Graduation date: 1955

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Wine quality is greatly influenced by the number of microorganisms, which occur throughout the winemaking process. Yeasts are responsible for the alcoholic fermentation, the lactic acid bacteria (LAB) are responsible for malolactic fermentation (MLF), while acetic acid bacteria (AAB) are responsible for converting ethanol to acetic acid. These microorganisms are present on the grapes and in the cellar and these consequently serve as gateways to the fermentation tanks where they will affect the wine quality. However, these microorganisms can be seen either as beneficial or as wine spoilage microorganisms, depending on the conditions that prevail throughout the winemaking process. It is thus very important to prevent any process that could lead to the lowering of the wine quality. In this regard, some of the factors that should always be evaluated include the quality of the grapes, winemaking techniques and quality control. One of the measures that have been implemented during winemaking to ensure the microbial stability is the use of chemical preservatives. Sulphur dioxide (502) has been, and is, used widely as primary preservative in winemaking. However, an ever-increasing consumer resistance against the use of chemical preservatives has developed as it poses possible health risks and decreases the sensorial quality of wine. An alternative approach to chemical preservation that has triggered numerous new investigations, is biological preservation or biopreservation. This is the use of the natural microbial flora and/or their antimicrobial products, such as bacteriocins, to inhibit or destroy the other sensitive microorganisms that are unwanted in the same environment. Evidence in the wine industry has shown that bacterial spoilage still is a very common problem in many wineries. This bacterial spoilage can lead to, amongst other, two main problems, which are of great concern to winemakers. This include high levels of volatile acidity, resulting in the wine having a vinegary off-flavour, and sluggish/stuck fermentation, which is the result of compounds such as acetic- and other fatty acids that causes inhibition of the yeast's growth. With acetic acid being the common link in both cases, it became evident that investigations should be performed on the main producer of acetic acid, namely AAB. As a result, AAB turned out to be one of the main spoilage microorganisms associated with winemaking. Most of the research on biopreservation in the food and beverage industry has been performed on the Gram-positive LAB. The fact that their spectrum of inhibition currently excludes most Gram-negative bacteria, specifically AAB, indicated that AAB should be screened in search of possible antimicrobial compounds that could be applied to control their cell numbers during winemaking. No evidence of antimicrobial action amongst AAB could be found in literature, therefore this work was considered novel. The main objectives of this study were to screen wine isolates of AAB for the production of antimicrobial compounds. This was followed by the isolation and preliminary characterisation of the antimicrobial substances produced. Various attempts to optimise the production of the antimicrobial compounds and isolation procedures, were also included. This study forms part of a larger research programme that has been initiated at the Institute for Wine Biotechnology at Stellenbosch University on the biopreservation in wine. Our results indicated that possible antimicrobial compounds of proteinaceous nature, produced by AAB isolated from wine, do exist. It was found that two different species of AAB, namely Acetobacter aeeti and Gluconobacter frateurii, produced antimicrobial compounds that inhibited other species of AAB. Preliminary results indicated that these compounds are heat sensitive and stable in a wide pH range. It was also shown that after the action of proteolytic enzymes, such as proteinase K and a-chemotrypsin, all inhibitory activity was lost. This study also revealed the existence of the species Gluconobacter frateurii, which have not yet been associated with the winemaking environment. This study made a valuable contribution to the limited amount of information and understanding of AAB, not only in the wine environment, but also elsewhere. The results and findings of this research would serve as platform for further projects. This might soon lead to the development of antimicrobial substances or tailored wine-yeasts with antimicrobial abilities, which can be applied during winemaking to assist the winemaker in combatting high cell numbers and subsequent spoilage by AAB. / AFRIKAANSE OPSOMMING: Wynkwaliteit word beïnvloed deur 'n verskeidenheid van mikroorganismes wat regdeur die wynrnaakproses teenwoordig is. Die giste is vir die alkoholiese fermentasie, die melksuurbakterieë (MSB) vir die appelmelksuurgisting, terwyl die asynsuurbakterieë (ASB) vir die omskakeling van etanol na asynsuur verantwoordelik is. AI hierdie mikroorganismes is teenwoordig op die druiwe en in die kelder, en dit dien gevolglik as 'n weg waardeur hulle in die fermentasietenke kan kom om sodoende die wynkwaliteit te beïnvloed. Hierdie mikroorganismes kan egter gesien word as óf voordelig óf as wynbederfmikroorganismes, afhangende van die heersende kondisies gedurende die wynrnaakproses. Dit is daarom baie belangrik om enige proses te voorkom wat tot 'n verlaging in wynkwaliteit kan lei. Wat laasgenoemde aanbetref, is daar sekere faktore wat altyd geëvalueer moet word, naamlik die druifkwaliteit, wynrnaaktegnieke en kwaliteitsbeheer. Een van die maatreëls wat geïmplementeer is om mikrobiologiese stabiliteit tydens die wynrnaakproses te handhaaf, is die gebruik van chemiese preserveermiddels. Swaweidioksied (S02) word algemeen gebruik as primêre preserveermiddel tydens wynrnaak. Daar is egter 'n toenemende verbruikersweerstand teen die gebruik van chemiese preserveermiddels, aangesien dit moontlike gesondheidsrisiko's kan inhou, asook tot 'n verlaging in sensoriese kwaliteit van die wyn kan lei. 