Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The influence of malolactic fermentation (MLF) in most red and some white wines is one of many factors that determine or influence wine quality, because it affects the flavour and sensory profile of wine. This process is a decarboxylation process conducted by lactic acid bacteria (LAB) such as Oenococcus, Lactobacillus, Pediococcus and Leuconostoc. Mostly Oenococcus oeni, but recently also Lactobacillus plantarum is used in commercial starter cultures and also the first mixed MLF starter culture (NT 202 Co-Inoculant) was commercialized in 2011. The reason for the predominant use of O. oeni and recently L. plantarum is due to their tolerance to the harsh wine environment. Malolactic fermentation leads to a decrease in acidity and an increase in pH that leaves the wine with a softer mouthfeel. Another reason to conduct MLF is the improvement of microbial stability by the removal of malic acid as carbon source. Research focus has recently shifted to the ability of LAB and MLF as well as the interaction of LAB with yeast to alter the wine aroma profile via the modification and/or production of certain aroma compounds. The main goal of this study was to assess the impact of yeast and nutrient addition on the ability of the NT 202 Co-Inoculant to conduct MLF during co-inoculation and to evaluate the aroma compound production in the final wine. The first aim was to evaluate the impact of different red and white wine yeast strains on the ability of the NT 202 Co-Inoculant to conduct MLF during co-inoculation in Chardonnay, Merlot and Shiraz. Malolactic fermentation was unsuccessful in the Chardonnay due to a low pH, but successful in Merlot and Shiraz. Based on the malic acid degradation ability of the NT 202 Co- Inoculant, the yeasts were grouped into three categories: inhibitory, neutral or stimulatory towards MLF. Co-inoculated MLF showed a clear decrease in total fermentation time while yeast strains such as WE 372 and Exotics showed positive compatibility with the NT 202 Co- Inoculant. The impact of the yeast-bacterial combinations on the aroma compound production in the final wine was evaluated. Co-inoculated MLF showed positive aroma changes in the red wines with a general increase in total esters (associated with fruity characters in wine) especially ethyl lactate and diethyl succinate that also contribute to the mouthfeel of the wine. Production of esters, volatile fatty acids and higher alcohols seemed to depend on the yeastand LAB strain used. The NT 202 Co-Inoculant contributed to the monoterpenes produced and MLF led to increased concentrations of diacetyl and acetoin, which are associated with buttery characters in wine. The second aim of this study was to evaluate the impact of wine additives (used during coinoculation) such as yeast- and bacterial nutrients, clarifying- and detoxifying agents on the ability of the NT 202 Co-Inoculant to conduct MLF and to assess their impact on the aroma compound production in the final wine. No negative or positive impact on the malic acid degradation of the NT 202 Co-Inoculant or the resulting aroma compound production was observed for the different wine additives used in this study. The results generated from this study showed that the selection of yeast strains is important as it will influence both the fermentation duration and final wine aroma. / AFRIKAANSE OPSOMMING: Die invloed van appelmelksuurgisting (AMG) in die meeste rooi- en witwyne is een van baie faktore wat wynkwaliteit beïnvloed, omrede dit die geur en sensoriese profiel van wyn beïnvloed. Hierdie proses is 'n dekarboksileringsaksie wat deur melksuurbakterieë (MSB), soos Oenococcus, Lactobacillus, Pediococcus en Leuconostoc, uitgevoer word. Die mees algemene bakterieë wat gebruik word, is Oenococcus oeni, maar onlangs het Lactobacillus plantarum ook na vore getree in die gebruik van kommersiële aanvangskulture. Die eerste gemengde AMGaanvangskultuur (NT 202 Co-Inoculant) is in 2011 gekommersialiseer. Die rede vir die oorheersende gebruik van O. oeni en L. plantarum word toegeskryf aan hul gehardhiedsgraad in ‘n uitdagende wynomgewing. Appelmelksuurgisting lei tot 'n afname in die suurheidsgraad en 'n toename in die pH van die wyn, wat 'n sagter mondgevoel tot gevolg het. Nog 'n rede waarom AMG deurgevoer word, is om die mikrobiese