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 (Food Science))--University of Stellenbosch, 2006. / The aim of the study was to provide the South African wine industry with guidelines for the production of Pinotage wines with optimal total antioxidant capacity (TAC), while retaining sensory quality. The contribution of individual phenolic compounds to the wine TAC is important in this regard. The wine TAC was measured with the 2,2 -azino-di(3-ethylbenzo-thiazoline-sulphonic acid radical cation) (ABTS +) scavenging assay. The contributions of individual phenolic compounds to the wine TAC were calculated from their content in the wines and the Trolox equivalent antioxidant capacity (TEAC) of pure phenolic standards. The effects of climate region, vine structure, enological techniques (pre-fermentation maceration, juice/skin mixing, addition of commercial tannins, extended maceration) and maturation (oak barrels, alternative oak products, oxygenation) on the phenolic composition, TAC and sensory quality of Pinotage wines were also investigated. The TEAC values of quercetin-3-galactoside, isorhamnetin and peonidin-3-glucoside were reported for the first time. TEAC values observed for most compounds were much lower than those reported previously, although TEAC values for gallic acid, caftaric acid, caffeic acid and kaempferol were consistent with some previous reports. Caftaric acid and malvidin-3-glucoside were the largest contributors to the wine TAC. The contents of monomeric phenolic compounds and procyanidin B1, however, only explained a small amount (between 11 and 24%) of the wine TAC, with the remaining TAC attributed to oligomeric and polymeric phenolic compounds and other unknown compounds. Some synergy between different monomeric phenolic compounds was also demonstrated. All the viticultural and enological factors investigated affected the phenolic composition of Pinotage wines, while the wine TAC was only affected by some treatments. Changes in wine TAC could not always be explained by changes in phenolic composition as the contribution of oligomeric, polymeric and unknown compounds could not be assessed, but could play a large role. Differences in wine colour were also difficult to explain due to the large number of factors involved and the dark wine colour, which made objective measurements difficult. The concentration of vitisin A, an orange-red pyranoanthocyanin, was increased consistently as a result of prefermentation maceration treatments and affected the wine colour of oxygenated wines. Increased wine TAC was observed when cultivating Pinotage grapes on bush vines and in cooler climatic regions, compared to cultivation on trellised vines in warmer climatic regions. All the climatic regions and vine structure treatments, however, resulted in wines with good sensory quality. In terms of enological techniques, pumping-over, as opposed to punching-down and rotor treatments, is not recommended as a juice/skin mixing technique, due to reduced wine TAC, colour and sensory quality. Pre-fermentation maceration, addition of commercial tannin preparations, and oak maturation using traditional and alternative treatments, resulted in improved sensory quality, but with no change in wine TAC. However, optimisation of the tannin addition protocol may result in increased wine TAC if additions are made after fermentation or higher dosages are used. Oxygenation of Pinotage wine needs further investigation to optimise the protocol, as improvements to the wine colour and fullness were observed for some treatments, but loss of sensory quality and TAC were observed in most cases.

