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.

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

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

No description available.

Two enzymatic systems from the lactic acid bacterium Oenococcus oeni, isolated from wine, have been studied. The first one is the H+-ATPase for which the activity was characterized under various conditions of growth. The activity gradually increased by l.6 to 1.9-fold upon inoculation at pH 3.5. The H+-ATPase activity did not vary significantly in function of the growth rate or with and without malic acid. However, acidification of the medium in the absence of malic acid induced the activity by 1.5 to 2.2-fold depending on the initial pH. The partially cloned H+-ATPase genes shared high homologies with those from other bacterial F0F1-ATPases. A mRNA of about 7 kb was detected by Northern blot and its size suggests that the genetic organization of O. oeni atp operon is similar to most F0F 1-ATPases. Furthermore, the amount of atp mRNA was shown to increase in acidic conditions. O. oeni H +-ATPase activity was pH-inducible and regulation of the expression seems to occur at the level of mRNA synthesis. Thus, the results confirmed the proposed role of the H+-ATPase in acid tolerance in O. oeni. / The second system studied was a chromosome-encoded P-type ATPase (CopB) and its putative transcriptional regulator (CopR). The copB gene encodes a protein showing great similarities with other Cu2+-ATPases of the CPx-type family of heavy-metal ATPases like Enterococcus hirae copB. Another gene (copR) was found 250 bp upstream of copB and displays great similarities with proteins of the MecI/BlaI family of transcriptional regulators, including En. hirae CopY repressor. O. oeni was shown to be highly resistant to copper and growth occurred in up to 30 mM CuSO4. Northern blot analyses indicated that the amount of copB mRNA increased upon a 0.2 to 4.0 mM copper stress suggesting that expression of the enzyme might be regulated at the level of mRNA synthesis. Whether CopR is involved in this regulation remains to be determined, but the results suggest that copRB genes might be involved in copper resistance in O. oeni.

This thesis presents a systematic investigation ofyeasts associated with wine grapes cultivated in several Australian vineyards during the 2001-2003 vintages. Using a combination of cultural and molecular methods, yeast populations of red (Cabernet sauvignon, Merlot, Tyrian) and white (Sauvignon blanc, Semilion) grape varieties were examined throughout grape cultivation. The yeast-like fungus, Aureobasidium pullulans, was the most prevalent species found on grapes. Various species of Cryptococcus, Rhodotorula and Sporobolomyces were frequently isolated throughout grape maturation. Ripe grapes showed an increased incidence of Hanseniaspora and Metschnikowia species for the 2001-2002 seasons, but not for the drought affected, 2002-2003 seasons. Atypical, hot and dry conditions may account for this difference in yeast flora and have limited comparisons of data to determine the influences of vineyard location, grape variety and pesticide applications on the yeast ecology. More systematic and controlled studies of these variables are required. Damaged grape berries harboured higher yeast populations and species diversity than intact healthy berries. PCR-DGGE analysis was less sensitive than plate culture for describing the diversity of yeast species on grapes; it detected prevalent species, but subdominant populations below 103 CFU/g were not detected. In some cases, PCR-DGGE revealed the presence ofyeasts (Candida galli, C. zemplinina) not isolated by culture. Fermentative wine species (Kluyveromyces, Torulaspora, Saccharomyces) were rarely isolated, and only detected by enrichment cultures. Significant morphological and genetic variability were detected among A. pullulans and other black yeasts isolates from grapes. Taxonomic characterization of 61 strains by ITS-RFLP and rDNA sequencing revealed that they belonged to several distinct species within the generic groupings ofAureobasidium, Hormonema and Kabatiella. Isolates were strong producers of extracellular enzymes and polysaccharides that could have oenological significance, and, using a plate assay, some were antagonistic towards Bacillus thuringiensis, several wine yeasts, and some spoilage and mycotoxigenic fungi found on grapes. Growth of Saccharomyces cerevisiae was not inhibited by these organisms in grape juice. A species-specific probe was developed for the identification of the wine spoilage yeast, Zygosaccharomyces bailii in a microtitre plate hybridization assay. The probe detected 102 cells/ml in wine, reliably differentiating Z. bailii from other Zygosaccharomyces and other wine-related yeasts.

Includes bibliographies. / 104, [64] leaves, [18] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / 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. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997?