stabiliteit van die wyn te verbeter deur die verwydering van appelsuur as koolstofbron. Die navorsingsfokus het onlangs verskuif na die vermoë van MSB en AMG, sowel as die interaksie van MSB met die gis, om die wynaromaprofiel te verander deur middel van die verandering en/of produksie van sekere aromaverbindings. Die hoofdoel van hierdie studie was om die impak van die gis en voedingstof te evalueer ten opsigte van die vermoë van die NT 202 Co-Inoculant om AMG uit te voer tydens koïnokulasie. Die produksie van aromakomponente in die finale wyn is ook geëvalueer. Die eerste doelwit was om die impak van verskillende rooi- en witwyngisrasse te evalueer ten opsigte van die vermoë van die NT 202 Co-Inoculant om AMG uit te voer tydens koïnokulasie in Chardonnay, Merlot en Shiraz. Appelmelksuurgisting was onsuksesvol in die Chardonnay weens 'n lae pH, maar suksesvol in Merlot en Shiraz. In terme van die appelsuurafbraakvermoë van die NT 202 Co-Inoculant, is die giste in drie kategorieë gegroepeer: inhiberend, neutraal of stimulerend teenoor AMG. Ge-koïnokuleerde AMG het 'n duidelike afname in die totale fermentasietyd getoon, terwyl gisrasse, soos WE 372 en Exotics, ‘n positiewe verenigbaarheid met die NT 202 Co-Inoculant getoon het. Die impak van die gisbakteriële kombinasies op die aromakomponentproduksie in die finale wyn is geëvalueer. Gekoïnokuleerde AMG het positiewe aromaveranderinge in die rooiwyne getoon met 'n algemene toename in die totale esters (wat geassosieer word met vrugtige karakters in wyn), veral etiellaktaat en dietielsuksinaat, wat ook bydra tot die mondgevoel van die wyn. Dit het voorgekom dat produksie van esters, vlugtige vetsure en hoër alkohole moontlik afhanklik kan wees van die gis- en bakteriële ras gebruik. Die NT 202 Co-Inoculant het bygedra tot die monoterpene wat geproduseer is en AMG het gelei tot verhoogde konsentrasies van diasetiel en asetoïen, wat geassosieer word met botteragtige karakters in wyn. Die tweede doelwit van hierdie studie was om die impak van wyntoevoegingsmiddels (wat tydens koïnokulasie gebruik word) bv. gis- en bakteriese voedingstowwe, verhelderingsagente, asook detoksifiserende agente, te evalueer ten opsigte van die vermoë van die NT 202 Co- Inoculant om AMG uit te voer en om hul impak op die produksie van die aromakomponente van die finale wyn te ontleed. Geen negatiewe of positiewe effekte is waargeneem vir die verskillende wyntoevoegingsmiddels, wat in hierdie studie gebruik is, in terme van die appelsuurafbraak van die NT 202 Co-Inoculant of die gevolglike produksie van aromakomponente nie. Hierdie studie se resultate toon dat die keuse van die gisras belangrik is, omdat dit die fermentasietydperk, asook die finale wynaroma, beïnvloed. / Anchor Yeast, Oenobrands, The National Research Foundation and THRIP, for financial support

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Lactic acid bacteria (LAB) play a vital role in reducing wine acidity and also contributing to its aroma and flavour. However, they can also be responsible for many wine spoilage problems that compromise the quality and value of wine. While Oenococcus oeni contributes positive characteristics to the sensory properties of wine, certain species of the genera, Lactobacillus and Pediococcus can affect the wholesomeness of wine by producing undesirable compounds, such as biogenic amines and ethyl carbamate. Chemical preservatives like sulphur dioxide (SO2) are used to prevent the growth of spoilage micro-organisms during the winemaking process. SO2 also acts as a reducing agent and maintains the benefits of antioxidant properties of the polyphenols of wine. However, there is a worldwide demand to reduce SO2 levels due to the increasing health related risks and other factors. All these considerations have increased the interest in research to look for new preservation strategies, and LAB-produced bacteriocins seem to be a potential alternative that has been explored in the last decade. Various types of bacteriocins have been identified and characterized. However, there are few reports on bacteriocins produced by LAB of oenological origin or on bacteriocins present in the finished wine. The present study screened 155 LAB isolates from the IWBT culture collection for bacteriocin production. The isolates originated from South African red wines undergoing spontenous