'n Alternatiewe benadering vir chemiese preservering, wat reeds tot verskeie nuwe ondersoeke gelei het, is biologiese preservering of biopreservering. Dit is die gebruik van die natuurlike mikroflora en/of hulle antimikrobiese produkte, soos bv. bakteriosiene, om die sensitiewe mikroorganismes wat in dieselfe omgewing voorkom, se groei te inhibeer óf om hulle dood te maak. Aanduidings vanuit die wynbedryf dui daarop dat bakteriese bederf steeds 'n algemene probleem is wat in baie kelders ondervind word. Hierdie bakteriese bederf kan onder andere twee hoofprobleme veroorsaak, wat 'n groot bekommernis vir verskeie wynmakers is. Dié probleme sluit in hoë vlakke van vlugtige suurheid, wat gevolglik die wyn 'n asyn-afgeur gee, en slepende/gestaakte fermentasies, wat die gevolg is van komponente soos asynsuur en ander vetsure, wat die gis se groei inhibeer. Die feit dat asynsuur die gemeenskaplike faktor in beide gevalle was, het daarop gedui dat 'n ondersoek rakende die hoofproduseerder van asynsuur, naamlik ASB, benodig word. ASB word gevolglik as een van die hoofbederforganismes wat met die wynrnaakproses geassosieer word, beskou. Die meeste navorsing oor biopreservering in die voedsel -en drank bedryf is op die Gram-positiewe MSB gedoen. Die spektrum van inhibisie van die bakteriosiene van MSB sluit egter die meeste Gram-negatiewe bakterieë uit, veral ASB, en dit dui daarop dat ASB gesif moet word in 'n soektog na antimikrobiese substanse wat moontlik gebruik kan word om hul getalle tydens die wynrnaakproses te beheer. Geen bewyse kon tot dusver uit die literatuur gekry word met betrekking tot antimikrobiese aktiwiteit teen ASB nie, daarom word hierdie navorsing dus as nuut beskou. Hierdie studie se hoofdoelwittewas om die wyn-isolate van ASB vir die produksie van antimikrobiese peptiede te sif. Dit is gevolg deur die isolasie en voorlopige karakterisering van die geproduseerde antimikrobiese komponente. Daar is ook verskeie pogings aangewend om die produksie van die antimikrobiese substanse, asook die isolasieprosedures, te optimiseer. Hierdie studie vorm deel van 'n groter navorsingsprogram oor biopreservering van wyn wat deur die Instituut vir Wynbiotegnologie by die Universiteit van Stellenbosch geïnisieer is. Die resultate het daarop gedui dat antimikrobiese substanse van proteïenagtige aard, afkomstig vanaf wyn-isolate van ASB, wel bestaan. Daar is gevind dat twee veskillende spesies, naamlik Aeefobaefer aeefi en Glueonobaefer frafeurii, antimikrobiese peptiede produseer, wat ander spesies van ASB kan inhibeer. Voorlopige resultate het getoon dat hierdie substanse hitte-sensitief is en ook stabiel is oor 'n wye pH-reeks. Daar was ook aanduidings dat, ná die aksie van proteolitiese ensieme, soos bv. proteïnase K en a-chemotripsien, al die inhibitoriese aktiwiteit verlore gegaan het. Hierdie studie het ook die voorkoms van die spesies Glueonobaefer frafeurii aangedui, wat nog nie tot dusver met die wynrnaakomgewing geassosieer is nie. Hierdie studie maak 'n waardevolle bydrae tot die beperkte hoeveelheid inligting oor en begrip van ASB, nie net in die wynomgewing nie, maar ook in die algemeen in die natuur. Die bevindinge en resultate van hierdie navorsing sal as basis dien vir verdere projekte wat sal volg. Dit kan moontlik binnekort lei tot die ontwikkeling van antimikrobiese substanse, en ook pasgemaakte wyngiste met antimikrobiese vermoëns, wat tydens die wynrnaakproses gebruik kan word om sodoende die wynmaker in staat te stelom die hoë bakteriese getalle en die gevolglike bederf deur ASB, te beheer.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Wine is the product of a complex biological and biochemical interaction between grapes and different microorganisms (fungi, yeasts, lactic acid bacteria and acetic acid bacteria, as well as the mycoviruses and bacteriophages affecting them) in which yeasts play the most important role regarding the alcoholic (primary) fermentation. These wine-associated yeasts can be divided into Saccharomyces and non-Saccharomyces yeasts. During fermentation, there is a sequence of dominance by the various non-Saccharomyces yeasts, followed by Saccharomyces cerevisiae, which then completes the fermentation. This is especially evident in spontaneously fermenting must, which has a low initial S. cerevisiae concentration. Some non- Saccharomyces yeasts can also be found throughout the fermentation. The non- Saccharomyces presence in the fermentation can affect wine quality, either positively or negatively. A positive contribution could be especially useful to improve wines produced from grape varieties with a neutral flavour profile due to non-optimal climatic conditions and/or soil types. As part of a comprehensive South African research programme, the specific objectives of this study were: the isolation of indigenous non-Saccharomyces yeasts from vineyards and musts; the identification of these isolates; the characterisation and evaluation of predominant species under winemaking conditions; and the development of a protocol for their use in enhancing wine quality. Initially, 720 isolates representing 24 different species, were isolated from grape (vineyard) and must samples taken over three vintages from four distinctly different wine producing regions. The isolates were characterised and grouped utilising biochemical profiles and DNA karyotyping, whereupon representative isolates were identified. The yeast species that had the