en O. oeni) het AMG in die kortste tyd voltooi. Die impak van die bakteriese rasse op die modifikasie van die aroma komponente was groter met ko- as sekwensiële inokulasies. Daar was ‘n toename in die totale esterkonsentrasies veral in etiellaktaat en etielasetaat. Die produksie van esters, vlugtige vetsure en hoër alkohole word beïnvloed deur die gisras en MSB rasse wat gebruik word. Die tweede doelwit was om die impak van NH4Cl (ammonium) en aminosure aanvullings op die gis- en MSB rasse gedurende ko- en sekwensiële inokulasie strategieë te bepaal. Melksuurbakterieë se groei was beter met die ammonium aanvulling. Appelmelksuurgisting was in die kortste tyd voltooi met O. oeni en die bakteriese kombinasie. Die ko-inokulasie praktyke in kombinasie met die kompleks aminosure aanvulling het die grootste impak op die produksie van aroma komponente gehad. Daar was weereens ‘n toename in die totale esterkonsentrasies vir die NH4Cl aanvulling, veral in etiellaktaat en etielasetaat. Die gis en MSB rasse speel ‘n rol by die produksie en konsentrasies van esters, vlugtige vetsure en hoër alkohole. Die resultate van hierdie studie bewys dat die chemiese samestelling van die fermentasie medium, die seleksie van gis- en MSB rasse is belangrik omdat hierdie faktore die aroma en geur profiele van wyne beïnvloed.

Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 92-103). Also available on the World Wide Web.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Yeast strains contend with numerous stresses during winemaking. An inability to perceive and initiate the physiological changes needed to adapt to stress, has been linked to slow or incomplete (residual sugar > 4 g/L) fermentations. Wine yeast strains differ in genotype; this is manifested as differences in their stress tolerance, and fermentation performance. The first goal of this study was to evaluate how the initial sugar (200 or 240 g/L) and nitrogen (50, 100, 250, or 400 mg/L) content, and the fermentation temperature (15°C or 20°C) affected the fermentation performance of 17 commercial wine yeast strains. Fermentation performance was evaluated based on the fermentation kinetics (lag phase, maximum fermentation rate and total weight loss by CO2 evolution), residual sugar content and yeast dry weight. The results demonstrate that the fermentation performances of commercial yeast cultures are significantly and differently affected by initial nitrogen and sugar levels, as well as the fermentation temperature. Additionally, excess nitrogen had a negative impact on the fermentation kinetics and sugar consumption. Nitrogen deficiency is a common cause of slow and incomplete fermentations, as it affects yeast growth and thus fermentation rates. Nitrogen supplements are routinely added at the onset of fermentation, reducing the risk of problematic fermentations. Therefore characterising the fermentative ability of a strain over a range of oenologically relevant conditions, could aid winemakers in selecting a yeast strain capable of fermenting a grape must (of known sugar and nitrogen levels) to completion at the desired fermentation temperature. Investigations on fermentation related stress generally focus on its influence on fermentation rate and sugar consumption. However, from a winemaking perspective, the strain’s ability to produce the desired volatile aroma compounds is equally important. Yet, literature provides little insight into the influence stress has on the volatile aroma profile; this is surprising as wine aroma is closely linked to wine quality and consumer liking. The final goal of this study was to evaluate changes to the volatile aroma profiles produced by five commercial yeast strains, in response to hyperosmotic and temperature stress. The concentrations of the aroma compounds were quantified using a gas chromatograph coupled to a flame ionization detector. The results show that hyperosmotic and temperature stress caused significant changes in the levels of a number of aroma compounds. Furthermore, the changes observed differed among the evaluated strains, as well as for the fermentation stress treatments studied. Future aims should be directed towards the potential application of yeast strain selection as a means to avoid problematic fermentations in grape must; in addition to the further characterisation of the relationship between stress and the resultant volatile aroma profile in wine. / AFRIKAANSE OPSOMMING: Gisrasse moet verskeie stresfaktore afweer tydens die wynmaak proses. Die onvermoë van ‘n wyngis om stres waar te neem en die nodige fisiologiese veranderinge te inisieer om aan te pas by die strestoestande word met slepende