Bibliography: p. 177-188. / xvi, 188 p. : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The effectiveness of methods to extract haze protective material from whole yeast cells was tested on three winemaking Saccharomyces cerevisiae strains. / Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 1998

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Wine is a complex medium. Wine aroma, flavour and colour are important quality factors, but these can be influenced by many factors, such as grape-derived compounds that exist as free volatiles and also as glycosidically bound. The chemical composition of wine is determined by factors such as grape variety, geographic position, viticulture condition, microbial ecology of the grape and the winemaking process. The varietals aroma is determined by both the volatile and the non-volatile compounds, such as monoterpenes, norisoprenoids and benzene derivatives, which are naturally present in the wine. Monoterpenes are very important in the flavour and aroma of grapes and wine. They can be found in grapes and wine either in the free, volatile and odorous form, or in the glycosidically-bound, non-volatile and non-odorous form. The ratio of glycosidically-bound compounds to free aroma compounds is very high in the Gewürztraminer, Muscat and Riesling cultivars in particular. The glycosidic bonds can be hydrolysed either by the acid method or by using enzymes. The acid method is disadvantageous because it can modify the monoterpenes, whereas enzymatic hydrolysis has the advantage of not modifying the aroma character. The enzyme method of breaking the glycosidic bonds occurs in two successive steps: initial separation of glucose from the terminal sugar by a hydrolase (a-L-arabinofuranosidase, a-L-rhamnosidase or β-apiosidase, depending on the aglycone moiety), followed by the breaking of the bond between the aglycone and glucose by β-glucosidase. The enzyme β-glucosidase can be obtained from many plant (Vitis vinifera), bacterial, yeast or fungal sources. Most of the enzymes produced by these sources are not functional under the winemaking conditions of low pH, low temperature, high glucose and high ethanol content. However, β-glucosidases from fungal origins, particularly from Aspergillus spp., are tolerant of winemaking conditions. The idea of using the β-glucosidase gene from the fungus Aspergillus kawachii (BGLA), which is linked to the cell wall and the free β-glucosidase, was to determine if anchoring the enzyme to the cell wall will increase the activity of the enzyme compared to the free enzyme. Four plasmids, pCEL 16, pCEL 24, pDLG 97 and pDLG 98, were used in this study. BGLA that was cloned into the plasmids pCEL 24 and pDLG 97 was linked to CWP2, and in pDLG 98 it was linked to AGa1 anchor domains. All the plasmids were genome-integrated and expressed in the reference strain Saccharomyces cerevisiae 303-1A. All the transformants were grown in 2% cellobiose and showed higher biomass production compared to the reference strain. β-Glucosidase activity was also assayed and transformed strain W16 showed a fourfold increase in activity compared to the reference strain. There was no significant increase in the activity of the other transformed strains, W24, W97 and W98. Enzymatic characterisation for optimum pH and temperature was done – for all strains the optimum pH was 4 and the optimum temperature was 40ºC. The recombinant strains together with the reference strain were used to make wine from Gewürztraminer grapes. The levels of numerous monoterpenes were enhanced in the resultant wines. The concentration of nerol was increased fourfold, that of citronellol twofold, and geraniol was 20% higher than in the wild type. There was also an increase in the levels of linalool and a-terpinol, but this was not significant. In wines produced with W97, W98 and W24, monoterpene levels did not show a significant difference. In future, the expression of the W16 expression cassette in an industrial wine yeast strain could be performed. In combination with the production of enzymes such as a-arabinofuranosidase, a-rhamnosidase and β-apiosidase, which are involved in the first step of enzymatic hydrolysis, this