highest incidence of predominance in the vineyard was Kloeckera apiculafa. However, some vineyard samples were characterised by low numbers or absence of this yeast, which is not according to generally accepted norms. Other species that also predominated in a few of the vineyard samples were Candida pulcherrima, Kluyveromyces thermofolerans, Rhodotorula sp. and Zygosaccharomyces bailii. Generally, there was a greater diversity of yeasts in the processed must than from the vineyard samples. Furthermore, while each sample showed a different yeast population, no pattern linking species to climatic zone was observed. Four species i.e. Candida collieulosa, Candida pulcherrima, Candida stel/ata and Kloeckera apiculata, were found to predominate in grape must samples. Representative strains consequently received further attention during laboratory and small-scale winemaking trials. A protocol was developed whereby individual species could be used in co-inoculated fermentations with S. cerevisiae in the small-scale production of wine. An improvement in wine quality was achieved and it was found that there was a link between specific species and grape cultivar. The ability of C. pulcherrima to improve Chenin blanc wine quality was investigated further. Results over three vintages showed that the wine produced by the co-inoculated fermentation was superior to that of a reference wine (produced by S. cerevisiae only). The improvement in wine quality was not linked to increased ester content nor were the standard chemical analyses adversely affected. The effects of pH and wine production parameters i.e. 802, fermentation temperature and use of di-ammonium phosphate (DAP), on this yeast followed the same pattern as that known for S. cerevisiae. This study was successfully completed and the developed protocol can be used for the improvement of Chenin blanc wine where additional aroma and quality is needed. / AFRIKAANSE OPSOMMING: Wyn is die produk van 'n komplekse biologiese en biochemiese interaksie tussen druiwe en mikroorganismes (swamme, giste, melksuurbakterieë, asynsuurbakterieë, asook die mikovirusse en bakteriofage wat hul beïnvloed) waar gis die belangrikste rol speel ten opsigte van die alkoholiese (primêre) fermentasie. Die betrokke giste kan in Saccharomyces- en nie-Saccharomyces-giste verdeel word. Tydens gisting vind daar 'n opeenvolging van dominansie deur die verskillende nie-Saccharomyces giste plaas, gevolg deur Saccharomyces cerevisiae, wat dan die gisting voltooi. Dit is veral in spontaan fermenterende mos, waarin aanvanklik lae konsentrasies S. cerevisiae-gisselle voorkom, waarneembaar. Sekere nie-Saccharomyces-giste kan ook regdeur die verloop van fermentasie gevind word. Die teenwoordigheid van nie-Saccharomyces-giste kan 'n bydrae maak tot wynkwaliteit, hetsy positief of negatief. 'n Positiewe bydrae kan veral nuttig wees vir die verbetering van wyn geproduseer van druifsoorte met neutrale geurprofiele as gevolg van nie-optimale klimaatstoestande en/of grondtipes. As deel van 'n uitgebreide Suid-Afrikaanse navorsingsprogram, was die doelwitte van hierdie studie soos volg: die isolasie van inheemse nie-Saccharomyces-giste vanuit wingerde en mos; die identifikasie van hierdie isolate; die karakterisering en evaluering van spesies wat tydens wynbereiding oorheers; en die ontwikkeling van 'n protokol waarin geselekteerde nie- Saccharomyces-giste gebruik kan word vir die verbetering van wynkwaliteit. Druif- en mosmonsters is oor drie oestye vanuit vier duidelik onderskeibare wynproduserende gebiede geneem en 720 isolate, verteenwoordigend van 24 verskillende spesies, is hieruit geïsoleer. Hierdie isolate is volgens biochemiese profiele en DNA-kariotipering gekarakteriseer en gegroepeer waarna verteenwoordigende isolate geïdentifiseer is. Die gisspesie wat die meeste in wingerde voorgekom het, was Kloeckera apiculata. Sommige wingerde is egter deur lae getalle of afwesigheid van dié gis gekenmerk, In feit wat afwyk van die algemeen aanvaarde norm. Ander spesies, nl. Candida pulcherrima, Kluyveromyces thermotolerans, Rhodotorula sp. en Zygosaccharomyces bailii, het ook in enkele gevalle in die wingerdmonsters oorheers. Oor die algemeen was daar 'n groter diversiteit van giste in die geprosesseerde mos as in die wingerdmonsters. Verder is elke monster gekenmerk deur verskillende gispopulasies, maar geen verband tussen gisspesie en klimaatsone is waargeneem nie. Vier spesies, nl. Candida collieulosa, Candida pulcherrima, Candida stel/ata en Kloeckera apiculata, het in hoë getalle in die druiwemosmonsters oorheers en verteenwoordigende rasse het verdere aandag tydens laboratorium- en kleinskaalse wynmaakproewe geniet. 'n Protokol, waar hierdie rasse individueel gebruik is in gesamentlike geïnokuleerde fermentasies met S. cerevisiae vir die kleinskaalse produksie van wyn, is ontwikkel. 'n Verbetering in wynkwaliteit is verkry en daar is 'n verband tussen spesifieke gisspesies en druifvariëteit gevind. Gevolglik is die vermoë van C. pulcherrima om die gehalte van Chenin blanc wyn te verbeter, verder ondersoek. Resultate oor drie oesjare het gewys dat die wyn wat met die C. pulcherrima / S. cerevisiae kombinasie geproduseer is, beter was as 'n verwysingswyn (deur slegs S. cerevisiae geproduseer). Die waargenome verbetering in wynkwaliteit was egter nie aan 'n verhoging in esterinhoud te danke nie en die standaard chemiese analises het geen negatiewe afwyking uitgewys nie. Verder is gevind dat die effek van pH en wynproduksieparameters, nl. die gebruik van S02, fermentasietemperatuur en die gebruik van di-ammoniumfosfaat (DAP), dieselfde patroon as die bekend vir S. cerevisiae gevolg het. Die ontwikkelde protokol kan nou aangewend word waar verhoogde Chen in blanc wynaroma en kwaliteit verlang word.

