Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Acids are one of the major components that originate largely from the berry, that are found in wine, and that influence the sensory perception. The presence of organic acids in adequate concentrations in the grape berry, of which tartaric- and malic acid are the main organic acids present, is important as this determines the potential of a must to produce a good and stable wine. The effect of temperature on the organic acid content of the must is widely discussed with higher temperatures in general being associated with lower quantities of organic acids present in the juice, and lower temperatures during ripening associated with higher quantities, specifically in the case of malic acid. Due to the topographical diversity of the Stellenbosch Wine of Origin district and the closeness of the ocean and the occurrence of sea breezes, the mesoclimate differs greatly over short distances. Sixteen sites, consisting of eight Sauvignon Blanc and Cabernet Sauvignon sites respectively, were selected from a broader terroir study site network. Three vintages with complete climatic datasets were selected for vintage comparisons. Climate in the study area was monitored on differing scales, and data from a weather station network, as well as from mesoclimatic dataloggers within the sites were available. The available data was firstly compared to determine the variability of the data, not only between the two climatic scales, but also between the sites. Different climate classification indices and parameters available in literature were thereafter compared and evaluated for the best representation in this area. The Huglin index was found to be a better representation regarding the thermal climatic indices. Due to the great differences between temperatures noted for the mesoclimatic loggers and the nearest automatic weather station, the use of mesoclimatic logger data was preferred, and is advised in future studies where this scale of data is available. Malic and tartaric acid has a definite synthesis period up until véraison, after which the content of tartaric acid remains constant in the berry and the content of malic acid decreases until harvest due to mainly respiration. The temperature data was therefore separated in a synthesis period from flowering to véraison, and a ripening period from véraison to harvest. In this study, clear differences were firstly seen in the climate as expected, not only between sites per vintage, but in addition between vintages and between vintages per site. The phenological differences between the sites could be largely attributed to the differences in temperature as phenology and temperature was found to be highly correlated in this study. Differences in the ripening parameters were noticed in addition to the contents of the organic acids between sites, although no definite contribution of temperature was shown to affect the contents of these compounds at either véraison or harvest. These differences may be attributed to other factors such as the soil water content and the canopy architecture. In addition, these factors all contribute in differing percentages to the differences found in the contents per site. It was found though that temperature can be used as an indicator of the organic acid content in the grape berry, considering that the temperature data is available on a mesoclimatic scale, separated in a synthesis and period of degradation, and the number of hours within the temperature thresholds are determined. Differences seen in the organic acid contents can however not only be attributed to the differences in topography and the temperature as discussed in this study. / AFRIKAANSE OPSOMMING: Sure is belangrike druifkomponente wat grootliks hul oorsprong in die korrel het, in die wyn voorkom, en die sensoriese persepsie van die wyn beïnvloed. Die voorkoms van organiese sure in genoegsame konsentrasies in die korrel, waarvan wynsteensuur en appelsuur die hoof organiese sure is, is belangrik aangesien dit die potensiaal van die sap om ʼn goeie en stabiele wyn te produseer, bepaal. Hoe temperatuur die inhoud van organiese sure in die druiwesap affekteer is gereeld onder bespreking, met hoër temperature in die algemeen geassosieer met ʼn laer inhoud van organiese sure, terwyl laer temperature geassosieer word met ʼn hoër inhoud van organiese sure in die