malolactic fermentation (MLF). Eight strains (5%) were identified to be producers, as evidenced by strong inhibition zones formed against sensitive organisms on agar plates. The producers demonstrated a broad spectrum of antimicrobial activity by inhibiting Lactobacillus spp., Leuconostoc mesenteroides, Listeria monocytogenes and Pediococcus pentosaceus strains. Some of these bacterial genera are important in winemaking since they are potential wine spoilage bacteria. Hence these strains and/or the bacteriocins they produce could possibly find application in the food fermentation industry. The physiological results, biochemical tests and sugar fermentation profiles all gave the same results for the seven isolates, which were indicative of enterococci. The identification through 16S rRNA gene sequencing revealed that the seven tested isolates were all Enterococccus faecium. RAPD-PCR fingerprinting gave the same profile for the seven strains confirming that they were all identical on genetic level. Determining the molecular weight using SDS-PAGE showed the peptides to be below 4.6 kDa in size. PCR amplification of the enterocin P gene, sequencing and BLAST search results confirmed that all eight strains contained the enterocin P gene from Ent. faecium. The enterocin tested in this study was heat stable at 100°C (30 min), but lost 50% of its activity at 121°C (15 min). Factors such as bacteriocin production and heat resistance are among many that enable enterococci to be dominant in fermented products such as dairy foods or meat. Therefore, enterococci producing bacteriocins have potential applications in various foods and fermented products. The pH tests showed enterocin to be active over a broad pH range (2-10). Enterocin activity over a wide pH range make them potentially more suitable as natural preservatives of foods and fermented products where products are acidified or pH decreases due to natural LAB present. They also have potential applications in oenological process where pH levels are as low as 3 and 4. Proteolytic enzyme treatments with lysozyme, lipase, lyticase and catalase could not inhibit enterocin activity. This indicated that their antimicrobial activity was independent of lipid or carbohydrate moieties or hydrogen peroxide. α-Chymotrypsin and proteinase K inactivated enterocin, which indicated that the compound was proteinaceous in nature. Bacteriocin production tested in two of the isolates, #16.3 and 128.1, coincided with the exponential growth phase which occurred after 6 hours of incubation at 30°C, which was an indication of primary metabolite kinetics. The highest production of 400 AU/ml was observed after eight hours and was maintained for several hours (46 hours) in the stationery phase. The bactericidal effect of the cell free supernatants from #16.3 and 128.1 against the sensitive culture of Lactobacillus pentosus DSM 20314 was clearly demonstrated by complete inhibition of growth for most of the experimental period, while the control increased exponentially throughout the experiment. In conclusion, this study has confirmed the isolation and identification of Ent. faecium strains from wine, a genus that is rarely found in the wine environment. Although one can speculate on the origin of this bacterium in the wine e.g. human handling and contaminated water, these bacterial isolates produced enterocin P which have antimicrobial action against wine-related LAB genera and therefore have a potential role in wine spoilage control. / AFRIKAANSE OPSOMMING: Melksuurbakterieë (MSB) speel ‘n belangrike rol in die redusering van die suurgehalte van wyn en dra ook by tot die aroma en smaak daarvan. Hulle kan egter ook verantwoordelik wees vir vele wynbederfprobleme wat die gehalte en waarde van wyn negatief beïnvloed. Hoewel Oenococcus oeni positiewe karaktertrekke aan die sensoriese eienskappe van wyn verleen, kan sekere spesies van die genus, Lactobacillus en Pediococcus, die heilsaamheid van wyn beïnvloed deur ongewenste verbindings, soos biogeniese amienes en etielkarbamaat, te produseer. Chemiese preserveermiddels, soos swaweldioksied (SO₂), word gebruik om die groei van bederfmikro-organismes tydens die wynbereidingsproses te voorkom. SO₂ fungeer ook as ‘n reduseermiddel en onderhou die voordele van die antioksidant eienskappe van die poli-fenole van wyn. Daar is egter ‘n wêreldwye vraag na die redusering van SO₂-vlakke as gevolg van die toename in gesondheidsverwante risiko’s en ander faktore. Al hierdie oorwegings het belangstelling in die navorsing van nuwe preserveringstrategieë laat toeneem en MSB-geproduseerde bakteriosiene lyk na ‘n potensiële alternatief wat in die laaste dekade ondersoek word. Verskeie tipes bakteriosiene is geïdentifiseer en getipeer. Daar is egter nog weinig gerapporteer oor bakteriosiene wat deur MSB van wynkundige oorsprong geproduseer is of oor bakteriosiene wat in afgeronde wyn teenwoordig is. Die huidige studie het 155 MSB isolate van die Instituut vir Wynbiotegnologie se kultuurversameling vir bakteriosien-produsering gegradeer. Agt stamme (5%) is as produseerders geïdentifiseer, soos gestaaf is deur sterk inhibisiesones wat teen sensitiewe organismes op agarplate gevorm het. Die produseerders het ‘n breë spektrum van antimikrobiese aktiwiteit by inhiberende Lactobacillus spp., Leuconostoc mesenteroides, Listeria monocytogenes en Pediococcus pentosaceus stamme gedemonstreer. Sommige van hierdie bakteriese genera is belangrik in wynbereiding, omdat dit potensiële wynbederfbakterieë is. Hierdie isolate en/of die bakteriosiene wat dit produseer, kan dus moontlik toepassing in die voedselfermentasiebedryf vind. Die fisiologiese resultate, biochemiese toetse en suikerfermentasieprofiele het almal dieselfde resultate vir die sewe isolate, wat indikatief van enterococci was, gelewer. Die identifisering deur 16S rRNA-basispaaropeenvolging het onthul dat die sewe getoetste isolate almal Enterococccus faecium was. RAPD-PKR-vingerafdrukke het dieselfde profiel vir die sewe rasse gelewer, wat bevestig dat die rasse almal identies op genetiese vlak was. Deur die molekulêre gewig vas te stel deur middel van SDSPAGE, het dit getoon dat die peptiede kleiner as 4.6 kDa in grootte is. PKR-amplifikasie van die enterosien-P geen, die bepaling van basispaaropeenvolging en BLAST-soekresultate het bevestig dat al agt rasse die enterosien-Pgeen van Ent. faecium bevat. Die enterosien wat in hierdie studie getoets is, was hitte-stabiel teen 100°C (30 min), maar het 50% van sy aktiwiteit teen 121°C (15 min) verloor. Faktore soos bakteriosienproduksie en hittebestandheid, is van die vele faktore wat enterococci in staat stel om dominant in gefermenteerde produkte, soos suiwelprodukte of vleis te wees. Enterococci wat bakteriosiene produseer het dus potensiële toepassings in verskeie kossoorte en gefermenteerde produkte. Die pH-toetse het getoon dat enterosien-P oor ‘n breë pH spektrum (2-10) aktief was. Enterosienaktiwiteit oor ‘n wye pH spektrum maak dit potensieel meer geskik as natuurlike preserveermiddels vir kossoorte en gefermenteerde produkte waar produkte versuur word of die pH afneem as gevolg van natuurlike MSB wat teenwoordig is. Dit het ook potensiële toepassings in enologiese prosessering waar pH-vlakke so laag as 3 en 4 is. Proteolitiese ensiembehandelings met lisosiem, lipase, litikase en katalase kon nie enterosienaktiwiteit inhibeer nie. Daar is getoon dat hul antimikrobiese aktiwiteit onafhanklik was van lipiede, koolhidraatdele óf waterstofperoksied. α-Chymotripsien en proteïenase-K het enterosien onaktief gemaak, wat getoon het dat die samestelling proteïenagtig van nature is. Bakteriosienproduksie wat in twee van die stamme #16.3 en 128.1 getoets is, het ooreengestem met die eksponensiële groeifase wat na 6 ure van inkubasie teen 30°C plaasgevind het, en wat ‘n aanduiding is van primêre metabolitiese kinetika. Die hoogste produksie van 400 AU/ml is na agt ure waargeneem en is vir etlike ure (46 uur) in die stasionêre fase gehandhaaf. Die bakterie-dodende effek van die selvrye supernatant van #16.3 en 128.1 teenoor die sensitiewe kultuur van Lactobacillus pentosus DSM 20314 is duidelik gedemonstreer deur totale inhibisie van groei vir die grootste deel van die eksperimentele periode, terwyl die kontrole eksponensieel deur die hele eksperiment toegeneem het. Hierdie studie het dus die isolering en identifisering van Ent. faecium-stamme, ‘n genus wat baie selde gevind word in ‘n wynomgewing, vanuit wyn bevestig. Alhoewel daar gespekuleer kan word oor die oorsprong van hierdie bakterie in wyn bv. menslike hantering en besmette water, het hierdie rasse wel enterosien geproduseer en daarom die potensiaal om ‘n rol te speel in beheer teen verskeie bederf-MSB-genera. / TIA, NRF and THRIP