Bibliography: leaves 200-214. / xi, 214 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Three structurally related compounds, 2-acetyltetrahydropyridine (ACTPY), 2-ethyltetrahydropyridine (ETPY) and N-heterocycle, 2-acetyl-1-pyrroline (ACPY), were quantified and found to be unique components of mousy wines. 35 lactic acid bacteria (LAB) were screened for the ability to produce mousy off-flavour. In addition to Lactobacillus brevis and L. cellobiosus, a diversity of LAB species, particularly heterofermentative Lactobacillus spp. and Oenococcus oeni exhibited this ability in a range of ethanolic and wine-based media. The substrates and metabolism of mousy compound formation by LAB were also investigated. A pathway for the formation of ACPY and ACTPY by heterofermentative LAB was proposed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture & Oenology, 1999

Bibliography: leaves 200-214. Three structurally related compounds, 2-acetyltetrahydropyridine (ACTPY), 2-ethyltetrahydropyridine (ETPY) and N-heterocycle, 2-acetyl-1-pyrroline (ACPY), were quantified and found to be unique components of mousy wines. 35 lactic acid bacteria (LAB) were screened for the ability to produce mousy off-flavour. In addition to Lactobacillus brevis and L. cellobiosus, a diversity of LAB species, particularly heterofermentative Lactobacillus spp. and Oenococcus oeni exhibited this ability in a range of ethanolic and wine-based media. The substrates and metabolism of mousy compound formation by LAB were also investigated. A pathway for the formation of ACPY and ACTPY by heterofermentative LAB was proposed.

Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: The importance of indigenous fruit wines is not well researched and documented. There is a need to develop and exploit these valuable food resources through improved production practices, storage, preservation and utilization technologies. The maruia fruit is beneficial in many ways, it can be used for making juice, jam, beer or can be eaten as a whole fruit. The highly nutritive nature of the fruit, its distinctive tropical flavor, its wild occurrence and demand by the local and international communities for the by-products of the fruit necessitated efforts to optimize the technological processes for the production of the possible by-products. This study focuses on the fermentation technology of the maruia fruit. The effect of enzymes prior to the fermentation process and post-fermentation was evaluated. For pre-fermentation processes we focused on the ability of commercial enzymes to increase juice yield, improve the clarification and filterability. For pre- and post-fermentation applications, aroma release was considered. The results indicated a significant increase in the yield depending on the enzyme used. An increase of at least 2% was recorded and a maximum of 12% yield increase was observed. The enzymes also had a phenomenal effect on the release of bound monoterpenes and hence enhancing the flavor of the juice. The panel of judges confirmed the results from the gas chromatography analyses by noting an increase in flavor intensity in the enzyme treated juice. The possibility of selecting a yeast strain that performs best during the fermentation of maruia pulp was also looked at. This study aimed at selecting a strain that produces wine and distillate with the typical maruia flavor complex. We showed the effect of the different yeast strains, in the wines and distillates, on the principal volatile compounds. We then correlated the performance of the different strains as perceived by the panel to the various volatile compounds. The effect of fermentation temperature on the performance of the different yeast strains was also considered. Fermenting the maruia pulp at different temperatures resulted in the production of wines and distillates with different volatile profiles for the different yeast strains. The wines and distillates fermented at a low temperature of 15°C were preferred to the wines and distillates fermented at 30°C. However, not all strains performed well at 15°C, strains like NT116 performed better at 30°C. The different commercial strains produced wines and distillates with significantly different flavor profiles. These differences in the flavor profiles were reflected in the sensory evaluation where, depending on the interaction of the volatile compounds some wines and distillates were preferred to others. The effect of the different commercial enzymes and yeast strains should thereof be further evaluated and optimized on a larger scale. This would greatly help prevent variation in quality of the fermented by-products of the maruia fruit. / AFRIKAANSE OPSOMMING: Die belang van inheemse vrugtewyne is nie goed nagevors en gedokumenteer nie. Daar is 'n behoefte om hierdie waardevolle voedselbronne te ontwikkel en te benut, deur verbeterde produksiepraktyke, storing, preservering en benuttingstegnologieë. Die maroelavrug is veelsydig op baie wyses, deurdat dit gebruik word vir die maak van sap, konfyt, bier, of as heel vrug geëet kan word. Die vrug is hoog in voedingswaarde, het In kenmerkende tropiese geur, kom wild voor, en is in aanvraag by plaaslike en internasionale gemeenskappe vir die by-produkte van die vrug. Dit maak dit essensieel om die tegnologiese prosesse vir die produksie van hierdie moontlike by-produkte te optimiseer. Hierdie studie fokus op die fermentasie-tegnologie van die maroelavrug. Die effek van ensieme voor en na die fermentasie-proses is geëvalueer. Vir prosesse wat voor fermentasie plaasvind, het ons gefokus op die vermoë van kommersiële ensieme om sapopbrengs te verhoog, asook om verheldering en filtrering te verbeter. Vir beide voor- en na-fermentasie toepassings is die vrystelling van aroma gemonitor. Die resultate dui op 'n betekenisvolle verhoging in die sapopbrengs, afhangende van die ensiem wat gebruik is. 'n Verhoging van ten minste 2% is opgeteken, en 'n maksimum van 12% opbrengsverhoging is waargeneem. Die ensieme het ook 'n geweldige effek op die vrystelling van gebonde monoterpene gehad, en dus die verhoging in die geur van die sap. Die proepaneel het die resultate bevestig van die gaschromatografie-analises, deur 'n verhoging in die geurintensiteit in die ensiembehandelde sap te bemerk. Daar is ook gekyk na die moontlikheid om 'n gisras te selekteer wat die beste presteer tydens die fermentasie van maroela-pulp. Hierdie studie het die doelstelling gehad om In gisras te selekteer wat wyn en distillaat produseer met In tipiese maroelageurkompleks. Ons het die effek van verskillende gisrasse aangedui in die wyne en distillate, op grond van van vlugtige komponente. Ons het dan die prestasie van die verskillende rasse, soos waargeneem deur die paneel, gekorrelleer met die verskeie vlugtige komponente. Die effek van fermentasie-temperatuur op die werkverrigting van die verskillende gisrasse is ook in ag geneem. Fermentasie van die maroela-pulp by verskillende temperature het gelei tot die produksie van wyne en distillate met verskillende vlugtige profiele vir die verskillende gisrasse. Die wyne en distillate wat by In laer temperatuur van 15°C gefermenteer is, is verkies bo die wyne en distillate wat by 30°C gefermenteer is. Alle rasse het egter nie baie goed presteer by 15°C nie, soos byvoorbeeld NT116 wat beter presteer het by 30°C. Die verskillende kommersiële rasse het wyne en distillate geproduseer met betekenisvol verskillende geurprofiele. Hierdie verskille in geurprofiele is gereflekteer in die sensoriese evaluering waar, afhangende van die interaksie van die vlugtige komponente, sommige wyne en distillate bo ander verkies is. Die effek van die verskillende kommersiële ensieme en gisrasse moet verkieslik verder op groter skaal geëvalueer en geoptimiseer word. Dit sal veral help om variasie in kwaliteit van die gefermenteerde by-produkte van die maroelavrug te voorkom.