wine strain could release the bound monoterpenes and enhance the aroma of the wine. / AFRIKAANSE OPSOMMING: Wyn is ‘n komplekse medium. Wynaroma, -geur en -kleur is belangrike kwaliteitsfaktore, hoewel hierdie kwaliteite deur verskeie faktore beïnvloed kan word, soos druifafgeleide verbindings wat as vry vlugtige stowwe teenwoordig kan wees of glikosidies gebind is. Die chemiese samestelling van wyn word bepaal deur faktore soos druifvariëteit, geografiese ligging, wingerdkundige toestande, mikrobiese ekologie van die druif en die wynbereidingsproses. Die variëteitsaroma word bepaal deur vlugtige en nie-vlugtige verbindings, soos monoterpene, norisoprenoïede en benseenderivate, wat natuurlik in die wyn voorkom. Monoterpene is baie belangrik vir die geur en aroma van druiwe en wyn. Monoterpene is teenwoordig in die druiwe en wyn in vry, vlugtige en geurige, of in glikosidiesgebinde, nie-vlugtige en nie-geurige vorms. Die verhouding van glikosidiesgebonde verbindings tot vry aromaverbindings is baie hoog, veral in die Gewürztraminer-, Muscat- en Riesling-kultivars. Glikosidiese verbindings kan deur óf die suurmetode óf die ensiemmetode gehidroliseer word. Die nadeel van die suurmetode is dat dit monoterpene kan modifiseer, terwyl die ensiemmetode die voordeel het dat dit nie die aromakarakter modifiseer nie. Die ensiemmetode waarmee die glikosidiese verbinding afgebreek word, vind in twee opeenvolgende stappe plaas: aanvanklike skeiding van glukose van die terminale suiker deur ‘n hidrolase (a-L-arabinofuranosidase, a-Lramnosidase of β-apiosidase, afhangende van die aglikoongedeelte), gevolg deur die verbreking van die verbinding tussen die aglikoon en glukose deur β- glukosidase. Die β-glukosidase-ensiem kan vanaf ‘n verskeidenheid plant- (Vitis vinifera), bakterie-, gis- en swambronne verkry word. Die meerderheid van die ensieme wat deur hierdie bronne geproduseer word, is nie onder die wynbereidingstoestande van lae pH, hoë temperatuur, hoë glukose en hoë etanol funksioneel nie. β- Glukosidase vanaf ‘n swamoorsprong, veral vanaf Aspergillus-spesies, kan egter wynbereidingstoestande verdra. Die idee agter die gebruik van die β-glukosidasegeen afkomstig van die swam Aspergillus kawachii (BGLA), wat aan die selwand en die vry β-glukosidase gekoppel is, was om te bepaal of die aktiwiteit van die ensiem in vergelyking met dié van die vry ensiem verhoog sou word indien die ensiem aan die selwand geanker is. Vier plasmiede, pCEL 16, pCEL 24, pDLG 97 en pDLG 98, is in hierdie studie gebruik. BGLA, wat in die plasmiede pCEL 24 en pDLG 97 gekloneer is, is gekoppel aan CWP2, en in pDLG 98 is dit aan AGa1-ankergebiede gekoppel. Al die plasmiede is in verwysingsras Saccharomyces cerevisiae 303-1A genoomgeïntegreer en uitgedruk. Al die transformante is in 2% sellobiose gegroei en het hoër biomassaproduksie as die verwysingsras getoon. β-Glukosidaseaktiwiteit is ook geëssaieer en die getransformeerde ras W16 het ‘n viervoudige verhoging in aktiwiteit in vergelyking met die verwysingsras getoon. Daar was geen noemenswaardige verhoging in die aktiwiteit van die ander getransformeerde rasse, W24, W97 en W98, nie. Ensimatiese karakterisering vir optimum-pH en - temperatuur is gedoen – vir al die rasse was die optimum-pH 4 en die optimumtemperatuur 40ºC. Die rekombinante rasse, tesame met die verwysingsras, is gebruik om wyn met Gewürtztraminer-druiwe te maak. Die vlakke van talryke monoterpene is in die gevolglike wyne verhoog. Die konsentrasie van nerol is viervoudig verhoog, dié van sitronellol tweevoudig, en geraniol was 20% hoër as in die wilde tipe. Daar was ook ‘n verhoging in die vlakke van linaloöl en a-terpinol, maar hierdie verhoging was nie noemenswaardig nie. In wyne wat met W97, W98 en W24 gemaak is, het die monoterpeenvlakke nie ‘n noemenswaardige verskil getoon nie. In die toekoms sal die uitdrukking van die W16-uitdrukkingskasset in ‘n industriële wyngisras uitgevoer kan word. In kombinasie met die produksie van ensieme soos a-arabinofuranosidase, a-ramnosidase, β-apiosidase, wat in die eerste stap van ensimatiese hidrolise betrokke is, sal hierdie wyngisras die gebonde monoterpene kan vrylaat en die aroma van die wyn kan verbeter.