sap, veral in die geval van appelsuur. As gevolg van die topografiese diversiteit van die Stellenbosch Wyn van Oorsprong distrik, asook die nabyheid van die oseaan met die gepaardgaande voorkoms van die seebries, verander die mesoklimaat aansienlik oor klein afstande in hierdie area. Vir die studie was sestien wingerde, wat bestaan het uit agt Sauvignon Blanc en agt Cabernet Sauvignon wingerde, geselekteer vanuit ʼn groter terroir studie. Verder was drie seisoene, met volledige klimaatsdatastelle, geselekteer vir die vergelyking van data tussen die seisoene. Klimaat was op verskillende skale binne die studie area gemonitor en data van ʼn weerstasie netwerk, sowel as van mesoklimaat dataversamelaars binne die wingerde, was beskikbaar. Die beskikbare datastelle was vergelyk, asook geëvalueer, om die mees verteenwoordigende datastel vir die area te bepaal. Met die oorweging van die termiese indekse was daar gevind dat die Huglin indeks beter verteenwoordigend van die area was. Verder, as gevolg van die groot verskille wat gevind is tussen die temperature gemeet met die mesoklimaat dataversamelaars en die naaste outomatiese weerstasie, was daar besluit dat die gebruik van die mesoklimaat data verkies is en is dit ook aan te beveel vir die gebruik in toekomstige navorsing indien die tipe data beskikbaar is. Wynsteen- en appelsuur het beide ʼn definitiewe sintese periode tot en met véraison, waarna die hoeveelheid wynsteensuur in die korrel relatief konstant bly en die hoeveelheid appelsuur afneem hoofsaaklik as gevolg van respirasie. Die temperatuur data was dus verdeel in ‘n periode van sintese vanaf blom tot en met véraison, en ʼn rypwordingsperiode vanaf véraison tot en met oes. In hierdie studie was daar eerstens groot verskille waargeneem in die klimaat soos wat daar verwag is. Hierdie verskille was nie net waargeneem as tussen die seisoene nie, maar ook tussen die wingerde binne ʼn seisoen. Die fenologiese veskille tussen die wingerde wat ook waargeneem is, kon hoofsaaklik aan die verskille in die temperatuur toegeskryf word en ʼn goeie korrelasie tussen temperatuur en fenologie is opgemerk. Merkwaardige verskille in die rypwordingsparameters, asook in die inhoud van die organiese sure, was waargeneem, alhoewel die bydrae van temperatuur op die inhoud van hierdie komponente by véraison of oes nie as definitief getoon is nie. Dit kan toegeskryf word aan die bydrae van ander faktore, soos byvoorbeeld die grondwaterinhoud en die lowerargitektuur, op die inhoud van hierdie komponente. Die addisionele faktore dra egter in verskillende persentasies by tot die verskille waargeneem tussen die wingerde.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Nitrogen composition of grape must is highly variable and impacts on the health of the fermenting yeast population as well as the formation of aroma and flavour compounds in wine. Insufficient yeast assimilable nitrogen (YAN), mostly consisting of amino acids and ammonium, can lead to stuck or sluggish fermentations as well as the formation of undesirable compounds such as H2S. Furthermore, it is well established that the total concentration of YAN and the specific amino acid composition have a significant impact on the final aroma and flavour of wines. However, the impact of individual amino acids and of specific amino acid compositions on fermentation kinetics and on the production of aroma and flavour impact compounds under winemaking conditions is not well understood. The first goal of this study was to evaluate the effect of single amino acids on growth kinetics and major volatile production of two industrial wine yeast strains under conditions resembling wine fermentations. To facilitate these fermentation conditions while also allowing for easy reproducibility and manipulation of the initial components, a synthetic grape media was utilized. Biomass formation, exponential growth rate, lag phase, and fermentation duration were utilized to evaluate the efficiency of single amino acids. The data show that previously observed trends in laboratory strains mostly apply to these conditions and strains. In general, the efficiency of amino acids to be used as nitrogen sources and the production of major volatiles due to their presence followed the same patterns for both industrial yeast strains. However, the production of the secondary metabolites butanol, propanol, acetic acid, and ethyl acetate