Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The microorganisms associated with grape berry surface can be influenced by numerous factors such as agronomic parameters. Hence, the focus of this study was comparison between three agronomic farming systems to evaluate their impact on yeast diversity. In addition, the dynamics of the yeast population throughout wine alcoholic fermentation were monitored. Three vineyards (conventional, biodynamic and integrated) were chosen and the experiment was carried out during the 2012 and 2013 vintages. A total of 600 yeast isolates including Saccharomyces and non-Saccharomyces were obtained from grape must and during different stages of fermentation including beginning, middle and end of alcoholic fermentation, from all three vineyards. Yeast species diversity in grape must and their population dynamics were evaluated by cultivating the yeasts in nutrient media and using “Polymerase Chain Reaction and sequence analysis of the ITS1-5.8S rRNA-ITS2 region. Eight, four and one species were detected from biodynamic, conventional and integrated must in 2012 vintage whereas, 2013 vintage displayed a higher diversity and 12, 11 and 9 different species were identified from biodynamic, conventional and integrated vineyard, respectively. Aureobasidium pullulans was the most frequent isolate in all three vineyards whereas Saccharomyces cerevisiae was below detection level in grape must and was only isolated in low frequencies in biodynamic must (3% of the total population) in both vintages. In general, the overlap of common yeast isolates (e.g. M. pulcherrima and H. uvarum) was observed in the musts obtained from different vineyards although unique minor species could be isolated and clearly demonstrated the distinction between the three vineyards. Moreover, biodynamic must displayed a higher degree of diversity in both 2012 and 2013 compared to the conventional and integrated vineyards. The beginning of all spontaneous fermentations was dominated by non-Saccharomyces yeast species (e.g. H. uvarum, C. zemplinina), as the fermentation proceeded, the population of non-Saccharomyces species were gradually decreased and strongly fermentative yeast S. cerevisiae dominated and completed the fermentations. The dynamics of S. cerevisiae strains was also evaluated during different stages of fermentation (beginning, middle and end), using interdelta PCR methods. A high diversity (10-18 strains per fermentation) and the sequential substitution of S. cerevisiae strains were observed throughout spontaneous fermentations. In addition, integrated vineyard displayed the highest S. cerevisiae strains compared to biodynamic and conventional vineyard. / AFRIKAANSE OPSOMMING: Die mikro-organismes wat met die oppervlak van druiwe bessies geassosieer word kan deur veskeie agronomiese faktore beїnvloed word. Gevolglik was die focus van die studie om ‘n vergelyking tussen die impak van drie verksillende boerdery sisteme op die invloed op gis diversiteit te bepaal. Die dinamiek van gis populasies tydens alkoholiese fermentasie is bykomstig bestudeer. Drie verskillende wingerde (konvesioneel, biodinamies en geïntegreerd) is gebruik vir die studie tydens die 2012 en 2013 oesjare. In total is 600 gis isolate, insluitend Saccharomyces en nie-Saccharomyces giste, verky van druiwe mos tydens verkillende fases van die fermentasie proses (begin, middle en einde) vir al drie wingerde. Die diversiteit en populasie dinamika van gis spesies in die druiwe mos is geëvalueer deur die giste in verskillendde media op te groei en ook deur die gebruik van die “polymerase ketting reaksie” (PKR) en DNS volgorde bepaling van die ITS1-5.8S rRNA-ITS2 gebied. Tydens die 2012 oesjaar is agt, vier en een afsonderlike spesies geїsoleer, in vergelyking met die 12, 11 en 9 verskillende spesies wat tydens 2013 geidentifiseer is is uit die biodinamiese, konsensionele en geïntegreerde onderskeidelik. Aureobasidium pullulans is teen die hoogste frekwensie geїsoleer in al drie wingerde, terwyl Saccharomyces cerevisiae onder die deteksie limiet was in druiwe mos en ook slegs in lae getalle in die biodinamiese mos (3% van die totale populasie) in beide oesjare. Oor die algemeen is ‘n oorvleuling tussen verwante spesies (bv. M. pulcherrima en H. uvarum) waargeneem en die mos vanaf verskillende wingerde, terwyl meer geringe spesies deurgans geїsoleer kon word en duidelik ‘n verkill tussen die drie wingerde uitgewys het. Druiwe mos uit die biodinamiese wingerd het verder ‘n hoёr graad van diversiteit en beide 2012 en 2013 vertoon as beide die konvesnionele en geïntegreerde wingerde. Die begin van alle spontane fermentasies was gedomineer deur die populasie van nie-Saccharomyces gis spesies (bv. H. uvarum, C. zemplinina), wat geleidelik afgeneem het met die verloop van die fermentasie. Die populasie van die sterk fermentatiewe, S. cerevisiae, het toegeneem tydens fermentasie en die fermentasie afgehanel as dominante gis. Die dinamika van S. cerevisiae rasse is ook geëvalueer tydens die verskillende fases van fermentasie (begin, middle en einde) deur gebruik te maak van interdelta PKR metodes. ‘n Hoё diversiteit (10-18 rasse per fermentasie) en die opeenvolgende verplasing van S. cerevisiae rasse was waargeneem deur die verloop van spontane fermentasies. Daarbenewens het die geïntegreerde wingerd die grootste getal S. cerevisiae rasse in vergelyking met die biodinamiese en konvensionele wingerde opgelewer.