Thesis (PhD (Genetics. Plant Biotechnology))--Stellenbosch University, 2008. / Wine quality is greatly influenced by the number of microorganisms, which occur throughout the winemaking process. These microorganisms are naturally present on the grapes and in the cellar from where they can be introduced to the winemaking process at any given time and consequently impart specific contributions to the wine quality. However, these microorganisms can be seen either as beneficial or as wine spoilage microorganisms, depending on the conditions under which they can proliferate during the winemaking process. Wine yeasts (Saccharomyces spp.) are typically responsible for the alcoholic fermentation; lactic acid bacteria (LAB) are responsible for malolactic fermentation (MLF), while acetic acid bacteria (AAB) and other wild yeasts (non-Saccharomyces spp.) are typically associated with the formation of off-flavours under poorly controlled winemaking conditions. In recent years, evidence from the wine industry has highlighted a specific group of non-Saccharomyces yeast species as a serious cause for wine spoilage that required more research investigations. Yeast of the genus Brettanomyces or its teleomorph Dekkera has been identified as one of the most controversial spoilage microorganisms during winemaking as they can produce several compounds that are detrimental to the organoleptic quality of wine. This has triggered the research initiative behind this doctoral study on the significance of Brettanomyces and Dekkera yeasts during winemaking. In this dissertation, various aspects of the detection, isolation and identification methods of Brettanomyces yeast from the winemaking environment were investigated. As a first objective, a culture collection of Brettanomyces bruxellensis wine isolates had to be established. This followed after the isolation of Brettanomyces yeasts from various red wine cultivars from South African wineries from different stages of the winemaking process. Different conventional microbiological methods such as plating on selective agar media and microscopy were investigated along with molecular identification techniques such as the polymerase chain reaction (PCR) in this regard. Other focus areas of this study aimed at performing genetic characterisation and differentiation studies of B. bruxellensis wine isolates. For this purpose, different intraspecific identification methods were investigated on several strains, including strains of European origin. The application of molecular techniques allowing strain identification aided in the selection of specific strains that were evaluated for volatile phenol production in synthetic media and wine. The results obtained from this work indicated that a large degree of genetic diversity exists among B. bruxellensis strains and that the volatile phenol production differed between the strains after evaluation in synthetic media and wine. In addition to the molecular intraspecific strain identification techniques that were investigated, a feasibility study was also performed that focused on evaluating Fourier transform infrared (FTIR) spectroscopy combined with chemometrics as an alternative approach for differentiating between B. bruxellensis strains. The two approaches of FTIR spectroscopy that were investigated involved the use of firstly, Fourier transform mid-infrared (FTMIR) spectroscopy to obtain spectral fingerprints of spoiled wines by different B. bruxellensis strains; and secondly, Attenuated total reflectance (FTIR-ATR) to obtain spectral fingerprints from whole cells of B. bruxellensis on microbiological agar media. The results of this study illustrated the potential of FTIR spectroscopy to become a reliable alternative to molecular based methods for differentiating between B. bruxellensis strains and for characterisation studies. The formation of volatile phenols in wine by species of the genera Brettanomyces and Dekkera is one of the primary reasons for their classification as wine spoilage yeasts. The enzymatic activities of this reaction have been identified and involve a phenyl acrylic (phenolic) acid decarboxylase (PAD) and a vinyl phenol reductase (VPR). However, only a limited amount of information is available about these enzymes from Brettanomyces/Dekkera yeasts and no