of onvolledige fermentasies (met ‘n residuele suiker van meer as 4 g/L) geassosieer. Wyngisrasse verkil in genotipe; wat as groot verskille in die graad van strestoleransie, en dus ook fermentasie sukses geopenbaar word. Die eerste doelwit van hierdie studie was om te evalueer hoe die suiker (200 of 240 g/L) en stikstof (50, 100, 250, of 400 mg/L), asook die fermentasie temperatuur (15°C of 20°C) die fermentasie prestasie van 17 kommersiële wyngiskulture beïnvloed. Die sukses van fermentasie is geëvalueer op grond van fermentasie kinetika (sloerfase, maksimum fermentasiespoed en totale gewigsverlies as CO2 verlies), die residuele suiker inhoud en die gis droë massa. Die resultate demonstreer dat die fermentasie sukses van kommersiële giskulture beduidend en verskillend beïnvloed word deur die aanvangsstikstof en – suikerkonsentrasies, asook die fermentasie temperatuur. Daarbenewens, wanneer stikstof in oormaat teenwoordig is kan dit ‘n negatiewe impak op fermentasietempo en suiker metabolisme hê. Beperkende vlakke van stikstof ‘n algemene oorsaak van slepende of onvolledige fermentasies, aangesien stikstof die groei en gevolglik ook die fermentasiespoed van gis beïnvloed. Stikstofaanvullings word dikwels tot druiwemos toegevoeg aan die begin van gisting, wat die risiko van probleemfermentasies verlaag. Dus kan die karakterisering van die fermentasievermoë van ‘n gisras vir ‘n reeks wynkundig relevante kondisies die wynmaker help om ‘n gisras te selekteer wat in staat is om ‘n druiwemos (waarvan die suiker en stikstofvlakke bekend is) droog te gis by die gewenste temperatuur. Meeste studies wat fermentasieverwante stress ondersoek, fokus op die die invloed daarvan op fermentasietempo en suikerverbruik. Van ‘n wynmaakperspektief is die gis se vermoë om die gewensde vlugtige aroma komponente te produseer egter ewe belangrik as die vermoë om fermentasie te voltooi. Tog verskaf die literatuur min insig tot die invloed van stres op die vlugtige aromaprofiel; wat verbasend is aangesien die aromaprofiel ‘n belangrike faktor is van die waargenome wynkwaliteit en daarom ook verbruikersvoorkeur. Die finale doelwit van hierdie projek was om die veranderinge tot die vlugtige aromaprofiel geproduseer deur vyf kommersiële gisrasse in reaksie op hiperosmotiese stres en temperatuur stres te evalueer. Die konsentrasies van die aromakomponente is gekwantifiseer deur gas chromatografie gekoppel aan vlam‐ioniserende deteksie. Die resultate wys dat hiperosmotiese‐ en temperatuur stres beduidende veranderinge meebring in die vlakke van ‘n aantal aromakomponente. Verder is die waargenome veranderinge ook verskillend vir die geëvalueerde gisrasse, asook vir die verskille stresbehandelings wat ondersoek is. Toekomstige studies behoort gerig te wees op die toepassing van gis seleksie om potensiële probleemfermentasies in druiwemos te voorkom; asook die verdere karakterisering van die verhouding tussen omgewingstresfaktore en die gevolglike vlugtige aromaprofiel in wyn.

La levure Saccharomyces cerevisiae joue un rôle essentiel dans la production de composés aromatiques, tels que les esters, les alcools supérieurs et les acides organiques, ainsi que dans la transformation de précurseurs d'arômes du raisin pendant la fermentation du vin. Afin d'identifier les bases génomiques et métaboliques de ces propriétés, un croisement a été réalisé entre deux souches de levures de vin, sélectionnées pour leurs besoins en azote différents lors de la fermentation. 130 ségrégants de génération F2 ont été génotypés par séquençage complet du génome et individuellement phénotypés pendant la fermentation en mesurant les métabolites extracellulaires par HPLC et GC-MS. Les flux métaboliques intracellulaires ont été estimés à l’aide d’un modèle à base de contraintes. Une analyse QTL (quantitative trait locus) a été utilisée pour identifier les allèles influençant les variations d'arômes et de flux métaboliques. Plus de 80 QTL expliquant la variation de 59 caractères quantitatifs ont été détectés. Ces caractères comprennent des paramètres fermentaires, de consommation de substrat, la production de principaux métabolites et d’arômes fermentaires, ainsi que le métabolisme de composés aromatiques du raisin. L’intérêt de la cartographie QTL pour identifier les déterminants génétiques de variations de flux intracellulaires (f-QTLs) a par ailleurs été démontrée. Les QTL détectés ont été disséqués et des gènes dont les allèles contribuent spécifiquement aux variations phénotypiques ont été identifiés. Ces résultats soulignent la complexité génomique et métabolique de la synthèse et de la transformation d'arômes par la levure. L'identification de ces déterminants génétiques permet de mieux comprendre les liens entre variation génétique des levures et traits technologiques et fournit une base précieuse pour le développement de souches optimisées par des stratégies génétiques de croisement assisté par marqueurs. / The yeast Saccharomyces cerevisiae plays a vital role in the production of aroma compounds, such as esters, higher alcohols and organic acids, and the conversion of grape-derived aroma precursors during wine fermentation. To identify the genomic and metabolic bases for these processes, a cross was performed between two wine yeast strains selected because of their different nitrogen requirement during fermentation. 