were found to be produced in different final concentrations dependent upon the yeast strain. The branched-chained and aromatic amino acids (BCAAs) treatments were observed to have the most dramatic effects on major volatile production. Investigating the relationships between the initial concentration of the BCAAs and the final concentrations of major volatile compounds, it was found that the production of fusel alcohols and fusel acids due to the degradation of BCAAs by S. cerevisiae could be predicted from the initial concentration of BCAAs. While under simple nitrogen conditions the production of several other secondary metabolites such as butanol, propionic acid, valeric acid, decanoic acid and 2-phenylethyl acetate were found to be correlated to the initial concentration of BCAAs in the media. Future studies should focus on the validation of these trends in aroma production in real grape musts under various fermentation temperatures for a number of industrial wine yeast strains. / AFRIKAANSE OPSOMMING: Die stikstof samestelling van druiwemos is hoogs veranderlik en impakteer op die kerngesondheid van die fermenterende gis populasie asook die produksie van aroma- en geurverbindings in wyn. Onvoldoende assimileerbare stikstof (ook genoem “yeast assimilable nitrogen” (YAN)), wat meestal bestaan uit aminosure en ammonium, kan aanleiding gee tot steek- of slepende fermentasies, asook die vorming van ongewensde verbindings soos H2S. Dit is alombekend dat die totale konsentrasie van YAN en dan ook die spesifieke aminosuur samestelling ‘n noemenswaardige impak op die finale aroma en smaak van wyn het. Die invloed van individuele am inosure en spesifieke aminosuur samestellings op die fermentasie kinetika, asook die produksie van verbindings met ‘n impak op die wyn aroma en smaak, word egter nie deeglik verstaan nie. Die eerste doelwit van die studie was om twee industriële gisrasse te gebruik om die effek van enkel aminosure op die groei-kinetika en produksie van die vername vlugtige verbindings onder wynmaak toestande te bepaal. Kunsmatige, gedefinieerde druiwermosmedium is gebruik om wynmaak toestande te simuleer en ook herhaalbaarheid en manipulering van die aanvanklike samestelling van die medium te verseker. Die studie het vorige tendense wat opgemerk is in die evaluasie van laboratorium rasse onder soortgelyke toestande bevestig. Die doeltreffendheid waarmee aminosure oor die algemeen gebruik word as stikstofbron, asook die produksie van die vernaamste vlugtige verbindings wat gekoppel is aan hulle teenwoordigheid, het ‘n vergelykbare patroon vir beide rasse gevolg. Die sekondêre metaboliete butanol, propanol, asetaat en etiel-asetaat is egter wel in verskillende eindkonsentrasies geproduseer deur die verskillende gisrasse. Die vertakte-ketting en aromatiese aminosuur (“branched-chained and aromatic amino acids” (BCAAs)) behandelings het die mees dramatiese effek op die produksie van die vernaamste vlugtige komponente gehad. Ondersoek na die verwantskap tussen die aanvanklike konsentraies van die BCAAs en die finale konsentrasies van dié verbindings het aangedui dat die produksie van hoër alkohole en sure, as gevolg van die afbraak van BCAAs deur S. cerevisiae, met behulp van die aanvanklike konsentrasie van die BCAAs voorspel kon word. Terselfdertyd is gevind dat onder eenvoudige stikstoftoestande, verskeie ander sekondêre metaboliete soos butanol, propionaat, valeriaat, dekanoësuur en 2-fenieletielasetaat, gekorreleer kan word met die aanvanklike BCAAs in die media. Verdere studies moet poog om hierdie tendense ten opsigte van aromaproduksie te bevestig en wel deur gebruik te maak van ware druiwemos, verskeie fermentasietemperature, asook ’n verskeidenheid van wyngisrasse.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The market for high quality lower alcohol wines is growing globally. Several factors are responsible for this trend, with socio-economic and health concerns being considered as being the most relevant. It is therefore no surprise that in the past three decades many systems have been developed to reduce wine ethanol levels, each with its own strengths and weaknesses. However, current systems are not always cost effective and frequently result in unwanted side-effects. Microbiological methods primarily based on redirecting carbon flux in existing, or novel Saccharomyces and non-Saccharomyces yeast strains, might