Two yeast/E.coli shuttle vector plasmids were studied in 1994, termed pIBIDB and pBP33. According to this study, each plasmid should contain at least one ADH2UAS (upstream activation sequence in the alcohol dehydrogenase 2 gene) insert. In the present study, the constructed plasmids were analysed and transformed into laboratory strain yeast. The aim of this project was to identify the orientation, quantity and quality of the insert in the selected plasmids. Methods such as restriction analysis, polymerase chained reaction (PCR), sequencing, plate assays and enzyme assays were used to identify and evaluate the novel inserts. The data presented in this thesis suggest the inserted ADH2UAS fragment did enhance the production of maltose permease and maltase when the transformants were cultivated in maltose and ethanol-glycerol medium. The results suggested that transformants containing two inserts of ADH2UAS had a greater influence on the transformants than a single insert. But the inserts within the vectors and in transformed laboratory stain yeast appeared unstable. This could be due to the method used for plasmid construction and the storage condition of the transformants / Master of Science (Hons)

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Grape must is a complex medium, and during wine production numerous biochemical pathways and metabolic reactions are taking place simultaneously to produce a specific taste and aroma. Microorganisms, specifically yeast, play a key role in the formation of metabolites formed during alcoholic fermentation. Sauvignon blanc, a well studied grape cultivar, is known to have a versatile range of aroma profiles ranging from “green” to “tropical”. It has been broadly stated that a “green” Sauvignon blanc can be created in the vineyard and a “tropical” Sauvignon blanc can be created by selecting a specific yeast strain, and that the balance between “green” and “tropical” characters is essential for the final aroma profile. Except for grape-derived varietal aromatic compounds such as methoxypyrazines (green), volatile thiols (tropical) and monoterpenes (floral), yeast derived volatile compounds including esters, higher alcohols, fatty acids and carbonyl compounds will also contribute to the final wine aroma. The main aim of this study was to assess how viticultural treatment-derived differences in grape must, can impact on aroma production when this grape must is fermented with different commercial wine yeast strains. The viticulture treatment focused on light intensity modulated through canopy treatment. Volatile aroma differences were compared for canopy and yeast treatments, specifically focusing on the fermentation derived bouquet (esters, higher alcohols, volatile fatty acids, carbonyl compounds and monoterpenes). Results showed significant differences between initial must compositions, including titratable acidity, malic acid and yeast assimilable nitrogen. The volatile aroma compounds were also significantly impacted although no noticeable effect on the overall fermentation kinetics was observed. Depending on the yeast strain differences in volatile compounds varied. A clear vintage effect is noticeable between volatile compounds affected by the treatments. Data generated in 2012 shows clear differences between ethyl- and acetate esters and could clearly be grouped according to yeast strain through multivariate analysis. Sensory evaluation results could clearly be distinguished according to canopy treatment and to a lesser degree according to yeast strain used. This indicates that although yeast has a more prominent impact on the fermentative bouquet that develops during alcoholic fermentation the overriding aroma is primarily derived from grape-derived metabolites which can be manipulated by canopy treatments. None the less the difference in fermentation bouquet does add to the complexity of the wine especially in the case of fermentation derived “tropical” aromas including guava and passion fruit. In some cases where shaded grapes had higher ester concentrations, the resultant wine also had higher aroma quality. This study has contributed to a better understanding of the complex relationships between canopy manipulation and yeast selection on aroma formation. The analysis of volatile aroma alone however is not enough to understand the