130 F2-segregants were genotyped by whole genome sequencing and individually phenotyped during wine fermentation by measuring extracellular metabolites using HPLC and GC-MS. Intracellular metabolic fluxes were estimated by constraint-based modeling. Quantitative trait locus (QTL) mapping was used to identify allelic variants influencing variations in the aroma profile and metabolic fluxes. More than 80 QTLs explaining variation in 59 quantitative traits were detected. These traits consisted of general fermentation parameters, substrate consumption, the production of main metabolites and fermentative aromas and the metabolism of grape aroma compounds. The applicability of QTL mapping to detect regions influencing intracellular fluxes (f-QTLs) was furthermore demonstrated. Found QTLs were dissected and genes with allele specific contributions to the phenotype were identified. These results emphasize the genomic and metabolic complexity of yeast aroma formation. In addition, the identification of genetic determinants increases knowledge about the links between genetic variation and industrial traits and provides a valuable foundation for the development of optimized strains by marker-assisted selection breeding strategies.

Les bases moléculaires responsables des propriétés technologiques des levures œnologiques sont pas ou peu connues. Or, ces connaissances sont nécessaires pour mettre en œuvre des stratégies d'amélioration génétique des capacités fermentaires ou de l'impact organoleptique des levures par croisements et une meilleure exploitation de la biodiversité. Ces travaux de thèse ont visé à l'identification des bases génétiques de plusieurs propriétés d'intérêt, telles que la production de sulfites, la capacité fermentaire en milieu carencé en azote ou la formation d'esters. Une approche de recherche de QTL a été mise en œuvre en s'appuyant sur la création d'une population de ségrégants méiotiques issus du croisement de deux souches œnologiques aux caractéristiques contrastées. Une caractérisation des phénotypes des ségrégants associé à leur génotypage a permis d'identifier des QTL pour les différents traits étudiés. L'implication d'une région du génome dans le contrôle de la voie d'assimilation des sulfates a été démontrée. Ce locus est responsable de la variation phénotypique de plusieurs métabolites de cette voie et de métabolites indirectement connectés. D'autres QTL ont également pu être mis en évidence pour les capacités fermentaires sur un milieu carencé en azote et la production de différents composés volatils. Leur analyse a permis d'identifier plusieurs gènes candidats dont les fonctions sont liées directement ou indirectement aux phénotypes étudiés. / The molecular basis of technological properties of wine yeasts are not or poorly known. However, this knowledge is required for the improvement of the fermentation capacity and of the organoleptic impact of wine yeasts by breeding strategies and a better exploitation of the yeast biodiversity. This thesis aimed at identifying the genetic bases of several traits of interest such as: sulfite production, fermentation ability in low nitrogen medium or esters formation. A QTL mapping approach has been implemented from a population of meiotic segregants derived from the cross of two strains with contrasting oenological characteristics. Phenotyping of the a population of meiotic segregants was combined with a genotyping to identify QTL for these traits. The involvement of a region of the genome in the control of sulphate assimilation pathway has been demonstrated. This locus is responsible for the phenotypic variation of several metabolites of this pathway and indirectly connected metabolites. Other QTLs have also been demonstrated for the fermentation capacities on a nitrogen limiting medium and production of various volatile compounds. Their analysis revealed several candidate genes whose functions are related directly or indirectly to the studied phenotypes.