have the potential to eliminate or reduce such shortcomings. However, little base-line information regarding differences in ethanol yields of existing wine yeast strains, and on the impact of fermentation conditions on such yields is currently available. In this study the ethanol yield of 15 wine yeast strains was investigated in synthetic wine must under varied wine fermentative conditions including changes in yeast assimilable nitrogen, sugar concentration, pH and fermenting temperatures to identify strains that produce lower ethanol yields and conditions that would favour such an outcome. Most strains and conditions resulted in very similar ethanol yields, however in some cases interesting differences were observed. Some of the strains showed significant differences between high and low nitrogen containing must. Results from synthetic must were confirmed in grape must (Sauvignon Blanc, Chardonnay, Shiraz and Cabernet Sauvignon), but no consistent response could be observed. Interestingly the Shiraz fermentations always showed a higher ethanol yield for all strains investigated. This may be due to a parameter (or combination thereof) which was not included as an experimental factor in our study. Glycerol yield was also studied in the grape must experiments and was found to be more significantly condition dependent than ethanol yield. Temperature and glycerol seemed to be directly proportional confirming the results of previous studies. While temperature did increase glycerol production, it was concluded that the redirection of carbon towards glycerol was not substantial enough to have measurable effect on the final ethanol concentration. The most notable differences which were observed were very specific to a particular yeast strain and condition pairing, thus no generally applicable treatment to achieve lower ethanol yields could be established. / AFRIKAANSE OPSOMMING: Deesdae is daar ‘n groeiende mark vir lae alkohol wyne van hoë gehalte. Verskeie faktore is verantwoordelik vir hierdie verskynsel, met sosio-ekonomiese en gesondheidskwessies as die hoof rolspelers. Vir hierdie rede is daar gedurende die laaste drie dekades baie stelsels ontwikkel om wyn etanol vlakke te verlaag, elkeen met voor- en nadele. Meeste van die huidige stelsels is nie koste effektief nie en lei gewoonlik tot ongewenste newe effekte. Mikrobiologiese metodes wat gebaseer is op koolstof vloei veranderinge in wyn gisrasse mag die potensiaal bied om hierdie tekortkominge te verminder of te oorbrug. ‘n Alternatief is om nuwe Saccharomyces en nie-Saccharomyces gisrasse te identifiseer wat laer etanol opbrengste lewer. In hierdie studie is die etanol opbrengste van 15 wyn gisrasse ondersoek in ‘n sintetiese mos in verskeie toestande, bv. veranderde stikstof vlakke, suiker vlakke, pH en temperatuur, om die rasse te identifiseer wat laer etanol opbrengste lewer (asook die toestande wat laer etanol opbrengste bevorder). Meeste rasse en toestande het soortgelyke etanol opbrengste getoon, alhoewel daar in sekere gevalle interessante verskille was rakende sekere rasse wat verskillende resultate lewer in mos met verskillende stikstof vlakke. Die resultate van die sintetiese mos eksperimente was bevestig in druiwe mos van vier kultivars (Sauvignon Blanc, Chardonnay, Shiraz en Cabernet Sauvignon), maar geen algemene tendens kon afgelei word nie. Wat interessant was is die feit dat die Shiraz fermentasies altyd hoër etanol opbrengste gelewer het vir al vier gisrasse wat gebruik is vir hierdie eksperimente. Dit mag wees weens ‘n eksperimentele faktor wat nie bestudeer was in die raamwerk van hierdie projek nie. Die opbrengs van gliserol was ook bepaal in die verskeie eksperimente en daar was gevind dat gliserol opbrengs baie meer kondisie-afhanklik is in vergelyking met etanol. Temperatuur en gliserol het ‘n direkte verbandskap met mekaar getoon, wat die bevindinge van vorige studies bevestig. Alhoewel verhogings in temperatuur wel gliserol produksie vermeerder het, was die effek nie genoeg om ‘n meetbare impak op die finale etanol konsentrasie te hê nie. Verskillende giste in verskeie verskillende fermentasie toestande het soortgelyke etanol opbrengste gelewer. Die mees merkbare verskille wat bevind is was spesifiek tot individuele gisras en kondisie kombinasies, maar geen algemene afleiding kon gemaak word rakende behandelings wat etanol opbrengste kan verlaag nie.