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The global wine industry has shifted to a more adopted ‘consumer-preference’ production. Modern wine consumers are more knowledgeable and cultivated in their understanding of wine quality, value and style. The quality of red wines mainly depends on grape composition, the wine making process and the ability of tasters to recognise sensory attributes. The harvest date/stage has an influence on the grape composition, thus making the decision on when to harvest an important factor in the production of quality wines or different wine styles. The traditional indicators used in the wine industry to determine time of harvest are more related to the perception of taste and mouthfeel and give little indication of the style of wine in terms of aromatic profile. A new physiological indicator using berry sugar accumulation for the purpose of sequential harvest is proposed to assist the winemaker in producing wines with possible different sensory profiles. This indicator can be used in association with the classical indicators of ripening to affect the diversity of wine styles from a single vineyard or a group of vineyards. The wines could thus have different potential aroma profiles, depending on when the grapes were harvested. The main aim of this study was to assess the effect of performing sequential harvest using a physiological indicator on red wine’s sensory composition. This was done to study the possible relation between harvest time (e.g. fruit composition evolution) and the wine styles/sensory attributes across the different harvest times, thereby possibly increasing the diversity of wine styles. A theoretical berry sugar loading concept was compiled and displays a phase of rapid sugar loading starting at véraison followed by a plateau phase. Depending on whether grapes were harvested in the beginning, mid or end of the plateau phase of fruit sugar accumulation the wines could have different potential aroma profiles. Three main stages: fresh fruit (FF), neutral (N) or pre ripe and mature fruit (MF) has been previously proposed using the sugar loading concept and in terms of harvesting dates. Cabernet Sauvignon and Merlot grapes form Eikendal Vineyards, Stellenbosch were used to make wines according to sequential harvest. Four harvest stages were considered, pre fresh fruit (Pre FF), fresh fruit (FF), mature fruit (MF) and over ripe (OR). The wines were tasted and analysed using two different sensory techniques. In both Merlot and Cabernet Sauvignon wines, the PreFF and OR stages could be more easily discriminated than the two harvest stages in-between, FF and MF. The results suggested that the wines made from the FF and MF stages could not be distinguished from each other in general when the attribute citation frequency method or sorting tasks were performed. However, a trend could be observed for both Cabernet Sauvignon and Merlot wines in terms of aroma attributes with attributes changing from green to ripe fruit during ripening using expert tasters. Relevant research should be engaged to refine sequential harvest in order to obtain more diverse wine styles from a single site or a group of vineyards. / AFRIKAANSE OPSOMMING: Die wêreldwye wynbedryf het ’n verskuiwing ondergaan na ’n verbruikersvoorkeurbenadering in produksie. Wynverbruikers is deesdae beter ingelig en meer ontwikkeld ten opsigte van hulle kennis van wyngehalte, wynstyl, asook die waarde van wyn. Die gehalte van rooiwyn hang hoofsaaklik af van die druifsamestelling, die wynmaakproses en die vermoë van proewers om sensoriese eienskappe te herken. Aangesien die oesdatum/-fase ’n invloed het op druifsamestelling, is die besluit oor wanneer daar geoes moet word ’n belangrike faktor in die vervaardiging van gehaltewyne of verskillende wynstyle. Die tradisionele aanwysers wat in die wynbedryf gebruik word om oestyd te bepaal, hou verband met die waarneming van smaak en mondgevoel en gee weinig aanduiding van die wynstyl op grond van die aromatiese profiel. ’n Nuwe fisiologiese aanwyser wat gebruik maak van suikerakkumulasie in die druiwekorrel in opeenvolgende oeste, het ten doel om die wynmaker te help om wyne met verskeie moontlike sensoriese profiele te vervaardig. Hierdie aanwyser kan saam met die klassieke aanwysers van rypwording gebruik word om ’n verskeidenheid wynstyle uit een wingerd of wingerdgroep te vervaardig. Die wyne kan dus potensieel oor verskillende aromatiese profiele beskik, afhangend van wanneer die druiwe geoes is. Die hoofdoel van die studie was om die invloed van opeenvolgende oes te toets deur ’n fisiologiese aanwyser op rooiwyn se sensoriese samestelling toe te pas. Dit word gedoen deur die moontlike verhouding tussen die oestyd (bv. ontwikkeling van vrugsamestelling) en die wynstyle of wyn se sensoriese kenmerke op verskillende oestye te bestudeer ten einde die verskeidenheid wynstyle potensieel te vermeerder. ’n Teoretiese konsep van druifsuikeropname is saamgestel wat dui op ’n fase van vinnige suikeropname wat by véraison begin, gevolg deur ’n plato-fase. Wyn kan oor verskillende moontlike aromatiese profiele beskik, afhangend daarvan of die druiwe aan die begin, middel of einde van die plato-fase van suikeropname geoes is. Drie hooffases is al voorheen voorgestel deur gebruik te maak van die konsep van suikeropname volgens oesdatum, te wete vars vrugte (VV) (“fresh fruit”, FF), neutraal (N) (“neutral”, N) of voor ryp (“pre ripe”), en ryp vrugte (RF) (“mature fruit”, MF). Cabernet Sauvignon- en Merlot-druiwe van Eikendal, Stellenbosch, se wingerde is gebruik om wyn volgens opeenvolgende oes te maak. Vier oesfases is oorweeg, te wete voor vars vrugte (VVV) (“pre fresh fruit”, Pre FF), vars vrugte (VV) (“fresh fruit”, FF), ryp vrugte (RV) (“mature fruit”, MF), en oorryp (OR) (“over ripe”, OR). Die wyn is geproe en geanaliseer deur gebruik te maak van twee verskille sensoriese tegnieke. In die geval van beide die Merlot- en Cabernet Sauvignon-wyn kon die VVV- en OR-fases makliker onderskei word as die twee tussenin-fases, VV en RV. Resultate dui daarop dat wyn wat van die VV- en RV-fases gemaak is, oor die algemeen nie van mekaar onderskei kan word wanneer die frekwensie van kenmerkaanhaling-metode en sorteringstaak uitgevoer word nie. ’n Tendens kon egter waargeneem word vir Cabernet Sauvignon- én Merlot-wyn ten opsigte van aromatiese kenmerke, deurdat kenmerke gedurende rypwording van groen na ryp vrugte verander het indien ekspertproewers gebruik is. Verdere navorsing moet gedoen word om opeenvolgende oes te verfyn ten einde ’n wyer verskeidenheid wynstyle van ’n enkele area of wingerdgroep te verkry.

Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: The present study deals in the first instance with the improvement of current analytical techniques for the analysis of the non-volatile content of wines. An improved sample preparation method, using solid phase extraction (SPE), was initially developed for the analysis of organic acids, sugars and phenolic compounds. Consequently, modem analytical methodologies were assessed to obtain optimal techniques for the separation of various non-volatile compounds. A capillary electrophoresis (CE) method, demonstrably more reliable than currently used high performance liquid chromatography (HPLC) and CE methods, is proposed for the analysis of organic acids. HPLC with refractive index (RI) or evaporative light scattering detection (ELSD) proved more suitable than CE for the analysis of sugars in dry wines. Liquid-chromatography-mass spectroscopy (LC-MS) offered superior sensitivity and resolution compared to the relatively new technique of CE-MS for the analysis of wine phenolics. LC-MS was further applied for the efficient and sensitive analysis of non-coloured phenolics and anthocyanins in wine. Negative- and positive electrospray ionisation, respectively, were used in conjunction with an ion-trap mass analyzer, for the identification of 34 phenolics and 31 anthocyanins in red wine samples. Complementary CE and LC methods were developed to allow the identification of artificial dyes in red wines, added illegally to improve their colour. Also, the application of stir bar sorptive extraction (SBSE) with liquid desorption and micellar electrokinetic chromatography (MEKC) for the analysis of bitter acids in beer is reported. In the second part of the thesis, the analytical results obtained for South African red and white wines were evaluated. Following comparison of the results with literature reports, several pattern recognition techniques were employed. A classification function obtained by linear discriminant analysis (LDA) was used to classify both red and white wines according to variety, based on their chemical composition. This classification is achieved independent of the factors of wine age or geographical origin, making it useful for authenticity evaluation. / AFRIKAANSE OPSOMMING: Hierdie studie het as primêre doel die verbetering van bestaande analitiese metodes vir die analise van nie-vlugtige komponente in wyn. In die lig hiervan, is eerstens 'n toepaslike monster-voorbereidingstegniek, gebasseer op soliede fase ekstraksie (SPE), ontwikkel vir die gelyktydige analise van organise sure, suikers en fenoliese komponente vanuit die wyn matriks. Vervolgens is moderne analitiese rtietodes ondersoek en gepastde skeidingstegnieke is ontwikkel vir die verskillende chemiese wyn-komponente. Kappillêre elektroforese (CE) en hoë-druk vloeistof-chromatografie (HPLC) in kombinasie met verskeie deteksie-metodes is vergelyk. Hieruit is 'n verbeterde CE metode vir die analise van organise sure is ontwikkel, terwyl HPLC in kombinasie met refraksie-indeks- en verdampings lig verstrooiings deteksie (ELSD) die beste resultate lewer vir die analise van suikers in droë wyne. Die toepasbaarheid van vloeistof-chromatografie met massa spektrometriese deteksie (LC-MS) vir die analise van fenoliese komponente is gedemonstreer, terwyl CE-MS onvoldoende resolusie en sensitiwiteit toon vir die analises. LC-MS is vervolgens ook gebruik vir die identifikasie van 34 fenoliese verbindings en 31 antosianiede in rooi wyn. Komplementêre HPLC en CE metodes is ontwikkel vir die identifikasie van onwettige sintetiese kleurstowwe in' rooi wyn. 'n Addisionele monstervoorbereidingsstap, roerstaaf sorptiewe ekstraksie (SBSE), is saam met vloeistof-desorpsie en misellêre elektrokinetiese chromatografie (MEKC) gebruik vir die analise van hops bitter sure in bier. In die tweede deel van die tesis word die resultate verkry m.b.v. bg. tegnieke vir die analise van Suid-Afrikaanse rooi- en wit wyne, bespreek. Die resultate is vergelyk met waardes uit die literatuur, en verskeie statistiese metodes is gebruik om die data te ondersoek. Met behulp van chemometriese metodes is 'n klassifikasie funksie verkry wat die klassifikasie van Suid-Afrikaanse wyne volgens druifsoort, gebasseer op die chemiese samestelling van die wyne, toelaat. Die klassifikasie is moontlik, onafhanklik van die ouderdom of geografiese oorsprong van die wyne.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The wine industry is an important sector of agriculture and wine analysis forms the basis of assessing compliance of its commodities with regulatory standards and research in this field. Liquid chromatography (LC) is extensively used for the determination of a wide range of nonvolatile wine components, but conventional detectors impose performance limitations on the technique that prevents its application to sophisticated analytical problems. In particular, conventional detectors for LC often lack the sensitivity and specificity for the determination of many wine compounds, especially trace level analytes, and furthermore, do not possess spectral capabilities for compound identification or structure elucidation. The hyphenation of mass spectrometry (MS) to LC has led to the introduction of a range of detectors that confers high levels of sensitivity and selectivity to the technique. In addition, a wide variety of MS architectures are available that are inherently suited for targeted analysis or structure elucidation studies. In this dissertation, the potential benefits of liquid chromatography – tandem quadrupole mass spectrometry (LC-MS/MS) to solve analytical problems relevant to the wine industry are explored. LC-MS/MS is a particularly versatile analytical technique because both mass analysers can be operated in full-spectrum mode or selected-ion monitoring, which, together with optional fragmentation, gives rise to four modes of operation that may be used for highly specific and sensitive targeted analysis or spectral investigations. In multiple reaction monitoring (MRM) mode, both analysers are set at single ion frequencies specific for the compound under investigation and one or more of its product fragments, respectively. MRM mode is ideally suited for trace level analysis in complex mixtures, even in cases where the target components are not resolved from interferences. In this study, MRM detection was used to solve challenges relevant to the wine industry for the selective quantitation of target analytes that could not be analysed by conventional LC methods. The application of this approach for the analysis of natamycin, ethyl carbamate (EC) and 3-alkyl-2- methoxypyrazines (MPs) in wine is demonstrated. Natamycin is an antimicrobial preservative that is not permitted in wine in the European Union. A rapid and sensitive method for the determination of natamycin was developed, and has been used since 2009 to regulate this vitally important sector of the South African wine export industry. EC is a natural carcinogen that occurs at trace level amounts in alcoholic products. It also has the potential to accumulate in wines and can