final perception of wine taste and further indepth studies of the viticultural and oenological factors is needed. In particular, this project has focused on a single vineyard over only two vintages. The general validity of the conclusions derived from this study therefore will require additional data sets. / AFRIKAANSE OPSOMMING: Druiwemos is ‘n komplekse medium en tydens wynbereiding is daar verskeie biochemiese weë en metaboliese reaksies wat gelyktydig plaasvind om ‘n spesifieke smaak en aroma te produseer. Mikro-organismes, veral gis, speel ‘n sleutelrol in die vorming van metaboliete tydens alkoholiese gisting. Sauvignon blanc, ‘n goed bestudeerde druifkultivar, besit ‘n veelsydige reeks aromaprofiele wat wissel van “groen” tot “tropies”. Oor die algemeen word dit voorgehou dat ‘n “groen” Sauvignon blanc in die wingerd geskep word, terwyl ‘n “tropiese” Sauvignon blanc geskep kan word deur ‘n spesifieke gisras te selekteer, en die balans tussen “groen” en “tropiese” karakters is noodsaaklik vir die finale aromaprofiel. Behalwe vir druifafgeleide kultivarafhanklike aromatiese verbindings soos metoksipirasiene (groen), vlugtige tiole (tropies) en monoterpene (blomagtig), sal gisafgeleide vlugtige komponente, waaronder esters, hoër alkohole, vetsure en karbonielverbindings, ook tot die finale wynaroma bydra. Die hoofdoelwit van hierdie studie was om te bepaal hoe verskille in druiwemos wat afkomstig is van wynkundige behandeling ‘n impak op aromaproduksie kan hê wanneer hierdie druiwemos met verskillende kommersiële wyngisrasse gegis word. Die wynkundige behandeling het gefokus op ligintensiteit wat deur lowerbehandeling gereguleer is. Vlugtige aromaverskille is op grond van lower- en gisbehandelings vergelyk, met ‘n spesifieke fokus op die gistingsafgeleide boeket (esters, hoër alkohole, vlugtige vetsure, karbonielverbindings en monoterpene). Die resultate het beduidende verskille getoon tussen aanvanklike mossamestellings, waaronder titreerbare suurheid, appelsuur en gis-assimileerbare stikstof. Daar was ook ‘n noemenswaardige impak op die vlugtige aromaverbindings, hoewel geen merkbare effek op die algehele gistingskinetika waargeneem kon word nie. Die verskille in vlugtige verbindings het gewissel op grond van die gisras. ‘n Duidelike oesjaareffek was merkbaar tussen vlugtige verbindings wat deur die behandelings geaffekteer is. Data wat in 2012 gegenereer is, toon duidelike verskille tussen etiel- en asetaatesters en kon duidelik m.b.v. meervariantanalise volgens gisras gegroepeer word. Die resultate van die sensoriese evaluering kon duidelik volgens lowerbehandeling onderskei word, en tot ‘n mindere mate volgens die gisras wat gebruik is. Dít dui daarop dat hoewel gis ‘n meer prominente impak het op die gistingsboeket wat tydens alkoholiese gisting ontwikkel, is die oorheersende aroma hoofsaaklik afgelei van druifafgeleide metaboliete wat deur lowerbehandelings gemanipuleer kan word. Nietemin dra die verskil in gistingsboeket by tot die kompleksiteit van die wyn, veral in die geval van gistingsafgeleide “tropiese” aromas, insluitend koejawel en grenadella. In sommige gevalle waar beskadude druiwe hoër esterkonsentrasies gehad het, het die gevolglike wyn ook ‘n hoër aromakwaliteit gehad. Hierdie studie dra by tot ‘n beter begrip van die effek van die komplekse verhoudings tussen lowermanipulasie en gisseleksie op aromavorming. ‘n Analise van vlugtige aroma alleen is egter nie voldoende om die finale persepsie van wynsmaak te begryp nie en bykomende diepgaande studies van die wingerdkundige en wynkundige faktore word benodig. Hierdie projek het in die besonder gefokus op ‘n enkele wingerd oor slegs twee oesjare. Die algemene geldigheid van die afleidings wat van hierdie studie gemaak word, sal dus bykomende datastelle vereis. / The National Research Foundation and Postgraduate Merit Bursary for financial support