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

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Brettanomyces bruxellensis has become of increasing interest over the past few decades yet this complex red wine spoilage yeast is still poorly understood and strain variance also leads to the contradictory results reported in literature. This yeast is responsible for the production of phenolic compounds, associated with off-flavours that render wine unpalatable. Sulphur dioxide (SO2) is the most commonly used antioxidant and antimicrobial preservative instrumental in the control of spoilage yeasts such as B. bruxellensis. However, its diploid/triploid genome is enriched for genes that provide the yeast a fortuitous advantage, under conditions permissive for growth, with genotype-dependent SO2 tolerance phenotypes observed among numerous strains. This study investigates the metabolic, physiological and genetic responses associated with SO2 exposure. It also explores the environmental cues responsible for the onset of non-SO2 induced morphological characteristics. These morphological characteristics were investigated using fluorescent probes and microscopy in the presence of SO2 and in the absence thereof, in YPD media. Pseudohyphae formation was observed to be a highly strain dependent feature and less pronounced in the presence of 0.6 mg/L molecular SO2. This study also reports on the metabolic response observed over a 3-week period, following exposure to SO2, in a synthetic wine medium. The following metabolites were consistently monitored during the course of the experiment: acetic acid, acetaldehyde, D-glucose and D-fructose. Utilization of sugars was retarded in the presence of SO2 for up to 10 days in the presence of 1.2 mg/L molecular SO2 and overproduction of acetaldehyde was prominent, with a peak at day 10. The study further highlights the expression profiles observed for the SSU1 gene (referring to SO2 tolerance) and the PAD gene (referring to production of volatile compounds) under SO2 induced conditions in SWM, using qRT-PCR. The co-involvement of increased acetaldehyde production and elevated gene expression were indicative of B. bruxellensis yeast adapting to the presence of molecular SO2, allowing survival of this fascinating yeast. Sequencing of the SSU1 and PAD genes suggests the probable existence of different alleles of these genes that could explicate SO2 tolerance and phenolic compound production associated differences among strains of this species. / AFRIKAANSE OPSOMMING: Hoewel Brettanomyces bruxellensis oor die afgelope paar dekades toenemende belangstelling gewek het, word hierdie komplekse rooiwynbederfgis steeds swak verstaan en lei rasvariasie ook tot teenstrydige resultate in die literatuur. Hierdie gis is verantwoordelik vir die produksie van fenoliese verbindings, wat geassosieer word met afgeure, wat die wyn onsmaaklik laat. Swaweldioksied (SO2) is die algemeenste preserveermiddel wat, weens antioksidant- en antimikrobiese eienskappe, instrumenteel in die beheer van bederforganismes, soos B. bruxellensis, gebruik word. Nogtans is die diploïede/triploïede genoom vir gene verryk, wat die gis ‘n toevallige voordeel bied tydens ongunstige toestande, met genotipe-afhanklike SO2 weerstandbiedende fenotipes wat onder verskeie rasse waargeneem word. Hierdie studie ondersoek die metaboliese, fisologiese en genetiese reaksies tydens SO2-blootstelling. Dit bestudeer verder die omgewingsleidrade wat vir die aanvang van die nie-SO2 geassosiseerde morfologiese eienskappe verantwoordelik is. Hierdie morfologiese eienskappe is ondersoek met behulp van fluoresserende bakens en mikroskopie in die teenwoordigheid van molekulêre SO2 en, in die afwesigheid daarvan, in YPD-medium. Pseudohyphae-vorming is as ʼn baie rasspesifieke eienskap waargeneem en is minder prominent in die teenwoordigheid van molekulêre SO2. Hierdie studie rappoteer ook oor die metaboliese reaksies waargeneem oor ‘n 3-weke tydperk, na blootstelling aan SO2, in ‘n sintetiese wynmedium. Die