Dissertation (PhD Agric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Malolactic fermentation (MLF) is conducted by lactic acid bacteria (LAB) and entails the decarboxylation of L-malate to L-Iactate through a reaction catalysed by the malolactic enzyme (MLE). The consequence of this conversion is a decrease in total acidity. MLF plays a part in microbial stabilisation and due to the metabolic activity of the bacteria the organoleptic profile of the wine is modified. In some wines MLF is considered as spoilage, especially in warm viticultural regions with grapes containing less malic acid. In addition to undesirable organoleptic changes, MLF can alter wine colour, and biogenic amines may be produced. To induce MLF we provided s. cerevisiae with the enzymatic activities required for MLF, which is then conducted by the yeast during alcoholic fermentation. The malolactic enzyme-encoding gene (mieD) was cloned from Pediococcus damnosus NCFB 1832, characterised and expressed in S. cerevisiae. The activity of this enzyme was compared to two other malolactic genes, mieS from Lactococcus lactis MG1363 and mleA from Oenococcus oeni La11, expressed in the same yeast strain. All three recombinant strains of S. cerevisiae converted L-malate to L-Iactate in synthetic grape must, reaching L-malate concentrations of below 0.3 gIL within 3 days. However, a lower conversion rate and a significant lower final L-Iactate level were observed with the yeast expressing mieD. In order to inhibit MLF, we show that the growth of O. oeni, the main organism responsible for MLF, could be safely repressed with a ribosomaly synthesised antimicrobial peptide, pediocin PD-1, produced by P. damnosus NCFB 1832, without effecting yeast growth. Pediocin PD-1 is stable in wine at 4°C-100°C, and ethanol or S02 does not affect its activity. The peptide was purified to homogeneity and sequence analysis suggests that the peptide is a member of the lantibiotic family of bacteriocins. The molecular mass was estimated by mass spectroscopy to be 2866.7 ± 0.4 Da. Pediocin PD-1 forms pores in sensitive cells, as indicated by the efflux of K+ from O. oeni, combined with inhibition of cell wall biosynthesis, leading to cell lysis. Loss of cell K+was reduced at low temperatures, presumably as a result of the increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane. Although pediocin PD-1 is active over a broad pH range, optimal activity was recorded at pH 5.0. The petide is, however, more stable between pH 2.0 and 5.0, with the best stability observed between pH 3.0 and 4.0. Pediocin PD-1 provides a safer biological alternative than chemical preservatives such as S02. / AFRIKAANSE OPSOMMING: Appelmelksuurgisting (AMG) word deur sekere melksuurbakterieë (MSB) uitgevoer en verwys na die dekarboksilering van L-malaat na L-Iaktaat, 'n reaksie gekataliseer deur die appelmelksuurensiem (AME). AMG verlaag die suurvlakke in wyn, speel 'n rol in mikrobiologiese stabiliteit, en verander die organoleptiese profiel van die wyn. In sommige wyne word AMG beskou as bederf, veral in warm wynbou streke met minder malaat in druiwe. AMG kan ongewenste organoleptiese veranderinge teweeg bring, die wyn se kleur beinvloed, en tot die produksie van biogene amiene lei. Vir die bevordering van AMG het ons S. eerevisiae met die ensiematiese aktiwiteit benodig vir AMG voorsien wat dan veilig deur die gis tydens alkoholiese fermentasie uitgevoer word. 'n AME-koderende geen (mIeD) is uit Pedioeoeeus damnosus NCFB 1832 gekloneer, gekarakteriseer en in S. Cerevisiae uitgedruk. Die aktiwiteit van die ensiem is vervolgens vergelyk met die aktiwitet van twee ander AME gene, mIeS van Laetoeoeeus laetis MG1363 en mleA van Oenoeoeeus oeni Lal1, uitgedruk in dieselfde gisras. AI drie rekombinante gisrasse het L-malaat binne die bestek van drie dae na L-Iaktaat omgeskakel en die finale L-malaat vlakke was minder as 0.3 gIL. Die tempo van omkakeling was egter laer in die gis wat die mIeD geen uitdruk en die finale L-Iaktaat vlakke was veel laer. Om AMG te inhibeer is die groei van O. oeni, die organisme hoofsaaklik verantwoordelik vir AMG, onderdruk deur die byvoeging van 'n ribosomaal gesintetiseerde antimikrobiese peptied, pediocin PD-1, geproduseer deur P. damnosus NCFB 1832. Gisgroei is nie geaffekteer nie. Pediocin PD-1 is stabiel in wyn by temperature wat wissel tussen 4°C en 100°C, en die aktiwiteit van die peptied word nie geaffekteer deur ethanol of S02 nie. Die peptied is gesuiwer volgens In eenvoudige metode wat amoniumsulfaat-presipitasie en katioon uitruilings-ehromatografie insluit. Aminosuur volgorde bepaling van gesuiwerde peptied dui daarop dat pediocin PD-1 tot die lantibiotiese familie van bakteriosiene behoort. Die molekulêre massa van die peptied, soos bepaal deur massa spektroskopie, is 2866.7 ± 0.4 Da. Pediocin PD-1 vorm porieë in selmembrane van sensitiewe selle soos aangedui deur die uitvloei van K+vanuit O. oeni selle. Die peptied kombineer hierdie aksie met die inhibisie van selwand biosintese wat lei tot sel lise. Verlies van sellulêre K+verminder by laer temperature, waarskynlik as gevolg van verandering in die lipied- en protein inhoud van die sitoplasmiese membraan. Alhoewel die peptied aktief is oor 'n breë pH grens, is die antimikrobiese aksie optimaal by pH 5.0. Die peptied is meer stabiel tussen pH 2.0 en 5.0 en toon die beste stabiliteit tussen pH 3.0 en 4.0. Peiocin PD-1 is 'n veilige biologiese alternatief vir chemiese preserveermiddels soos S02.

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.

Hen egg white lysozyme is a hydrolytic enzyme effective at preventing the growth of Gram positive bacteria by degrading the bacterial cell wall to a point of cell lysis. Investigating lysozyme as a processing tool in wine to control the growth of lactic acid bacteria and malolactic fermentation has significant commercial interest. In this project, the interactions of lysozyme with wine components and wine was evaluated along with the efficacy of lysozyme in preventing malolactic fermentation (MLF) in Oregon Pinot Noir and Chardonnay. The information from this work, together with results from similar projects, will allow the development of guidelines for lysozyme use in commercial wine. Interactions of lysozyme with wine components were evaluated by measurement of enzymatic activity in the presence of wine acids, ethanol, and phenolics. Enzyme inhibition was observed, to various degrees, with all wine components. Crude grape tannin altered the availability of free enzyme by complexing to lysozyme and forming a precipitate. In a model wine system, lysozyme activity was reduced by 50% when tannin was present. Lysozyme addition to red wine resulted in a reduction in pigmented compounds and detectable sensory differences. Wine trials evaluated the efficacy of lysozyme in completely preventing malolactic fermentation (MLF) and terminating MLF midway through fermentation in Oregon Pinot Noir and Chardonnay. Vintages from 1993 and 1994 were treated without SO₂, with SO₂, with SO₂ plus a starter culture of Leuconostoc oenos. Each lot was divided into 0 ppm lysozyme (control), 250 ppm lysozyme, 500 ppm lysozyme, and 1000 ppm lysozyme. Lactic acid bacteria were enumerated monthly, for ten months. Lysozyme prevented malolactic fermentation in all wines at the treatment levels of 500 and 1000 ppm. In the 1993 Pinot Noir, 250 ppm lysozyme prevented MLF but only delayed MLF in the 1994 vintage. Lysozyme effectively terminated MLF at a concentration between 200 and 300 ppm in both Pinot Noir and Chardonnay. / Graduation date: 1996

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.