occur in very high concentrations in some fruit brandies. The determination of EC is complicated by its physicochemical properties, and available analytical methods suffer from drawbacks such as the requirement for elaborate extraction procedures and high solvent consumption. A novel method for the determination of EC in wines, fortified wines and spirits is described and it was applied to perform an audit of the South African industry as well as to investigate factors responsible for its accumulation in alcoholic beverages. This work forms an integral part of the food safety mandate of the State and it ensures that export products comply with international norms for trade. MPs are ultra-trace-level aroma compounds that contribute to the varietal character of Sauvignon blanc wines. Their analytical determination is challenging due to their low levels of occurrence. The loading capacity of LC combined with the sensitivity and resolving power of MS was exploited to analyse concentrated extracts, in order to achieve very low limits of detection. The performance of the LC-MS/MS method enabled the quantitation of these compounds at their natural levels of occurrence, including the first quantitation and spectral confirmation of 3- ethyl-2-methoxypyrazine in wine. Extensive data pertaining to South African Sauvignon blanc wines are reported and statistical analysis is performed, reporting the correlation of variables such as vintage and origin as well as wine parameters such as malic acid with wine MPs. Furthermore, the application of LC-MS/MS for structural elucidation and screening of target classes of analytes was demonstrated for the analysis of red wine anthocyanins. The anthocyanidin-glycosides are responsible for the colour of red grapes and wine, contribute to the sensory properties of wine, and are also of interest due to their beneficial biological properties. Their determination is complicated by their large numbers and structural diversity, further exacerbated by diverse reactions during wine ageing as well as the lack of reference standards for most members of this class of compounds. Tandem MS in scan mode was used for the highly selective detection of glycosylated anthocyanins and derivatives, exploiting the predictable elimination of the sugar moiety in neutral loss mode. Concurrent survey scan experiments were used to unambiguously identify neutral loss detected compounds. The method therefore follows a simplified and structured approach for unambiguous peak identification based on elution order and mass spectral information to impart a high level of certainty in compound identification. In summary, the work presented in this dissertation demonstrates that LC-MS/MS is a versatile and powerful analytical approach for the analysis of diverse compounds of relevance to the wine industry. The sensitivity and specificity of MRM mode, and the selectivity and spectral capabilities of neutral loss and survey scan modes of MS/MS detection, is amply demonstrated by the applications presented in the dissertation. / AFRIKAANSE OPSOMMING: Die wynbedryf is ‘n belangrike komponent van landbou en wyn-analise vorm ‘n integrale deel van gehalteversekering ten opsigte van toepaslike wetlike standaarde. Wyn-analise is ook belangrik in navorsing oor die samestelling van wyn. Vloeistofchromatografie word dikwels aangewend vir die bepaling van ‘n wye verskeidenheid nie-vlugtige wynkomponente, maar konvensionele detektors plaas beperkinge op die aanwending van die tegniek tot gesofistikeerde analitiese toepassings. Meer spesifiek, konvensionele detektors vir vloeistofchromatografie beskik nie oor die sensitiwiteit en selektiwiteit vir die bepaling van baie wynkomponente nie, veral in die geval van spoorvlakanalise, en beskik boonop ook nie oor spektrale vermoëns vir identifikasie van komponente en struktuurbepaling nie. Die koppeling van vloeistofchromatografie met massaspektrometrie het ‘n reeks detektors tot die tegniek toegevoeg wat hoë vlakke van sensitiwiteit en selektiwiteit bied. Verder bied die verskeidenheid van massaspektrometrie-konfigurasies ook instrumente wat inherent geskik is vir geteikende analise of struktuurbepaling, afhangende van die doel van die ondersoek. In hierdie dissertasie word die voordele ondersoek wat verbonde is aan die aanwending van vloeistofchromatografie – tandem kwadrupool massaspektrometrie om relevante analitiese vraagstukke in die wynbedryf op te los. Hiedie tegniek is besonder toepaslik aangesien beide massa-analiseerders in geselekteerde-ioon modus of in volle skandering gebruik kan word. Tesame met opsionele fragmentasie, gee hierdie uitleg aanleiding tot vier funksionaliteite wat vir hoogs sensitiewe geteikende analise of spektrale onledings gebruik kan word. Eerstens word beide massa analiseerders vir enkel-ioon frekwensies opgestel, spesifiek tot die teikenkomponent en een of meer van sy produkfragmente, wat verkry word deur komponentspesifieke fragmentasie. Hierdie modus is by uitstek geskik vir spoorvlakontleding van komplekse monsters, selfs wanneer die teikenkomponente nie chromatografies van die matriks geskei is nie. In hierdie studie is die tegniek aangewend vir die hoogs sensitiewe bepaling van spoorvlak komponente wat nie met konvensionele detektors gemeet kon word nie. Die aanwending van hierdie tegniek word gedemonstreer vir die spoorvlakbepaling van natamycin, etielkarbamaat en 3-alkiel-2-metoksiepierasiene in wyn. Natamycin is ‘n antimikrobiese preserveermiddel wat ontoelaatbaar is in wyn in die Europese Unie. ‘n Vinnige en sensitiewe metode vir die bepaling van natamycin is ontwikkel, en word reeds sedert 2009 aangewend om hierdie uiters belangrike sektor van die Suid-Afrikaanse wyn uitvoerbedryf te reguleer. Etielkarbamaat is ‘n karsinogeen wat natuurlik voorkom in spoorhoeveelhede in alkoholiese produkte. Dit kan ook onder sekere omstandighede akkumuleer in wyn en in hoë konsentrasies voorkom in vrugtebrandewyne. Die bepaling van etielkarbamaat word bemoeilik deur sy chemiese eienskappe, en gevolglik word analitiese metodes gekenmerk deur uitgebreide, arbeidsintensiewe monstervoorbereiding en die gebruik van groot hoeveelhede, meestal giftige, oplosmiddels. ‘n Nuwe metode vir die bepaling van etielkarbamaat in wyn, gefortifiseerde wyn en spiritualië word beskryf en word aangewend om die faktore vir vorming daarvan te ondersoek. Die metode word aangewend om die Suid-Afrikaanse bedryf te ouditeer in terme van die voedselveiligheid mandaat van die Staat, en om te verseker dat uitvoere voldoen aan standaarde vir internasionale handel. Metoksiepierasiene is vlugtige, ultraspoorvlak wynaromakomponente wat verantwoordelik is vir die kenmerkede kultivarkarakter van Sauvignon blanc wyne. Hul analitiese bepaling word bemoeilik deur hulle lae konsentrasies in wyn. Die ladingskapasiteit van vloeistofchromatografie tesame met die sensitiwiteit en selektiwiteit van massaspektrometrie was benut om hoogs gekonsentreerde ekstrakte te ontleed. Baie hoë vlakke van sensitiwiteit word sodoende verkry. Die verrigting van die metode was voldoende om hierdie komponente teen hulle natuurlike