Thesis (MScAgric)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The quality of wine is influenced by numerous factors. These factors include the quality of the grapes, winemaking techniques and quality control throughout the winemaking process. It is thus very important that any process leading to the lowering of the quality of the wine be prevented. Evidence in the wine industry shows that bacterial spoilage is still very much a common problem in many wineries. The spoilage of wine by bacteria can lead to amongst other problems, elevated volatile acidity levels, of which only a certain concentration limit in wine is permitted. Usually more than 90% of the volatile acidity of wine consists of acetic acid. Different yeast strains, heterofermentative lactic acid bacteria and acetic acid bacteria (which can all be spoilage microorganisms) can produce acetic acid in high concentrations. It is thus important to be able to prevent the formation of this acid by controling the unwanted growth of these spoilage microorganisms. Acetic acid and other medium chain fatty acids, octanoic- and decanoic acid, can also lead to stuck or sluggish fermentations. A stuck or sluggish fermentation can also lead to wine spoilage, due to sugar remaining in the fermentation which can be utilized by spoilage microorganisms. Acetic- and other fatty acids enter the yeast cell by passive diffussion and releases its proton in the cytoplasm, thereby acidifying the cytoplasm and inhibiting some enzymes. These acids can also work synergistically with ethanol and its inhibitory effect is also dependent on the temperature. Yeast strains can also differ in their resistance to acetic and other medium chain fatty acids and these acids can also influence the growth of lactic acid bacteria. How acetic acid bacteria influence the winemaking process and the used measures to keep these bacteria from spoiling wine have been the subject of very little attention in the past. This was due to the belief that the anaerobic conditions prevailing in wine and the use of sulfur dioxide are enough to control these bacteria, since acetic acid bacteria were always described as being strictly aerobic microorganisms. Recently, some evidence showed that acetic acid bacteria can survive and even overcome the limits that the winemaking process places on its growth. These bacteria are also known to inhibit the yeasts growth and fermentation ability due to the production of acetic acid and other factors. A research programme on the origin of volatile acidity in South African wines had been initiated at the Department of Viticulture and Enology and at the Institute for Wine Biotechnology at the University of Stellenbosch after increases in volatile acidity in different South African wines had been reported. This spurred us to investigate the occurrence of acetic acid bacteria in South African red wine fermentations, which forms part of this study, and to identify the dominant acetic acid bacterial strains. The sulfur dioxide resistance of five representative strains were also determined, as well as the effect of metabolites which were produced by these bacteria on yeast growth and fermentation ability. Our results indicate that acetic acid bacteria can occur in high concentrations in the fresh must and during alcoholic fermentation. In the 1998 harvesting season acetic acid bacteria occurred at 106-107 cfu per ml in the fresh must. In 1999 these numbers were 104-105 cfu/ml. Acetic acid bacteria numbers decreased in 1998 to 102-103 cfulml during fermentation. The survival of these bacteria in 1999 correlated with the pH of the must, as well as sulfur dioxide dosages in the must. In must with a low pH and higher sulfur dioxide the number of acetic acid bacterial numbers decreased more drastically than in the high pH, low sulfur dioxide musts. This was also true for acetic acid bacterial counts during cold soaking of musts, with the number of acetic acid bacteria increasing during the cold soaking period in musts with a high pH. In musts with a low pH and higher S02 dosages acetic acid bacterial counts did not, however, increase during cold soaking. Gluconobacter oxydans dominated in the fresh must with Acetobacter liquefaciens and especially Acetobacter pasteurianus dominating during the