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Grape must is a complex medium, and during wine production numerous biochemical pathways and metabolic reactions are taking place simultaneously to produce a specific taste and aroma. Microorganisms, specifically yeast, play a key role in the formation of metabolites formed during alcoholic fermentation. Sauvignon blanc, a well studied grape cultivar, is known to have a versatile range of aroma profiles ranging from “green” to “tropical”. It has been broadly stated that a “green” Sauvignon blanc can be created in the vineyard and a “tropical” Sauvignon blanc can be created by selecting a specific yeast strain, and that the balance between “green” and “tropical” characters is essential for the final aroma profile. Except for grape-derived varietal aromatic compounds such as methoxypyrazines (green), volatile thiols (tropical) and monoterpenes (floral), yeast derived volatile compounds including esters, higher alcohols, fatty acids and carbonyl compounds will also contribute to the final wine aroma. The main aim of this study was to assess how viticultural treatment-derived differences in grape must, can impact on aroma production when this grape must is fermented with different commercial wine yeast strains. The viticulture treatment focused on light intensity modulated through canopy treatment. Volatile aroma differences were compared for canopy and yeast treatments, specifically focusing on the fermentation derived bouquet (esters, higher alcohols, volatile fatty acids, carbonyl compounds and monoterpenes). Results showed significant differences between initial must compositions, including titratable acidity, malic acid and yeast assimilable nitrogen. The volatile aroma compounds were also significantly impacted although no noticeable effect on the overall fermentation kinetics was observed. Depending on the yeast strain differences in volatile compounds varied. A clear vintage effect is noticeable between volatile compounds affected by the treatments. Data generated in 2012 shows clear differences between ethyl- and acetate esters and could clearly be grouped according to yeast strain through multivariate analysis. Sensory evaluation results could clearly be distinguished according to canopy treatment and to a lesser degree according to yeast strain used. This indicates that although yeast has a more prominent impact on the fermentative bouquet that develops during alcoholic fermentation the overriding aroma is primarily derived from grape-derived metabolites which can be manipulated by canopy treatments. None the less the difference in fermentation bouquet does add to the complexity of the wine especially in the case of fermentation derived “tropical” aromas including guava and passion fruit. In some cases where shaded grapes had higher ester concentrations, the resultant wine also had higher aroma quality. This study has contributed to a better understanding of the complex relationships between canopy manipulation and yeast selection on aroma formation. The analysis of volatile aroma alone however is not enough to understand the final perception of wine taste and further indepth studies of the viticultural and oenological factors is needed. In particular, this project has focused on a single vineyard over only two vintages. The general validity of the conclusions derived from this study therefore will require additional data sets. / AFRIKAANSE OPSOMMING: Druiwemos is ‘n komplekse medium en tydens wynbereiding is daar verskeie biochemiese weë en metaboliese reaksies wat gelyktydig plaasvind om ‘n spesifieke smaak en aroma te produseer. Mikro-organismes, veral gis, speel ‘n sleutelrol in die vorming van metaboliete tydens alkoholiese gisting. Sauvignon blanc, ‘n goed bestudeerde druifkultivar, besit ‘n veelsydige reeks aromaprofiele wat wissel van “groen” tot “tropies”. Oor die algemeen word dit voorgehou dat ‘n “groen” Sauvignon blanc in die wingerd geskep word, terwyl ‘n “tropiese” Sauvignon blanc geskep kan word deur ‘n spesifieke gisras te selekteer, en die balans tussen “groen” en “tropiese” karakters is noodsaaklik vir die finale aromaprofiel. Behalwe vir druifafgeleide kultivarafhanklike aromatiese verbindings soos metoksipirasiene (groen), vlugtige tiole (tropies) en monoterpene (blomagtig), sal gisafgeleide vlugtige komponente, waaronder esters, hoër alkohole, vetsure en karbonielverbindings, ook tot die finale wynaroma bydra. Die hoofdoelwit van hierdie studie was om te bepaal hoe verskille in druiwemos wat afkomstig is van wynkundige behandeling ‘n impak op aromaproduksie kan hê wanneer hierdie druiwemos met verskillende kommersiële wyngisrasse gegis word. Die wynkundige behandeling het gefokus op ligintensiteit wat deur lowerbehandeling gereguleer is. Vlugtige aromaverskille is op grond van lower- en gisbehandelings vergelyk, met ‘n spesifieke fokus op die gistingsafgeleide boeket (esters, hoër alkohole, vlugtige vetsure, karbonielverbindings en monoterpene). Die resultate het beduidende verskille getoon tussen aanvanklike