konsentrasies te kwantifiseer, insluitende die eerste kwantifisering en spektrale bevestiging van 3-etiel-2-metoksiepierasien. Omvattende data van die vlakke van hierdie komponente in Suid- Afrikaanse Sauvignon blanc wyne word getoon en statistiese ontleding is gedoen om korrelasies tussen veranderlikes soos oorsprong en oesjaar sowel as basiese wyn veranderlikes soos byvoorbeeld appelsuur, met metoksiepierasienvlakke te ondersoek. Verder was die toepassing van vloeistofchromatografie – tandem massaspektrometrie tot struktuurbepaling en skandering vir groepe van komponente gedemonstreer vir die ontleding van rooiwyn antosianiene. Die antosianien-glukosiede is verantwoordelik vir die kleur van rooi druiwe en wyn, dra by tot die sensoriese eienskappe daarvan, en is ook relevant as gevolg van die voordelige biologiese eienskappe daarvan. Die bepaling van hierdie komponente word gekompliseer deur hulle groot getalle en strukturele diversiteit, verder bemoeilik deur die wye verskeidenheid van reaksies wat hulle ondergaan tydens veroudering. Daar is ook ‘n gebrek aan beskikbaarheid van standaarde vir die meeste van die lede van hierdie klas van komponente. Tandem massaspektrometrie was in skanderingsmodus gebruik vir hoogs selektiewe deteksie van die antosianien-glukosiede deur die voorspelbare eliminasie van die suiker komponent in neutrale verliesskandering te benut. Gelyktydige skanderings van die komponente wat met neutraleverliesskandering waargeneem word, is gebruik vir ondubbelsinnige komponent identifikasie. Die metode volg daarom ‘n eenvoudige en gestruktureerde benadering vir piek identifikasie wat gebaseer is op chromatografiese orde, sowel as massaspektrale inligting, om ‘n hoë vlak van sekerheid aan die identifikasie van komponente te verleen. Samevattend, word daar getoon deur die werk wat in hierdie dissertasie uiteengesit is dat vloeistofchromatografie – tandem massaspektrometrie ‘n veelsydige en kragtige tegniek bied vir chemiese analise relevant tot die wynbedryf. Die sensitiwiteit, selektiwiteit en spektrale vermoëns van die tegniek word duidelik deur toepassings in die dissertasie getoon.

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Carnitine plays an essential role in eukaryotic metabolism by mediating the shuttling of activated acyl residues between intracellular compartments. This function of carnitine, referred to as the carnitine shuttle, is supported by the activities of carnitine acyltransferases and carnitine/acylcarnitine transporters, and is reasonably well studied and understood. While this function remains the only metabolically well established role of carnitine, several studies have been reporting beneficial effects associated with dietary carnitine supplementation, and some of those beneficial impacts appear not to be directly linked to shuttle activity. This study makes use of the yeast Saccharomyces cerevisiae as a cellular model system in order to study the impact of carnitine and of the carnitine shuttle on cellular physiology, and also investigates the eukaryotic carnitine biosynthesis pathway. The carnitine shuttle of S. cerevisiae relies on the activity of three carnitine acetyltransferases (CATs), namely Cat2p (located in the peroxisome and mitochondria), Yat1p (on the outer mitochondrial membrane) and Yat2p (in the cytosol), which catalyze the reversible transfer of activated acetyl units between CoA and carnitine. The acetylcarnitine moieties can be transferred across the intracellular membranes of the peroxisomes and mitochondria by the activity of the carnitine/acetylcarnitine translocases. The activated acetyl groups can be transferred back to free CoA-SH and further metabolised. In addition to the carnitine shuttle, yeast can also utilize the glyoxylate cycle for further metabolisation of in particular peroxisomally generated acetyl-CoA. This cycle results in the net production of succinate from two molecules of acetyl-CoA. This dicarboxylic acid can then enter the mitochondria for further metabolism. Partial disruption of the glyoxylate cycle, by deletion of the citrate synthase 2 (CIT2) gene, generates a yeast strain that is completely dependent on the activity of the carnitine shuttle and, as a consequence, on carnitine supplementation for growth on fatty acids and other non-fermentable carbon sources. In this study, we show that all three CATs are required for the function of the carnitine shuttle. Furthermore, overexpression of any of the three enzymes is unable to crosscomplement deletion of any one of the remaining two, suggesting a highly specific role for each CAT in the function of the shuttle. In addition, a role for carnitine that is independent of the carnitine shuttle is described. The data show that carnitine can influence the cellular response to oxidative stresses. Interestingly, carnitine supplementation has a protective effect against certain ROS generating oxidants, but detrimentally impacts cellular survival when combined with thiol modifying agents. Although carnitine is shown to behave like an antioxidant within a cellular context, the molecule is unable to scavenge free radicals. The protective and detrimental impacts are dependent on the general regulators of the cells protection against oxidative stress such as Yap1p and Skn7p. Furthermore, from the results of a microarray based screen, a role for the cytochrome c heme lyase (Cyc3p) in both the protective and detrimental effects of carnitine is described. The requirement of cytochrome c is suggestive of an involvement in apoptotic processes, a hypothesis that is supported by the analysis of the impact of carnitine on genome wide transcription levels. A separate aim of this project involved the cloning and expression in S. cerevisiae of the four genes encoding the enzymes from the eukaryotic carnitine biosynthesis pathway. The cloned genes, expressed from the constitutive PGK1 promoter, were sequentially integrated into the yeast genome, thereby reconstituting the pathway. The results of a plate based screen for carnitine production indicate that the engineered laboratory strains of S. cerevisiae are able to convert trimethyllysine to L-carnitine. This work forms the basis for a larger study that aims to generate carnitine producing industrial yeast strains, which could be used in commercial applications. / AFRIKAANSE OPSOMMING: Karnitien vervul ‘n noodsaaklike rol in eukariotiese metabolisme deur die pendel van asiel residue tussen intersellulêre kompartemente te medieer. Hierdie funksie van karnitien heet “die karnitien-pendel“ en word ondersteun deur verskeie karnitien asieltransferases en karnitine/asielkarnitien oordragsprotiëne. Die rol van die karnitien-pendel is redelik goed gekarakteriseer en is tot op hede die enigste bevestigde rol van karnitien in eukariotiese metabolisme. Verskeie onlangse studies dui egter op voordele geasosieer met karnitien aanvulling, wat in sommige gevalle blyk om onafhanklik te wees van die pendel aktiwiteit van karnitien. Hierdie studie maak gebruik van die gis, Saccharomyces cerevisiae, as ‘n sellulêre model sisteem om die impak van karnitien op sel fisiologie