fermentation. Different biochemical and physiological tests revealed that 52% of the 115 isolates tested belong to A. pasteurianus. The high occurrence of A. liquefaciens with A. pasteurianus during fermentation showed that the dominant acetic acid bacterial species in South Africa differed from reports from other wine producing countries. The sulfur dioxide resistance of the acetic acid bacteria tested also differed in white grape juice, with a molecular sulfur dioxide concentration of 0.64 mg/I being necessary to eliminate all the acetic acid bacterial strains tested. The A. hansenii strain was found to be the most resistant to sulfur dioxide and G. oxydans the least resistant. The latter strain was eliminated by only 0.05 mg/I molecular sulfur dioxide, while A. hansenii was only eliminated by 0.64 mg/I molecular sulfur dioxide. The A. pasteurianus, A. liquefaciens and A. aceti strains tested displayed varying degrees of resistance to sulfur dioxide. The volatile acidity produced by these bacteria profoundly influenced the growth and fermentation ability of yeast, which led to slow/stuck fermentation. The A. hansenii and A. pasteurianus strains produced the most volatile acidity in grape juice, with up to 4.02 g/I for A. hansenii within 4 days, which led to a stuck alcoholic fermentation. This was, however, prevented by inhibiting or eliminating the acetic acid bacteria with sufficient sulfur dioxide additions prior to yeast inoculation. Compounds produced by acetic acid bacteria can also influence wine quality. Certain organic acids were produced and metabolized by acetic acid bacteria, as well as acetoin. We could not, however, detect any other fatty acids that are inhibitory to yeast (produced by these bacteria). This study clearly showed that acetic acid bacteria could occur during fermentation and that certain winemaking techniques, like the maintenance of a low pH in the must and sulfur dioxide additions can influence the growth and survival of acetic acid bacteria. Acetic acid bacteria also influence both the winemaking process by inhibiting yeast as well as the quality of the wine by producing acetic acid and/or other compounds. This study also shed some light on the occurrence of acetic acid bacterial species in the South African context and could be important in assisting the winemaker, as well as the scientific reseacher, in finding ways to inhibit acetic acid bacteria in the ongoing battle against these spoilage microorganisms of wine. / AFRIKAANSE OPSOMMING: Wynkwaliteit word deur verskillende faktore beinvloed. Dit sluit die druifkwaliteit, wynmaak tegnieke en kwaliteitsbeheer deur die wynmaakproses in. Enige prosesse en faktore wat tot die verlaging in wynkwaliteit kan lei moet dus ten alle koste voorkom word. Die bederf van wyn deur bakterieë kan en is 'n algemene probleem in enige kelder. Bakteriese bederf kan, onder andere, lei tot verhoogte vlugtige suurheid, waarvan 'n sekere konsentrasie limiet in wyn toegelaat word. Asynsuur maak gewoonlik 90% van die vlugtige suurheid uit. Asynsuur kan deur verskillende gisrasse, heterofermantatiewe melksuurbakterieë en asynsuurbakterieë (wat almal wyn kan bederf) gevorm word. Die vorming van asynsuur in wyn kan dus voorkom word deur die ongewenste groei van dié organismes te voorkom. Asynsuur en ander medium ketting vetsure, soos oktanoë- en dekanoësuur, kan ook tot slepende of gestaakte gistings lei. Suiker wat in die wyn agterbly wat In slepende/gestaakte fermentasie ondergaan kan deur bederf bakterieë gebruik word om die wyn te bederf. Ongedissosieerde asynsuur en ander vetsure dring die gissel binne deur passiewe diffussie en stel 'n proton vry in die sitoplasma wat sitoplasma versuur en sekere ensieme inhibeer. Hierdie sure werk ook sinergisties met etanol en hul inhiberede effek is ook temperatuur afhanklik. Gisrasse verskil in hul weerstandbiedendheid teen asynsuur- en ander mediumketting vetsure en dié vetsure kan ook melksuurbakterieë se groei beïnvloed. Hoe asynsuurbakterieë wyn bederf en die aksies wat geneem kan word om dit te verhoed is in die verlede nie baie ondrsoek nie. Dit is hoofsaaklik daaraan toe te skryf dat geglo is dat die anaerobiese kondisies in wyn en die gebruik van swaweidioksied die groei van asynsuurbakterieë, wat altyd beskryf is as streng aerobe mikroorganismes, kan