Thesis (MSc)--Stellenbosch University, 1990. / ENGLISH ABSTRACT: To remain competitive in the market place, the South African wine industry will have to direct well-planned yeast strain-development programmes. However, the winemaker can only benefit from the extensive biochemical and molecular information of the yeast cell and the impressive arsenal of genetic techniques available, if the wine industry defines its requirements in genetic terms. The successful application of these genetic and recombinant deoxyribonucleic acid (DNA) techniques in breeding programmes depends on the availability of rapid and reliable techniques to differentiate between parental and hybrid strains. Ten strains of Saccharomyces cerevisiae used for commercial production of wine in South Africa, were characterised by electrophoretic banding patterns of total soluble cell proteins, DNA restriction fragments and chromosomal DNA. Variations in the protein and DNA profiles of strains N6, N21, N66, N76, N95 and N97 were apparent in the number, position and intensity of the bands. Strains N93 and N181 originated from the same culture and, as expected, displayed the same characteristic protein, DNA restriction fragment and chromosomal banding patterns. Similar protein and DNA profiles were also obtained for killer strain N96 and strain N91. Strain N91 is a derivative of strain N96, cured of the K2 killer character. Results obtained by electrophoretic fingerprinting and karyotyping corresponded well, indicating that these techniques are valuable in the identification and quality control of industrial wine yeasts. The value of electrophoretic fingerprinting and karyotyping was also demonstrated in a breeding programme. The aim of this breeding programme was to obtain hybrids that combine the desired oenological characteristics of strains N76 and N96, and of strains N96 and N181. The protein banding patterns of hybrids USM21, USM22 and USM23 were identical and contained a combination of prominent unique bands present in the profiles of parental strains, N76 and N96H (N96H is a haploid derived from N96). The DNA restriction fragment profiles of hybrids USM21, USM22 and USM23 contained slight variations, whereas their profiles were quite different from those of their parental strains, N76 and N96H. The contour clamped homogeneous electric field (CHEF) karyotypes of hybrids USM21, USM22 and USM23 were identical but differed from those of their parental strains, N76 and N96H. The protein profiles of hybrid USM30 and its parental strains, N96H and N181, were similar, whereas their DNA restriction fragment banding patterns and CHEF karyotypes showed discrete differences. In conclusion, protein and DNA fingerprinting techniques were found to be valuable in selecting four hybrid killer strains after mass spore-cell mating. These four killer hybrids contain desirable oenological properties long sought after by the South African wine industry. Fermentation trials and evaluation of these hybrids were conducted independently by the Deparment of Oenology, University of Stellenbosch and by Stellenbosch Farmers' Winery and they have now been released for commercial wine production. / AFRIKAANSE OPSOMMING: Om mededingend in die handel te bly, sal die Suid-Afrikaanse wynbedryf weloorwoe gisras-ontwikkelingsprogramme moet loads. Die wynmaker sal egter slegs voordeel kan trek uit die omvattende biochemiese en molekul...Lre inligting oor die gissel en die indrukwekkende arsenaal van genetiese tegnieke wat beskikbaar is, indien die wynbedryf sy vereistes in genetiese terme definieer. Die suksesvolle toepassing van hierdie genetiese en rekombinante deoksiribonuklei"ensuur (DNA) tegnieke in telingsprogramme sal afhang van die beskikbaarheid van vinnige en betroubare tegnieke om tussen ouerlike en hibried-rasse te onderskei. Tien rasse van Saccharomyces cerevisiae wat vir kommersiele wynproduksie in Suid-Afrika gebruik word, is met behulp van elektroforetiese bandpatrone van totale oplosbare selprotei"ene, DNA-restriksiefragmente en chromosomale DNA gekarakteriseer. Variasies in die protei"en- en DNA-profiele van rasse N6, N21, N66, N76, N95 en N97 het geblyk uit die aantal, posisie en intensiteit van die bande. Rasse N93 en N181 het uit dieselfde kultuur ontstaan en het, soos verwag, dieselfde karakteristieke protei"en-, DNA-restriksiefragmenten chromosomale bandpatrone getoon. Soortgelyke protei"en en DNA profiele is ook vir killerras N96 en ras N91 verkry. Ras N91 is 'n variant van ras N96 wat die K2 killerkenmerk verloor het. Resultate wat met behulp van elektroforetiese vingermerking en kariotipering verkry is, het goed ooreengestem en dui daarop dat hierdie tegnieke waardevol is vir die identifisering en beheer van industriele giste. Die waarde van elektroforetiese vingermerking en kariotipering in telingsprogramme is ook gedemonstreer. Die doel van hierdie telingsprogram was om hibriede te kry waarin die gewenste kenmerke van rasse N76 en N96, en van rasse N96 en N181, gekombineer is. Die protei"en-bandpatrone van hibriede USM21, USM22 en USM23 was identies en het 'n kombinasie van prominente unieke bande, teenwoordig in die profiele van hul ourlike rasse, N76 en N96H (N96H is 'n haploi"de afstammeling van N96), bevat. Die DNArestriksiefragment- profiele van hibriede USM21, USM22 en USM23 toon geringe onderlinge verskille, maar hul profiele het wesenlik van die van hul ouerlike rasse, N76 en N96H, verskil. Die kontoergeklampde-homogene-elektriese-veld (CHEF) elektroforetiese kariotipes van hibriede USM21, USM22 en USM23 was identies, maar het verskil van die van hul ouerlike rasse, N76 en N96H. Die protei"enprofiele van hibried USM30 en sy ouerlike rasse, N96H en N181, was soortgelyk, terwyl hul DNA-restriksiefragment-bandpatrone en CHEF-kariotipes diskrete verskille getoon het. Ten slotte is gevind dat prote'ien- en DNAvingermerkingstegnieke waardevol was in die seleksie van vier hibried-killerrasse na massa spoor-sel paring. Hierdie vier killerhibriede beskik oor gewenste wynkundige eienskappe waarna die Suid-Afrikaanse wynbedryf reeds lank soek. Fermentasie-proewe en evaluering is onafhanklik deur die Departement Wynkunde, Universitiet van Stellenbosch en deur Stellenbosch-Boerewynmakery gedoen en hulle is nou vir kommersiele wynproduksie vrygestel.