asook die eukariotiese karnitien biosintese pad te bestudeer. Die karnitien-pendel van S. Cerevisiae is afhanklik van die aktiwiteite van drie afsonderlike karnitien asetieltransferases (CATs), naamlik Cat2p (gelokaliseer in die peroksisoom en die mitochondria), Yat1p (op die buitenste membraan van die mitochondria) en Yat2p (in die sitosol). Die drie ensieme kataliseer die omkeerbare oordrag van asetielgroepe tussen CoA en karnitien. Die terugwaartse reaksie stel CoA-SH vry om sodoende verbruik te word in verdere metaboliese reaksies. Gis is in staat om, afsonderlik van die karnitien-pendel, gebruik te maak van die glioksilaat siklus vir verdere metabolisme van asetiel-CoA wat gevorm word in die peroksisoom. Gedeeltelike onderbreking van hierdie siklus deur uitwissing van die sitraat sintase (CIT2) geen, genereer ’n gisras wat afhanklik is van die funksie van die karnitienpendel en ook van karnitien aanvulling vir groei op vetsure en nie-fermenteerbare koolstofbronne. Hierdie studie dui daarop dat al drie CATs noodsaaklik is vir die funksionering van die karnitien-pendel. Ooruitdrukking van enige van die drie ensieme lei slegs tot selfkomplementasie en nie tot kruis-komplementasie van die ander twee CATs nie. Hieruit word ’n hoogs spesifieke rol vir elk van die drie ensieme afgelei. ’n Pendel-onafhanklike rol vir karnitien word ook in hierdie werk uitgewys in die bevordering van weerstand teen oksidatiewe stres. Dit is noemenswaardig dat karnitien ’n beskermende effek het in kombinasie met oksidante wat ROS genereer en ’n nadelige effek in kombinasie met sulfhidriel modifiserende agente. Dit word aangedui dat karnitien antioksidant funksie naboots in die konteks van ’n gis sel terwyl die molekuul nie in staat is om vry radikale te deaktiveer nie. Beide die beskermende asook die nadelige inwerking van karnitien is afhanklik van Yap1p en Skn7p, wat reguleerders is in die algemene beskerming teen oksidatiewe stres. Die resultate van ’n “microarray“ gebaseerde studie dui op ’n rol vir die sitokroom c heem liase (Cyc3p) in beide die beskermende en nadelige gevolge van karnitien aanvulling. Die vereiste vir sitochroom c dui op ’n moontlike rol vir apoptotiese prosesse. Hierdie hipotese word verder versterk deur ‘n analise van die impak van karnitien op genoomwye transkripsievlakke. ’n Afsonderlike doelwit van hierdie studie was toegespits op die klonering en uitdrukking van die vier ensieme betrokke in eukariotiese karnitien biosintese in S. cerevisiae. Die gekloneerde gene, uitgedruk vanaf die konstitutiewe PGK1 promotor, was geïntigreer in die gisgenoom om die pad op te bou. Die resultate van ’n plaat gebaseerde karnitien produksie toets dui aan dat die geneties gemanipuleerde gisrasse wel in staat is om trimetiellisien oor te skakel in Lkarnitien. Hierdie werk vorm die hoeksteen van ’n studie wat die ontwikkeling van karnitien produserende kommersiële gisrasse as doelwit stel.

Thesis (PhD (Microbiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Saccharomyces cerevisiae is the most commonly used organism in many fermentation-based industries including baking and the production of single cell proteins, biofuel and alcoholic beverages. In the wine industry, a consumer driven demand for new and improved products has focussed yeast research on developing strains with new qualities. Tremendous progress in the understanding of yeast genetics has promoted the development of yeast biotechnology and subsequently of genetically modified (GM) wine yeast strains. The potential benefits of such GM wine yeast are numerous, benefitting both wine makers and consumers. However, the safety considerations require intense evaluation before launching such strains into commercial production. Such assessments consider the possibility of the transfer of newly engineered DNA from the originally modified host to an unrelated organism. This process of horizontal gene transfer (HGT) creates a potential hazard in the use of such organisms. Although HGT has been extensively studied within the prokaryotic domain, there is an urgent need for similar studies on their eukaryotic counterparts. This study was therefore undertaken to help improve our understanding of this issue by investigating HGT in a model eukaryotic organism through a step-by-step approach. In a first step, this study attempted to determine whether large DNA fragments are released from fermenting wine yeast strains and, in a second step, to assess the stability of released DNA within such a fermenting background. The third step investigated in this study was to establish whether “free floating” DNA within this fermenting environment could be accepted and functionally expressed by the fermenting yeast cultures. Finally, whole plasmid transfer was also investigated as a unified event. Biofilms were also incorporated into this study as they constitute a possibly conducive environment for the observation of such HGT events. The results obtained during this study help to answer most of the above questions. Firstly, during an investigation into the possible release of large DNA fragments (>500 bp) from a GM commercial wine yeast strain (Parental strain: Vin13), no DNA could be detected within the fermenting background, suggesting that such DNA fragments were not released in large numbers. Secondly, the study revealed remarkable stability of free “floating DNA” under these fermentation conditions, identifying intact DNA of up to ~1kb in fermenting media for up to 62 days after it had been added. Thirdly, the data demonstrate the uptake and functional expression of spiked DNA by fermenting Vin13 cultures in grape must. Here, another interesting discovery was made, since it appears that the fermenting natural grape must favours DNA uptake when compared to synthetic must, suggesting the presence of carrier molecules. Additionally, we found that spiked plasmid DNA was not maintained as a circular unit, but that only the antibiotic resistance marker was maintained through genomic integration. Identification of the sites of integration showed the sites varied from one HGT event to the next, indicating that integration occurred through a process known as illegitimate recombination. Finally, we provide evidence for the direct transfer of whole plasmids between Vin13 strains. The overall outcome of this study is that HGT does indeed occur under the conditions investigated. To our knowledge, this is the first report of direct horizontal DNA transfer between organisms of the same species in eukaryotes. Furthermore, while the occurences of such events appears low in number, it cannot be assumed that HGT will not occur more frequently within an industrial scenario, making industrial scale studies similar to this one paramount before drawing further conclusions. / NO AFRIKAANS SUMMARY AVAILABLE