beheer. Daar is onlangs aangetoon dat asynsuurbakterieë kan oorleef in wyn en selfs die ongunstige kondisies daarin kan oorkom. Hierdie bakterieë is ook in staat om gisgroei en fermentasie vermoë te inhibeer deur die produksie van asynsuur en ander faktore. In Navorsingsprogram om die oorsprong van verhoogde vlugtige suurheid in Suid-Afrikaanse wyne te bepaal is deur die Departement Wingerd- en Wynkunde en die Instituut vir Wynbiotegnologie van die Universiteit van Stellenbosch geinisieer. Dit het ons aangemoedig om die voorkoms van asynsuurbakterieë in Suid-Afrikaanse rooiwyngistings, wat deel vorm van hierdie ondersoek, en ook die dominante asynsuurbakterie rasse te identifiseer. Die swaweidioksied bestandheid van vyf verteenwoordegende rasse en die effek wat metaboliete wat deur dié bakterieë geproduseer is op gisgroei en gisitingsvermoë is bepaal. Ons resultate bewys dat asynsuurbakterieë teen hoë getalle in vars mos en gedurende alkoholiese gisting kan voorkom. Asynsuurbakterieë het gedurende die 1998 seisoen teen 106-107 kve/ml en in 1999 teen 104-105 kve/ml in die vars mos voorgekom. Gedurende fermentasie het hierdie getalle in die 1998 seisoen gedaal na 102-103 kve/ml. Die oorlewing van hierdie bakterieë het gedurende die 1999 seisoen gekorrelleer met die pH en swaweidioksied konsentrasies van die mos. In die lae pH, hoë swaweidioksied moste het asynsuurbakterie getalle vinniger en meer dramaties gedaal as in die hoë pH, lae swaweidioksied moste. Asynsuurbakterie getalle het dieselfde tendens getoon in moste gedurende dopkontak by lae temperature. In moste met 'n hoë pH het asynsuurbakterie getalle toegeneem gedurende koue dopkontak, terwyl dit nie gebeur het nie in moste met 'n lae pH en hoë swaweidioksied konsentrasies. In die vars mos het Gluconobacter oxydans en gedurende die fermentasie Acetobacter liquefaciens en veral Acetobacter pasteurianus oorheers. Verskillende biochemiese en fisiologiese toetse het bepaal dat 52% van die 115 isolate A. pasteurianus is. Die hoë voorkoms van A. liquefaciens saam met A. pasteurianus gedurende gisting bewys dat die voorkoms en dominansie van asynsuurbakterieë in Suid- Afrika verskil van ander wynproduserende lande. Die swaweidioksied weerstandbiedendheid van die asynsuurbakterieë wat getoets het, het ook verskil, met 0.64 mg/I molekulêre swaweIdioksied nodig om hul almal te elimineer in wit druiwesap. Die A. hansenii en G. oxydans rasse was die mees weerstandbiedend en sensitiefste onderskeidelik ten opsigte van swaweidioksied. Slegs 0.05 mg/I molekulêre swaweidioksied was voldoende om G. oxydans te elimineer, terwyl A. hansenii deur 0.64 mg/I molekulêre swaweidioksied geëlimineer is. Die A. pasteurianus, A. liquefaciens en A. aceti rasse het verskillende swaweidioksied weerstandbiedendheid getoon. Die vlugtige suurheid wat deur dié bakterieë geproduseer is het die groei en gistingvermoë van gis drasties beinvloed, wat tot slepende/gestaakte fermentasies gelei het. Die A. hansenii en A. pasteurianus rasse het die meeste vlugtige suurheid geproduseer, met tot 4.02 g/I geproduseer deur A. hansenii na vier dae se groei, wat tot 'n gestaakte fermentasie gelei het. Dit is egter voorkom deur die asynsuurbakterieë te elimineer deur genoegsame swaweidioksied toevoegings tot die mos voor gisinokulasie te doen. Verbindings wat deur asynsuurbakterieë geproduseer word kan ook wynkwaliteit beinvloed. Sekere anorganiese sure is deur hierdie bakterieë geproduseer, terwyl ander gemetaboliseer is. Asetoïen is geproduseer deur die getoetse asynsuurbakterieë. Ons kon egter nie ander vetsure wat gis inhibeer opspoor nie. (Geproduseer deur die bakterieë). Hierdie studie bewys dat asynsuurbakterieë gedurende alkoholiese fermentasie kan voorkom en dat sekere wynmaaktegnieke, soos die verkryging van moste met 'n lae pH en genoegsame swaweidioksied toevoegings die groei en oorlewing van asynsuurbakterieë kan beivloed. Asynsuurbakterieë kan ook beide die wynmaakproses, deur giste te inhibeer, en die wynkwaliteit beivloed deur die produksie van asynsuur en/of ander verbindings. Hierdie studie het ook kennis oor die voorkoms van asynsuurbakterieë in Suid-Afrikaanse moste verbeter en kan ook as 'n nuttige hulpmiddel dien vir die wynmaker en navorser in die stryd om hierdie bederf organismes van wyn te elimineer.