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: In the current study, two isolates showing killer activity against several wine yeast species in a previous study were identified to strain level and found to belong to the yeast species Candida pyralidae. The identified yeast strains and a Kluyveromyces wickerhamii yeast strain used as a control exhibited killer activity against B. bruxellensis known for its spoilage characteristics in red wine, and against several strains of the genus Brettanomyces on white and red grape juice medium. The killer yeasts inhibited neither the growth of S. cerevisiae nor that of the lactic acid bacteria Oenococcus oeni and Lactobacillus plantarum strains. Yeasts are reported to secrete killer toxins, which can play a role in yeast microbial interactions under winemaking conditions. The C. pyralidae strains were found to secrete two novel killer toxins, designated CpKT1 and CpKT2. These killer toxins were stable and active under winemaking conditions, pH 3.5 - 4.5 and temperature ranges between 15 and 25°C. Ethanol and sugar concentrations found during winemaking did not affect the activity and stability of these killer toxins. Although, the killer toxins differed with regards to their biochemical and environmental stability and activity, they were found to have a similar mode of action. The killer toxins induced a fungistatic effect on B. bruxellensis sensitive cells in addition to binding to the cell wall of the sensitive cells, inducing cell surface and plasma membrane damage as did the Kwkt killer toxin secreted by K. wickerhamii. According to the author’s knowledge this is the first report on the identification of novel killer toxins secreted by C. pyralidae strains isolated from a wine environment as well as the identification of the mode of action of killer toxins on B. bruxellensis cells. This indeed provides great research scope in this field. The exoproteomes consisting of the killer toxins Kwkt, CpKT1 and CpKT2 revealed the presence of exo-glucanases and glucosidases, respectively. The enzymes KwExg1 (exoglucanase) and KwSun4 (glucosidase) retrieved from K. wickerhamii’s exoproteome were identified as the potential toxins, but their killer activity could not be confirmed. These findings suggest that hydrolytic enzymes possess killer activity, as previously reported in literature. However, further investigation is needed to identify the killer toxins characterized in this study. / AFRIKAANSE OPSOMMING: In die huidige studie is twee isolate wat in ’n vorige studie “killer” aktiwiteit teenoor verskeie wyngisspesies vertoon het, tot op rasvlak geïdentifiseer en daar is gevind dat hulle aan die gisspesie Candida pyralidae behoort. Die geïdentifiseerde gisrasse en ’n Kluyveromyces wickerhamii gisras wat as kontrole gebruik is, het “killer” aktiwiteit getoon teenoor B. bruxellensis, wat bekend is vir sy bederfkarakter in rooi wyn, en ook teenoor verskeie rasse van die genus Brettanomyces in wit en rooi druiwesapmedium. Die “killer” giste het nie die groei van óf S. cerevisiae óf van die melksuurbakteria Oenococcus oeni en Lactobacillus plantarum-rasse geïnhibeer nie. Giste word berig om “killer” gifstowwe uit te skei, wat ’n rol kan speel in gis mikrobiese interaksies onder wynbereidingstoestande. Die C. pyralidae-rasse is gevind om twee nuwe “killer” gifstowwe af te skei, wat CpKT1 en CpKT2 genoem is. Hierdie “killer” gifstowwe was stabiel en aktief onder wynbereidingstoestande, pH 3.5 - 4.5 en temperatuur tussen 15 en 25°C. Die etanol- en suikerkonsentrasies wat onder wynbereiding voorkom, het nie die aktiwiteit en stabiliteit van hierdie “killer” gifstowwe beïnvloed nie. Hoewel die “killer” gifstowwe met betrekking tot hulle biochemiese en omgewingstabiliteit en aktiwiteit verskil het, is daar gevind dat hulle ’n eenderse modus van aksie het. Die “killer” gifstowwe het ’n fungistatiese effek op B. bruxellensis sensitiewe selle geïnduseer, buiten dat dit aan die selwand van die sensitiewe selle gebind het, en het seloppervlak- en plasma-membraanskade geïnduseer, net soos die Kwkt “killer” gifstof wat deur K. wickerhamii afgeskei is. So ver die skrywer weet, is hierdie die eerste verslag van die identifisering van nuwe “killer” gifstowwe wat deur C. pyralidae rasse afgeskei word wat uit ’n wynomgewing geïsoleer is, asook van die identifikasie van die modus van aksie van “killer” gifstof op B. bruxellensis selle. Dit verbreed dus beslis die navorsingsomvang van hierdie gebied. Die eksoproteome, bestaande uit die “killer” gifstowwe Kwkt, CpKT1 en CpKT2, het die teenwoordigheid van ekso-glukanases en glukosidases onderskeidelik onthul. Die ensieme KwExg1 (eksoglukanase) en KwSun4 (glukosidase) wat vanuit K. wickerhamii se eksoproteoom herwin is, is as die potensiële gifstowwe geïdentifiseer, maar hulle “killer” aktiwiteit kon nie bevestig word nie. Hierdie bevindings suggereer dat hidrolitiese ensieme “killer” aktiwiteit besit, soos voorheen in die literatuur berig is. Verdere ondersoeke word egter benodig om die “killer” gifstowwe wat in hierdie studie gekarakteriseer is, te identifiseer.