Dissertation (DPhil)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Xylose is the second most abundant sugar present in plant biomass. Plant biomass is the only potential renewable and sustainable source of energy available to mankind at present, especially in the production of transportation fuels. Transportation fuels such as gasoline can be blended with or completely replaced by ethanol produced exclusively from plant biomass, known as bio-ethanol. Bio-ethanol has the potential to reduce carbon emissions and also the dependence on foreign oil (mostly from the Middle East and Africa) for many countries. Bio-ethanol can be produced from both starch and cellulose present in plants, even though cellulosic ethanol has been suggested to be the more feasible option. Lignocellulose can be broken down to cellulose and hemicellulose by the hydrolytic action of acids or enzymes, which can, in turn, be broken down to monosaccharides such as hexoses and pentoses. These simple sugars can then be fermented to ethanol by microorganisms. Among the innumerable microorganisms present in nature, the yeast Saccharomyces cerevisiae is the most efficient ethanol producer on an industrial scale. Its unique ability to efficiently synthesise and tolerate alcohol has made it the ‘workhorse’ of the alcohol industry. Although S. cerevisiae has arguably a relatively wide substrate utilisation range, it cannot assimilate pentose sugars such as xylose and arabinose. Since xylose constitutes at least one-third of the sugars present in lignocellulose, the ethanol yield from fermentation using S. cerevisiae would be inefficient due to the non-utilisation of this sugar. Thus, several attempts towards xylose fermentation by S. cerevisiae have been made. Through molecular cloning methods, xylose pathway genes from the natural xylose-utilising yeast Pichia stipitis and an anaerobic fungus, Piromyces, have been cloned and expressed separately in various S. cerevisiae strains. However, recombinant S. cerevisiae strains expressing P. stipitis genes encoding xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) had poor growth on xylose and fermented this pentose sugar to xylitol. The main focus of this study was to improve xylose utilisation by a recombinant S. cerevisiae expressing the P. stipitis XYL1 and XYL2 genes under anaerobic fermentation conditions. This has been approached at three different levels: (i) by creating constitutive carbon catabolite repression mutants in the recombinant S. cerevisiae background so that a glucose-like environment is mimicked for the yeast cells during xylose fermentation; (ii) by isolating and cloning a novel xylose reductase gene from the natural xylose-degrading fungus Neurospora crassa through functional complementation in S. cerevisiae; and (iii) by random mutagenesis of a recombinant XYL1 and XYL2 expressing S. cerevisiae strain to create haploid xylose-fermenting mutant that showed an altered product profile after anaerobic xylose fermentation. From the data obtained, it has been shown that it is possible to improve the anaerobic xylose utilisation of recombinant S. cerevisiae to varying degrees using the strategies followed, although ethanol formation appears to be a highly regulated process in the cell. In summary, this work exposits three different methods of improving xylose utilisation under anaerobic conditions through manipulations at the molecular level and metabolic level. The novel S. cerevisiae strains developed and described in this study show improved xylose utilisation. These strains, in turn, could be developed further to encompass other polysaccharide degradation properties to be used in the so-called consolidated bioprocess. / AFRIKAANSE OPSOMMING: Xilose is die tweede volopste suiker wat in plantbiomassa teenwoordig is. Plantbiomassa is die enigste potensiële hernubare en volhoubare bron van energie wat tans vir die mensdom beskikbaar is, veral vir die produksie van vervoerbrandstowwe. Vervoerbrandstowwe soos petrol kan vermeng word met etanol wat uitsluitlik van plantbiomassa vervaardig is, bekend as bio-etanol, of heeltemal daardeur vervang word. Bio-etanol het die potensiaal om koolstofuitlatings te verminder en vir baie lande ook afhanklikheid op buitelandse olie (hoofsaaklik afkomstig van die Midde-Ooste en Afrika) te verminder. Bio-etanol kan vanaf beide die stysel en sellulose in plante vervaardig word, maar sellulosiese etanol word as die meer praktiese opsie beskou. Lignosellulose kan deur die hidrolitiese aksie van sure of ensieme in sellulose en hemisellulose afgebreek word en dit kan op hulle beurt weer in monosakkariede soos heksoses en pentoses afgebreek word. Hierdie eenvoudige suikers kan dan deur mikro-organismes tot etanol gegis word. Onder die tallose mikro-organismes wat in die natuur teenwoordig is, is die gis Saccharomyces cerevisiae die doeltreffendste etanolprodusent in die bedryf. Sy unieke vermoë om alkohol te vervaardig en te weerstaan het dit die werksperd van die alkoholbedryf gemaak. Hoewel S. cerevisiae ‘n taamlike breë spektrum van substrate kan benut, kan dit nie pentosesuikers soos xilose en arabinose assimileer nie. Aangesien xilose ten minste ‘n derde van die suikers wat in lignosellulose teenwoordig is, uitmaak, sou die etanolopbrengs uit gisting met S. cerevisiae onvoldoende wees omdat hierdie suiker nie benut word nie. Verskeie pogings is dus aangewend om xilosegisting deur S. cerevisiae te bewerkstellig. Deur middel van molekulêre kloneringsmetodes is gene van die xiloseweg uit ‘n gis wat xilose natuurlik benut, Pichia stipitis, en ‘n anaërobiese swam, Piromyces, afsonderlik in S. cerevisiae-rasse gekloneer en uitgedruk. ‘n Rekombinante ras wat P. stipitis- se XYL1-xilosereduktase- en XYL2-xilitoldehidrogenase gene uitdruk, het egter swak groei op xilose getoon en het dié pentosesuiker tot xilitol gegis. Die hooffokus van hierdie ondersoek was om die benutting van xilose deur ‘n rekombinante S. cerevisiae-ras wat P. stipitis se XYL1 en XYL2-gene uitdruk onder anaërobiese gistingstoestande te verbeter. Dit is op drie verskillende vlakke benader: (i) deur konstitutiewe koolstofkataboliet-onderdrukkende mutante in die rekombinante S. cerevisiae-agtergrond te skep sodat ‘n glukose-agtige omgewing tydens xilosegisting vir die gisselle nageboots word; (ii) deur ‘n nuwe xilose-reduktasegeen uit die natuurlike xilose-afbrekende swam Neurospora crassa te isoleer en deur funksionele komplementasie in S. cerevisiae te kloneer; en (iii) deur willekeurige mutagenese van die rekombinante S. cerevisiae-ras ‘n haploïede xilose-gistende mutant te skep wat ‘n gewysigde produkprofiel ná anaërobiese xilosegisting vertoon. Deur hierdie drieledige benadering te volg, is dit bewys dat dit moontlik is om die anaërobiese xilosebenutting van rekombinante S. cerevisiae-rasse in wisselende mate deur die aangepaste metodes te verbeter, hoewel etanolvorming ‘n hoogs gereguleerde proses in die sel blyk te wees. Opsommend kan gesê word dat hierdie werk drie verskillende metodes uiteensit om xilosebenutting onder anaërobiese toestande te verbeter deur manipulasies op die molekulêre en metaboliese vlak. Die nuwe S. cerevisiae-rasse wat in hierdie studie ontwikkel en beskryf word, toon verbeterde xilosebenutting. Hierdie rasse kan op hulle beurt verder ontwikkel word om ander polisakkariedafbrekende eienskappe in te sluit wat in die sogenaamde gekonsolideerde bioproses gebruik kan word.