volgende metaboliete was voordurend gemonitor tydens die verloop van die eksperiment: asynsuur, asetaldehied, D-glukose en D-fruktose. Benutting van die suikers is in die teenwoordigheid van SO2 vertraag en oorproduksie van asetaldehied is prominent waargeneem. Hierdie studie beklemtoon verder die uitdrukkingsprofiele vir die SSU1-geen (verwys na SO2-weerstandbiedendheid) en die PAD-geen (verwys na die produksie van vlugtige verbindings) in SO2-geïnduseerde toestande in SWM, met behulp van qRT-PCR. Die gesamentlike invloed van beide verhoogde asetaldehied produksie en verhoogde uitdrukking van gene, was beduidend van B. bruxellensis-gis wat aanpas in die teenwoordigheid van molekulêre SO2, wat die oorlewing van hierdie fassinerende gis verseker. Volgordebepaling van die SSU1- en PAD-geen dui daarop dat daar waarskynlik meer as een verskillende alleel vir dié gene bestaan, wat die SO2-verdraagsaamheid en produksie van fenoliese verbindings, wat tans tussen verskeie spesies teenwoordig is, kan verduidelik.

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

Thesis (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.

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 (MSc (Institute for Wine Biotechnology))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: The process of winemaking involves a number of microorganisms, contributing both positively and negatively to the final product. Lactic acid bacteria (LAB) are present at all stages of vinification and therefore play a major role in the production of wine, especially red wine. LAB are responsible for malolactic fermentation (MLF), which can be desirable or unwanted depending on the style of wine. LAB can also be responsible for spoilage, and production of off flavours resulting in a decrease in the quality of the finished wine. Spoilage occurs if the wrong species are present at the wrong time and can also occur as a result of spontaneous MLF. It is therefore necessary to control the population of indigenous LAB present in the wine. Plantaricins are bacteriocins produced by Lactobacillus plantarum strains and have the potential to inhibit closely related strains that occupy the same ecological niche. This makes them promising for the control of LAB during the winemaking process. Inhibition of the indigenous LAB microflora could help to prevent the formation of undesirable off-flavours, as well as allowing for control over MLF. The use of plantaricin-producing starter cultures could also lead to a reduction in the amount of sulphur dioxide used in wine. The purpose of this study was to investigate the potential of L. plantarum strains isolated from wine to produce plantaricins under winemaking conditions. This potential was evaluated by investigating the expression of plantaricin genes under winemaking conditions. The first objective was to screen nineteen strains of L. plantarum isolated from South African red wines, as well as a commercial strain; for various genes responsible for the production of plantaricins, including structural, transport and regulatory genes. Results showed that the twenty strains contained at least 16 of the 24 genes (previously reported to be associated with the plantaricin locus for various L. plantarum strains) screened for. Only orfZ123 and orf345 genes yielded no positive results in any of the strains. The second objective was to sequence selected plantaricin genes (plnE, plnF, plnN, plnG and plnB) to determine the variation in nucleotide and amino acid sequences of these genes among the different wine L. plantarum isolates. High homology was found between the nucleotide sequences of the strains and none of the amino acid substitutions in the protein sequences occurred in conserved regions. The nucleotide sequence of plnN was identical in all but one of the strains and similarity of the plnB sequence ranged from 96% to 100%. Similarity of the plnG nucleotide sequence ranged from 99% to 100%. The plnE nucleotide sequence was identical in all but two strains and there were only two groups in terms