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology / Solid-state fermentation (SFF) has emerged as a good method for the production of microbial enzymes such as inulinases. The use of low-cost agricultural plants and agro-industrial residues as substrates in SSF processes provides a value adding alternative to these otherwise under/or un-utilised vegetation. Production of inulinases, using various inulin-containing plant materials as carbon sources was studied using pure and mixed cultures of yeast strains. All substrates resulted in different levels of enzyme activity. A mixed culture of Kluyveromyces marxianus and Saccharomyces cerevisiae produced an extracellular exoinulinase when grown on different types of inulin-containing plant materials. Initial inulinase production was achieved as follows: 10 IU/gds (garlic cloves), 15 IU/gds (parsnips), 10 IU/gds (wheat bran) and 7 IU/gds (amadumbe) by K. marxianus and S. cerevisiae in a mixed culture. The production of inulinases by a mixed culture of K. marxianus and S. cerevisiae under SSF was further optimized by investigating initial moisture content, temperature, carbon source, nitrogen source, inoculum volume and inoculum ratio. The highest inulinase activity attained was in garlic cloves (85 IU/gds), followed by parsnips (65 IU/gds), wheat bran (37 IU/gds) and amadumbe (25 U/gds). The activities yielded 5.6 fold higher inulinase than in preliminary studies. The optimum pH and temperature of the crude enzyme were 5.0 and 50 oC, respectively. The pH and temperature stability of the enzyme was steady for 1 hour retaining about 64% activity. The average inulinase/invertase activity (I/S) ratio of 1.0 by crude inulinases was also observed after 48 hours. The crude extracellular enzyme extracts from the garlic cloves, parsnips, amadumbe and wheat bran were partially purified by ammonium sulphate precipitation and showed a specific activity of 9.03 U/mg, 0.08 U/mg, 4.12 U/mg and 0.133 U/mg respectively. The Km and Vmax values of the inulinase were 21.95 mM and 2.09 μM/min; 19.79 mM and 1.38 μM/min; 31.59 mM and 0.51 μM/min; and 25.74 mM and 0.23 μM/min, respectively. All extracts demonstrated potential for large-.scale production of inulinase and fructose syrup.

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2018 / Beer is one of the most widely consumed alcoholic beverages in the world. The brewing process is based on natural enzymatic activities that take place during the malting of barley grain, mashing of grist and fermentation of wort. Insufficient malt enzyme activity during the mashing process leads to high levels of barley β-glucan, arabinoxylan (AX) and dextrins in the wort as well as in the final beer. It was reported that high levels of β-glucan and AX increase wort and beer viscosity which lower the rate of beer filtration and this negatively affect the production rate in the brewery. During beer fermentation, brewing yeast catalyses the conversion of wort sugars to ethanol, carbon dioxide and other metabolic products. However, non-fermentable carbohydrates i.e., limit dextrins remain in the wort and final beer. These non-fermentable carbohydrates are known to contribute to the caloric value of beer which might lead to weight gain in consumers. The objectives of this study were to evaluate the effect of recombinant yeast strains expressing an endo-β-1,4-glucanase or an endo-β-1,4-xylanase on beer viscosity (as an indicator of filterability) and an α-amylase on residual sugars levels. The effect of the above mentioned enzymes on the aroma, appearance, flavour, mouth-feel and overall quality of the beer was also determined. Wort was produced in the University of Limpopo micro-brewery and the wort was pitched with different recombinant strains. The wild-type strain served as control. The results obtained showed that the xylanase expressing strain produced a measurable decrease in viscosity over the course of the fermentation, but endo-glucanase did not have any effect on the beer viscosity. The α-amylase producing strain, did not show a measurable reduction of residual sugars in the final beer probably as a result of very low activity on α-1,6 glycosidic bonds in dextrins during fermentation. The xylanase and α-amylase producing strain fermented effectively with good attenuation (decrease in wort specific gravity). The beer produced by the α-amylase and control strains were preferred in terms of taste and had similar qualities. The secreted amylolytic activity was not sufficient to significantly reduce residual sugar in the final beer. Although the xylanase secreting strain produced a beer with lower viscosity, the enzyme had a negative impact on the taste of the beer. Key words: Brewer’s yeast, beer fermentation, low calorie beer, amylase, xylanase, endo-glucanase.