Dissertation (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: L-Malic and tartaric acid are the most prominent organic acids in wine and playa crucial role in winemaking processes and wine quality, including the organoleptic quality and the physical, biochemical and microbial stability of wine. The production of premium wines depends on the oenologist's skill to accurately adjust wine acidity to obtain the optimum balance with other wine components to produce wine with optimum colour and flavour. Strains of Saccharomyces, in general, rarely degrade L-malic acid completely in grape must during alcoholic fermentation, with relatively minor modifications in total acidity during vinification. The degree of L-malic acid degradation, however, varies from strain to strain. Some strains of Saccharomyces are known to be able to degrade a higher percentage of L-malic acid, but the underlying reason for this phenomenon is unknown. The underlying mechanisms of this phenomenon have been partially revealed during preliminary transcriptional regulation research during this study. In contrast, S. pombe cells can effectively degrade up to 29 gil L-malic acid via the malo-ethanolic pathway that converts L-malic acid to pyruvate and CO2, and ultimately to ethanol under fermentative conditions. A number of reasons for the weak degradation of L-malic acid in Saccharomyces cerevisiae have been postulated. Firstly, S. cerevisiae lacks the machinery for the active transport of L-malic acid found in S. pombe and relies on rate-limiting simple diffusion for the uptake of extracellular L-malic acid. Secondly, the malic enzyme of S. cerevisiae has a significantly lower substrate affinity for L-malic acid (Km = 50 mM) than that of S. pombe (Km = 3.2 mM), which contributes to the weaker degradation of L-malic acid in S. cerevisiae. Lastly, the mitochondrial location of the malic enzyme of S. cerevisiae, in contrast to the cytosolic S. pombe malic enzyme, suggests that the S. cerevisiae malic enzyme is inherently subject to the regulatory effects of fermentative metabolism. The malate permease gene tmael) and the malic enzyme gene (mae2) of S. pombe was therefore cloned and co-expressed in single or multi-copy under regulation of the constitutive S. cerevisiae 3-phosphoglycerate kinase (PGK1) promoter and terminator sequences in a laboratory strain of S. cerevisiae. This introduced a strong malo-ethanolic phenotype in S. cerevisiae where L-malic acid was rapidly and efficiently degraded in synthetic and Chardonnay grape must with the concurrent production of higher levels of ethanol. Functional expression of the malo-ethanolic pathway genes of S. pombe in a laboratory strain of S. cerevisiae paved the way for the genetic modification of industrial wine yeast strains of Saccharomyces for commercial winemaking. A prerequisite for becoming an inherited component of yeast is the stable integration of the malo-ethanolic genes into the genome of industrial wine yeast strains. Genetic engineering of wine yeasts strains of Saccharomyces is, however, complicated by the homothallic, multiple ploidy and prototrophic nature of industrial strains of Saccharomyces. Transformation and integration of heterologous genes into industrial strains of Saccharomyces require the use of dominant selectable markers, i.e. antibiotic or toxic compound resistance markers. Integration of these markers into the yeast genome is, however, not acceptable for commercial application due to the absence of long-term risk assessment and consumer resistance. A unique strategy for the integration of the S. pombe mae} and mae2 expression cassettes without the incorporation of any non-yeast derived DNA sequences was. The malo-ethanolic cassette, containing the S. cerevisiae PGK} promoter and terminator regions together with the S. pombe mae] and mae2 open reading frames, was integrated into the VRA3 locus of an industrial strain of Saccharomyces bayanus EC 1118 during co-transformation with a phleomycin-resistance plasmid, pUT332. After initial screening for phleomycin resistance, S. bayanus EC1118 transformants were cured of the phleomycin-resistance plasmid, resulting in the loss of non-yeast derived DNA sequences. After correct integration of the mae] and mae2 expression cassettes was verified, small-scale vinification in synthetic and Chardonnay grape must with stable transformants resulted in rapid and complete degradation of L-malic acid during the early stages of alcoholic fermentation. Integration and expression of the malo-ethanolic genes in S. bayanus ECll18 had no adverse effect on the fermentation ability of the yeast, while sensory evaluation and chemical analysis of the Chardonnay wines indicated an improvement in wine flavour compared to the control wines, without the production of any off-flavours. / AFRIKAANSE OPSOMMING: L-Appelsuur en wynsteensuur is die mees prominente organiese sure in wyn en