mossamestellings, waaronder titreerbare suurheid, appelsuur en gis-assimileerbare stikstof. Daar was ook ‘n noemenswaardige impak op die vlugtige aromaverbindings, hoewel geen merkbare effek op die algehele gistingskinetika waargeneem kon word nie. Die verskille in vlugtige verbindings het gewissel op grond van die gisras. ‘n Duidelike oesjaareffek was merkbaar tussen vlugtige verbindings wat deur die behandelings geaffekteer is. Data wat in 2012 gegenereer is, toon duidelike verskille tussen etiel- en asetaatesters en kon duidelik m.b.v. meervariantanalise volgens gisras gegroepeer word. Die resultate van die sensoriese evaluering kon duidelik volgens lowerbehandeling onderskei word, en tot ‘n mindere mate volgens die gisras wat gebruik is. Dít dui daarop dat hoewel gis ‘n meer prominente impak het op die gistingsboeket wat tydens alkoholiese gisting ontwikkel, is die oorheersende aroma hoofsaaklik afgelei van druifafgeleide metaboliete wat deur lowerbehandelings gemanipuleer kan word. Nietemin dra die verskil in gistingsboeket by tot die kompleksiteit van die wyn, veral in die geval van gistingsafgeleide “tropiese” aromas, insluitend koejawel en grenadella. In sommige gevalle waar beskadude druiwe hoër esterkonsentrasies gehad het, het die gevolglike wyn ook ‘n hoër aromakwaliteit gehad. Hierdie studie dra by tot ‘n beter begrip van die effek van die komplekse verhoudings tussen lowermanipulasie en gisseleksie op aromavorming. ‘n Analise van vlugtige aroma alleen is egter nie voldoende om die finale persepsie van wynsmaak te begryp nie en bykomende diepgaande studies van die wingerdkundige en wynkundige faktore word benodig. Hierdie projek het in die besonder gefokus op ‘n enkele wingerd oor slegs twee oesjare. Die algemene geldigheid van die afleidings wat van hierdie studie gemaak word, sal dus bykomende datastelle vereis. / The National Research Foundation and Postgraduate Merit Bursary for financial support

Thesis (PhD (Viticulture and Oenology. Wine Biotechnology))--Stellenbosch University, 2008. / The occurrence of sluggish and stuck fermentations continues to be a serious problem in the global wine industry, leading to loss of product, low quality wines, cellar management problems and consequently to significant financial losses. Comprehensive research has shown that many different factors can act either in isolation, or more commonly synergistically, to negatively affect fermentative activity of wine yeast strains of the species Saccharomyces cerevisiae. The individual factors most commonly referred to in the literature are various nutrient and oxygen limitations. However, other factors have been shown to contribute to the problem. Because of the mostly synergistic nature of the impacts, no single factor can usually be identified as the primary cause of stuck fermentation. In this study, several strategies to evolutionarily engineer wine yeast strains that are expected to reduce the occurrence of stuck and sluggish fermentations are investigated. In particular, the investigations focus on improving the ability of wine yeast to better respond to two of the factors that commonly contribute to the occurrence of such fermentations, nitrogen limitation and the development of an unfavorable ratio of glucose and fructose during fermentation. The evolutionary engineering strategies relied on mass-mating or mutagenesis of successful commercial wine yeast strains to generate yeast populations of diverse genetic backgrounds. These culture populations were then exposed to enrichment procedures either in continuous or sequential batch cultivation conditions while applying specific evolutionary selection pressures. In one of the stragegies, yeast populations were subjected to continuous cultivation under hexose, and especially fructose, limitation. The data show that the strains selected after this procedure were usually able to out-compete the parental strains in these selective conditions. However, the improved phenotype was not detectable when strains were evaluated in laboratory scale wine fermentations. In contrast, the selection procedure in continuous cultivation in nitrogen limiting conditions proved to be highly efficient for the generation of yeast strains with higher total fermentative capacity in low nitrogen musts. Furthermore, yeast strains selected after mutagenesis and sequential batch cultivation in synthetic musts with a very low glucose on fructose ratio showed a fructose specific improvement in fermentative capacity. This phenotype, which corresponds to the desired outcome, was also present in laboratory scale wine fermentations, where the discrepancy between glucose and fructose utilization of the selected strains was significantly reduced when compared to the parents. Finally, a novel strategy for the rectification of stuck fermentations was adjusted to industrial conditions. The strategy is based on the use of a natural isolate of the yeast species Zygosaccharomyces bailii, which is known for its preference of fructose. This process was successfully established and implemented in the wine industry.