of nucleotide sequence for plnF, with only two changes between the groups. The third objective was the evaluation of plantaricin production using plate assays mimicking certain wine parameters (pH and ethanol concentration). All twenty strains showed inhibitory activity to varying degrees against a panel of nine indicator microorganisms, including Enterococcus faecalis, Listeria monocytogenes and potential wine spoilage organisms, Lactobacillus spp, Pediococcus spp and Leuconostoc mesenteroides. Addition of 10% ethanol and a low pH of 3.5 decreased both the bacteriocin production as well as the spectrum of activity. Seven of the twenty strains, however, showed good bacteriocin activity under all conditions. The fourth objective was to investigate the expression of two plantaricin structural genes (plnEF and plnJK) and the transporter gene (plnG) under winemaking conditions. Two strains (R1122 and 113.1) were chosen, based on the results from the previous objectives, as starter cultures for MLF in synthetic wine media and Riesling wine. Low wine pH (3.2) and high wine pH (3.8) levels were investigated in conjunction with ethanol concentrations of 0%, 12% and 15%. All three of the genes were expressed to varying degrees depending on the fermentation condition. High ethanol and low pH generally decreased expression of the structural plantaricin genes. The influence on expression of the transporter gene was different, with low pH and presence of ethanol resulting in an increase in gene expression. The genes were also expressed in wine, although at a lower level relative to expression in the synthetic wine media. The presence of sensitive bacteria in the wine seemed to increase expression of the structural genes. Furthermore, expression of the mle gene responsible for MLF was investigated under the same winemaking conditions. Expression was shown to be inducible by malic acid, and negatively affected by the presence of ethanol but positively influenced by a lowering in pH from 3.8 to 3.2. This study confirms that plantaricin genes are expressed under winemaking conditions, which in turn indicates that the plantaricins could be produced under winemaking conditions. This confirms the potential use of these plantaricin-producing strains as starter cultures for MLF with the ability to inhibit indigenous LAB, however, presence of the plantaricin protein in wine still needs to be confirmed. It will also need to be established whether the protein is biologically active and not inhibited by wine-related factors. / AFRIKAANSE OPSOMMING: Die proses van wynmaak bevat 'n verskeidenheid mikroorganismes, wat postiewe en negatiewe bydrae kan lewer tot die finale produk. Melksuurbakterieë is teenwoordig by alle stadiums van wynmaak en speel 'n belangrike rol in die produksie van wyn. Melksuurbakterieë is verantwoordelik vir appelmelksuur gisting (AMG), wat gewens of ongewens kan wees, afhangende van die styl van die wyn. Melksuurbakterieë kan ook verantwoordelik wees vir bederf van wyn, asook die produksie van ongewenste geure wat bydrae tot ʼn toename in die kwaliteit van die wyn. Bederf van wyn kan gebeur as die verkeerde spesies voorkom op die verkeerde tyd en kan ook gebeur as ʼn gevolg van spontane AMG. Dit is dus nodig om die populasie van natuurlike melksuurbakterieë in wyn te beheer. Plantarisiene, geproduseer deur Lactobacillus plantarum wyn-isolate, het die potensiaal om naby verwante stamme se groei te inhibeer wat in dieselfde nis voorkom. Hierdie eienskap maak hul belowend vir die beheer van melksuurbakterieë se groei gedurende die wynmaakproses. Inhibering van die natuurlike mikroflora kan help om die vorming van ongewenste geure te verhoed, sowel as om AMG te beheer. Die gebruik van aanvangskulture, wat plantarisiene kan produseer, kan lei tot ’n vermindering in die gebruik van swaweldioksied in die wynindustrie. Die doel van hierdie studie was om die potensiaal van L. plantarum stamme, geïsoleer vanuit wyn, te ondersoek vir hul vermoë om plantaricins te produseer in toestande wat die