speel 'n kritiese rol in die wynbereidingsproses en organoleptiese wynkwaliteit, insluitende die fisiese, biochemiese en mikrobiese stabiliteit van wyn. Die produksie van hoë-kwaliteit wyne berus op die vermoë van 'n wynmaker om die suurinhoud korrek aan te pas om sodoende 'n gebalanseerde produk met optimale geur en kleur te produseer. Saccharomyces rasse kan gewoonlik nie appelsuur volledig tydens alkoholiese gisting benut nie en dra dus nie noemenswaardig tot 'n verlaging van die totale suurinhoud van wyn by nie. Die mate van appelsuur afbraak deur Saccharomyces wissel egter van ras tot ras. Sekere Saccharomyces rasse kan 'n groter persentasie appelsuur afbreek, maar die onderliggende rede vir hierdie verskynsel is onbekend. Die onderliggende meganismes vir hierdie verskynsel is gedurende hierdie studie uitgelig na afloop van voorlopige transkripsionele regulerings studies op die malaatensiemgeen. In teenstelling hiermee kan S. pombe tot 29 gIl appelsuur via die malo-alkoholiese padweg afbreek waartydens appelsuur na pirodruiwesuur en CO2, en uiteindelik na alkoholonder fermentatiewe toestande omgeskakel word. Verskeie redes vir die swak afbraak van appelsuur deur Saccharomyces cerevisiae is voorgestel. Eerstens beskik S. cerevisiae nie oor 'n meganisme vir die aktiewe transport van appelsuur, soos in die geval van S. pombe nie, en is aangewese op die stadige opname van appelsuur deur eenvoudige diffusie. Tweedens het die S. cerevisiae malaatensiem 'n baie laer substraataffiniteit vir appelsuur (Km = 50 mM) in vergelyking met die van S. pombe (Km = 3.2 mM), wat verder bydra tot die swak afbraak van appelsuur in S. cerevisiae. Laastens dra die mitochondriale ligging van die S. cerevisiae malaatensiem in teenstelling met die sitoplasmiese ligging van die S. pombe malaatensiem, verder by tot die swak afbraak van appelsuur, aangesien die mitochondria onder fermentatiewe toestande negatief gereguleer word. Die malaatpermease geen (maely en malaatensiem geen (mae2) van S. pombe is gevolglik gekloneer en heteroloog in 'n laboratoriumras van S. cerevisiae onder die beheer van die konstitutiewe 3-fosfogliseraat kinase (PGKI) promoter- en termineerdervolgordes uitgedruk. 'n Sterk malo-alkoholiese fenotipe was duidelik tydens fermentasies met die rekombinante gis in sintetiese en Chardonnay druiwemos, met 'n gepaardgaande verhoging in alkoholvlakke. Funksionele uitdrukking van die malo-alkoholiese gene van S. pombe in 'n S. cerevisiae laboratoriumras het die weg vir die genetiese modifisering van industriële wynrasse van S. cerevisiae vir kommersiële wynfermentasie gebaan. Om 'n integrale deel van die gis te word, moet die malo-alkoholiese gene stabiel in die genoom van industriële wynrasse geïntegreer word. Genetiese manipulering van industriële wynrasse word egter bemoeilik deur die homotalliese, multi-ploïediese en prototrofiese aard van industriële Saccharomyces rasse. Transformasie en integrasie van heteroloë gene in industriële Saccharomyces rasse vereis die gebruik van dominante merkers, bv. weerstandbiedendheid teen antibiotika of ander gifstowwe. Integrasie van hierdie merkers in die gisgenoom is egter nie vir kommersiële toepassing aanvaarbaar nie weens die afwesigheid van langtermyn risikobepalings en verbruikersweerstand. Tydens hierdie studie is daar dus gepoog om industriële wynrasse met 'n unieke strategie geneties te verbeter sodat slegs gis-DNA tydens die integrasie van die S. pombe mae1 en mae2 uitdrukkingskassette in die gisgenoom opgeneem word. Die Malo-alkoholiese integrasiekasset wat slegs die S. pombe mae1, mae2 oopleesrame en die S. cerevisiae PGK1 promoter en termineerdervolgordes bevat, is in die URA3 lokus van Saccharomyces bayanus ECll18 geïntegreer tydens parallelle transformasie met 'n 'phleomycin' weerstandbiedendheidsplasmied. Na seleksie van transformante op 'phleomycin' -bevattende media, is die S. bayanus EC 1118 transformante in nieselektiewe kondisies opgegroei sodat verlies van die 'phleomycin' plasmied kon plaasvind. Integrasie van die mae1 en mae2 uitdrukkingskassette is bevestig en kleinskaalse fermentasies in sintetiese en druiwemos het 'n vinnige en doeltreffende afbraak van appelsuur in die vroeë fases van die alkoholiese fermentasie getoon. Integrasie en uitdrukking van die malo-alkoholiese gene in S. bayanus ECl118 het geen nadelige effek op die fermentasievermoë van die gis getoon nie, terwyl sensoriese en chemiese ontleding van die Chardonnay wyne 'n verbetering in aroma relatief tot die kontrole wyne getoon het, met die afwesigheid van enige afgeure.

Includes bibliographies. This thesis describes the isolation of putative promoter sequences that can produce differential expression of a gene during anaerobic wine fermentations, the use of these sequences in the development of expression vectors and the application of this work to the production of genetically engineered wine yeasts for commercial purposes.