Pinot noir has a reputation for lower color stability than other red wine varieties. Because it has relatively low anthocyanin and phenolic content and lacks acylated anthocyanin pigments compared to other red vinifera varieties, color extraction and stability are particularly important. Varying the processing during fermentation of red wine can produce high quality wines. Pectolytic enzymes are used in wine processing for many purposes from increasing juice yield and filtering rates to improving color and phenolic extraction. Macerating enzymes used in this study, Scottzyme Color Pro (Scott Laboratories), Scottzyme Color X (Scott Laboratories), Lallzyme EX (Lallemand), GB Rapidase EX Color (Gist Brocades), and Vinozyme G (Cellulo) were added to 1997 Oregon Pinot noir must prior to fermentation to observe color, aroma and flavor changes. These commercial enzymes have been reported to increase color and improve aroma and flavor of red wines. The effect of these enzymes had not previously been investigated on Oregon Pinot noir but the manufacturers reported increased polymeric phenols, polymeric anthocyanins, tannins, color stability, red hue and saturation of red wine varieties. These enzymes have varying manufacturer recommended usage levels and it is not known how the dosage levels and the enzymes themselves affect Oregon Pinot noir. Sensory evaluations of these wines, at a high and low dosage level, were conducted through free-choice profiling by winemakers and descriptive analysis from a trained panel. In addition, chemical analyses were performed and related to sensory panel results. Overall the addition of these enzymes to Oregon Pinot noir produced wines with greater purple, red descriptors and higher color intensity than the control wine from trained descriptive panel and winemaker panel results. In aroma, the enzyme treated wines were higher in vegetative and earthy descriptors compared to the control. GB Rapidase EX Color (Gist Brocades) was higher in bitterness flavor compared with other samples. Low enzyme wine treatments separated wine samples more from the control then high enzyme wine treatments. The color and appearance, aroma and flavor axes of the profile maps were not significant in the high enzyme treated wines as determined from the winemaker panel. Furthermore, the winemaker panel found acidity the only aroma or flavor descriptor significant in the high dosage ANOVA results. Whereas six descriptors in the low enzyme ANOVA results were significant in separating aroma and flavor samples. Except for Lallzyme EX (Lallemand) treated wine, the hunter colorimeter results showed all low enzyme treated wines were significantly (p<0.05) more red-purple (lower hues) than the high enzyme treated wines. / Graduation date: 1999

This thesis aims to provide an insight into the legal rules governing viticulture and winemaking, and their historical development. Furthermore, the current legal and factual situation as well as the current issues associated with the legislation in these fields are more closely examined in the thesis. An anonymous questionnaire had been created and sent to a selected group of small and medium-size viniculturists and wine producers in the Slovacko wine subregion in order to supplement the research and its aim of presenting the current legislation from de lege ferenda point of view.

M.Sc. / The origin of a wine plays a key role in establishing the quality and the price the consumer is prepared to pay. Fingerprinting techniques based on multi-element data combined with multivariate statistical analysis as well as isotope ratio data for certain elements such as boron (11B/10B) and strontium (87Sr/86Sr) are being developed and have been used for provenance determination of wine with varying degrees of success. The aim of this study was to develop a method to determine boron isotope ratios (11B/10B) with the required precision using ICP-MS (inductively coupled plasma mass spectrometry) in soil and wine samples and applying this method to establish the origin of South African wines. Analytical difficulties such as the boron memory effect, dead time, mass bias drift and matrix effects were investigated. Although the memory effect, dead time and mass bias drift were satisfactorily resolved, it was not possible to determine what the cause of all the observed matrix effects was during this study. The method was used to categorise wines from the Robertson, Swartland and Stellenbosch regions and an attempt was made to link the measured boron isotope ratios with that obtained from the corresponding provenance soils. The 11B/10B isotope ratios for the wine samples (Robertson: 4.202 ± 0.014, Swartland: 4.173 ± 0.013 and Stellenbosch: 4.174 ± 0.028) were, however, higher than the ratios obtained for the soil samples (Robertson: 4.108 ± 0.020, Swartland: 4.070 ± 0.023 and Stellenbosch: 4.124 ± 0.039). It was possible to distinguish, using the boron isotope ratios (wine and soil samples), between the Robertson area (Breede River region) and the Swartland area (Coastal region). The wine and soil 11B/10B isotope ratios obtained for the Stellenbosch area (Coastal region) overlapped with the 11B/10B isotope ratios of the Robertson and Swartland regions making it impossible to differentiate it from these two regions.