wynmaakproses naboots. Die potensiaal was ondersoek deur te kyk na die uitdrukking van plantarisien-produserende gene onder wynmaak toestande. Die eerste objektief was om die 19 L. plantarum stamme, geïsoleer vanuit Suid-Afrikaanse rooi wyne, asook n kommersiele stam, te ondersoek vir die teenwoordigheid van verskeie gene wat verantwoordelik is vir die produksie van plantarisiene, sowel as strukturele, transporter en regulerende gene. Al twintig van hierdie stamme het ten minste 16 uit die 24 gene bevat waarvoor ondersoek was. OrfZ123 en orf345 het egter geen positiewe resultate opgelewer in enige van die stamme nie. Die tweede objektief was om die DNA-volgorde te bepaal van spesifieke gene (plnE, plnF, plnN, plnG, sowel as plnB) en sodoende die variasie in nukleotied en aminosuur volgorde van hierdie gene in die verskillende L. plantarum wyn-isolate te bepaal. Hoë vlakke van homologie was gevind en geen van die aminosuur veranderings het in behoue gebiede plaasgevind nie. Die nukleotied volgorde van plnN was identies in al die stamme, behalwe vir een, en die ooreenkomste tussen die plnB volgorde het varieër van 96% tot 100%. Die ooreenkomste tussen die plnG nukleotied volgorde het varieër van 99% to 100%. Die plnE nukleotied volgorde was identies in al die stamme, behalwe vir twee, en daar was net twee groepe in terme van nukleotied volgorde vir plnF, met net twee veranderinge tussen die groepe. Die derde objektief was om die vermoë van die stamme om plantaricins the produseer, deur gebruik te maak van plaat assays, onder verskillende wyntoestande te ondersoek. Die twinting stamme het verskillende vlakke van inhibering teenoor die nege toets-organismes getoon, wat Enterococcus faecalis, Listeria monocytogenes sowel as potensiele wyn bederf organismes, Lactobacillus spp, Pediococcus spp and Leuconostoc mesenteroides insluit. Die byvoeging van 10% etanol en ’n lae pH van 3.5, het beide bakteriosien produksie inhibeer, sowel as die spektrum van aktiwiteit verminder. Sewe van die stamme het egter steeds goeie aktiwiteit getoon onder al die kondisies wat getoets was. Die vierde objektief was om die uitdrukking van twee plantaricin strukturele gene (plnEF en plnJK), sowel as die transporter geen (plnG) onder wynmaak omstandighede te ondersoek. Twee stamme (R1122 en 113.1) was gekies as aanvangskulture vir AMG in sintesiese wyn media, sowel as Riesling wyn. Hierdie twee stamme was gekies op grond van die resultate wat van die vorige objektiewe verkry was. Lae wyn pH (3.2) en hoë wyn pH (3.8) was ondersoek in samewerking met verskillende etanol konsentrasies wat 0%, 12% en 15% etanol insluit. Al drie hierdie gene was uitgedruk teen verskillende vlakke, afhangende van die verskeie fermentasie kondisies. Hoë etanol en lae pH lei oor die algemeen tot ʼn toename in uitdrukking van die strukturele plantarisien gene. Die invloed op uitdrukking van die transporter geen was verskillend, want lae pH en die teenwoordigheid van etanol het gelei tot ʼn verhoging in geen uitdrukking. Die gene was uitegdruk in wyn, maar was teen laer vlakke relatief tot uitdrukking in die sintetiese wyn media. Dit blyk dat die teenwoordigheid van sensitiewe bakterieë in die wyn tot ‘n hoër uitdrukking van die strukturele gene lei. Die uitdrukking van die mle geen, verantwoordelik vir AMG, was ook onder dieselfde wynmaak kondisies ondersoek. Die uitdrukking was geïnduseer deur appelsuur, negatief beïnvloed deur die teenwoordigheid van etanol, maar positief beïnvloed deur ’n verlaging in pH van 3.8 tot 3.2. Hierdie studie toon dat plantaricin gene uitegedruk word onder wynmaak toestande en dat plantaricins moontlik onder hierdie toestande geproduseer kan word. Die potensiaal van hierdie stamme word getoon om as aanvangskulture gebruik te word vir AMG, om sodoende die groei van natuurlike melksuur bakterieë te inhibeer. Die teenwoordigheid van die plantarisien peptied in die wyn moet egter nog bewys word. Daar sal ook vasgestel moet word of die peptied biologies aktief is en nie deur wynverwante faktore geïnhibeer word nie.