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.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Wine production involves complex interactions between a variety of yeasts and bacteria. Conventional microbiological methods can be used to identify the different microorganisms present in wine, but prove to be time-consuming and certain microbial species may not grow on synthetic isolation media. The aim of this study was to evaluate the microbial population present in two South African red wines, Pinotage and Merlot, as well as five spoilt commercial South African wines by using a non-culturable approach, polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE). The results from the non-culturable approach were compared to conventional platings. Unique PCR-based DGGE fingerprints were obtained for the Bacteria and yeasts present in the South African Pinotage and Merlot wines. Using yeast specific primers the Pinotage wine showed the presence of non-Saccharomyces yeasts at the beginning of the alcoholic fermentation, while Saccharomyces cerevisiae was present until the completion of the malo-lactic fermentation (MLF). This yeast was also identified during both the alcoholic fermentation and MLF of the Merlot wine using PCR-based DGGE and conventional plating. Using Bacteria specific primers, Lactobacillus plantarum and Lactobacillus sp. was identified in the Pinotage wine using PCR-based DGGE, while Lactobacillus brevis were isolated from Merlot wine using conventional platings. Although the presence of S. cerevisiae is expected during wine fermentation, the presence of this microbe in bottled wine could lead to spoilage. Four of the spoilt commercial wine samples (RW1, RW2, RoW1 and WW1) were found to be spoilt by S. cerevisiae, while a fifth wine sample (RW3) was found to be spoilt by an Acetobacter sp. using PCR-based DGGE. Members of the family Enterobacteriaceae were identified from all the wines using PCR-based DGGE, while Enterobacter sakazakii was identified from RW1 using PCR-based DGGE and conventional plating. The members of the family Enterobacteriaceae could possibly have contributed to the spoilage of the wine by producing undesirable secondary metabolites. PCR-based DGGE proved to be an alternative to conventional microbiological methods for the identification of the microbial species in South African red grape must and wine. This method also proved to be useful in the identification of spoilage microbes in spoilt commercial South African wines. / AFRIKAANSE OPSOMMING: Die produksie van rooi wyn behels komplekse interaksies tussen ‘n verskeidenheid van giste en bakterieë. Konvensionele mikrobiologiese metodes kan gebruik word om die verskillende mikro-organismes wat in rooi wyn teenwoordig is te identifiseer, maar dit blyk tydrowend te wees, terwyl sekere mikro-organismes nie groei op sintetiese media nie. Die doel van hierdie studie was om die mikrobiologiese populasie wat in twee Suid-Afrikaanse rooi wyne, Pinotage en Merlot, en vyf bederfde kommersiële wyne teenwoordig is, te evalueer met die gebruik van ‘n kultuur-onafhanklike benadering, polimerase ketting-reaksie (PKR)-gebaseerde denaturerende gradiënt jel elektroforese (DGJE). Die resultaat van die kultuur-onhafhanklike benadering was vergelyk met konvensionele uitplating tegnieke. Unieke, ongeëwenaarde PKR-gebaseerde DGGE vingerafdrukke was verkry van die Bakterieë en giste aanwesig in die Pinotage en Merlot wyne. Deur gebruik te maak van gis-spesifieke inleiers het die Pinotage wyn die teenwoordigheid van nie-Saccharomyces giste getoon, terwyl Saccharomyces cerevisiae teenwoordig was tot en met die afhandeling van die appel-melksuur gisting (AMG). Hierdie gis is ook geïsoleer gedurende beide die alkoholiese gisting en AMG van die Merlot wyn deur gebruik te maak van PKR-gebaseerde DGGE en konvensionele uitplating tegnieke. Met Bakterieë-spesifieke inleiers, was Lactobacillus plantarum en Lactobacillus sp. geïdentifiseer in die Pinotage wyn deur gebruik te maak van PKR-gebaseerde DGGE, terwyl Lactobacillus brevis geïsoleer is uit Merlot wyn deur gebruik te maak van konvensionele uitplatings. Alhoewel die teenwoordigheid van S. cerevisiae verwag word gedurende wynfermentasie, kan die teenwoordigheid van hierdie mikrobe in gebottelde wyn tot bederwing lei. Vier van die bedorwe kommersiële wynmonsters (RW1, RW2, RoW1 en WW1) was bederf deur S. cerevisiae, terwyl ‘n vyfde wynmonster (RW3) bederf was deur ‘n Acetobacter sp. deur die gebruik van PKR-gebaseerde DGGE. Van al die wyne is lede van die Enterobacteriaceae familie geïdentifiseer deur gebruik gemaak te maak van PKR-gebaseerde DGGE, terwyl Enterobacter sakazakii geïsoleer is van RW1 met konvensionele uitplating. Die lede van die familie Enterobacteriaceae kon moontlik bygedra het tot die bederwing van die wyn deur ongewenste sekondêre metaboliete te produseer. PKR-gebaseerde DGGE bewys ‘n alternatief tot die konvensionele mikrobiologiese metodes vir die identifikasie van die mikrobiese spesies in Suid-Afrikaanse rooi druif mos en wyn te wees. Hierdie metode het ook die bruikbaarheid in die identifikasie van mikrobes wat kommersiële Suid-Afrikaanse wyne bederf, bewys.