genetic data have been described. It was therefore imperative that this dissertation should include a genetic investigation into the phenylacrylic (hydroxycinnamic) acid decarboxylase from the species B. bruxellensis involved in the formation of volatile phenols. Strategies that were investigated included various molecular DNA techniques and protein purification procedures to obtain either genetic or protein sequence data. The decarboxylase activity of this yeast species towards p-coumaric acid was demonstrated and substantial genetic sequence data was obtained. The results from this dissertation made a substantial contribution to the current available knowledge about Brettanomyces/Dekkera spp. and led to a better understanding of this wine spoilage yeast. This research developed a platform from which further investigations could follow and the knowledge gained will be invaluable for future Brettanomyces research projects at the Institute for Wine Biotechnology at Stellenbosch University.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Wine protein haze formation is a major challenge for wine makers, and several wine clarifying agents such as bentonite are used in the industry to protect wine from this occurrence. However, clarifying agents may have an undesirable impact on wine quality. Yeast mannoproteins have been shown to possess haze-protective properties, while also positively impacting on the sensorial properties of the product. However, while such mannoproteins are released into the wine during the wine making process, the amounts are low and therefore of limited oenological significance. However, and although commercial wine yeast strains display significant genotypic and phenotypic diversity, no broader assessment of haze protective activity and of mannoproteins release by different wine yeast strains has been undertaken. In this study, several yeast strains were screened for their impact on wine haze formation in Chardonnay must and in a grape juice model system. The data show that strains of the species Saccharomyces paradoxus possess better haze protective properties than the common Saccharomyces cerevisiae wine yeast strains. Differences in the nature of the proteins released by these two species were investigated, and indicated that several mannoproteins were released at significantly higher levels by S. paradoxus, and that some of these proteins might indeed contribute to the haze-protective activity. A further exploration of yeast cell wall properties indicated that the cell walls of haze-protective S. paradoxus strains contained higher levels of chitin than non-haze protective strains. Grape chitinases are likely to be primarily responsible for wine haze formation, and the data clearly demonstrate that these enzymes are able to bind to the yeast cell walls, and that strains with higher amounts of chitin in the cell wall will bind more chitinases. This finding suggests that the haze-protective nature of the strains is at least in part linked to the chitin levels of the strains. Furthermore, the impact of some genetic modifications in two wine strains (namely S. cerevisiae VIN13 and S. paradoxus RO88) suggests that several proteins contribute to wine haze protection. However, none of the mannoprotein-encoding flocculation genes, FLO1, FLO5, and FLO11 showed any impact on this property. Further studies are required to assess the full impact of the S. paradoxus strains on haze protection. In particular, the possible use of such strains as starter cultures or the use of S. paradoxus yeast hulls as clarifying agent needs to be further explored. / AFRIKAANSE OPSOMMING: Wyn proteïen-waas vorming is 'n groot uitdaging vir wynmakers en verskeie wyn verhelderings agente soos bentoniet word in die wynbedryf gebruik om wyn te beskerm teen die vorming van waas. Hierdie verheldering agente het egter 'n ongewenste impak op wynkwaliteit. Gis mannoproteïene is uitgewys as proteïene met moontlike waas-beskermende eienskappe wat ook 'n positiewe uitwerking op die sensoriese eienskappe van die produk het. Al word hierdie mannoproteïene egter vrygestel in die wyn tydens die wynmaak proses, is die hoeveelhede oor die algemeen te laag om van wynkundige belang te wees. Verder, ten spyte van die beduidende genotipiese en fenotipiese diversiteit van kommersiële wyngisrasse is daar nog geen breër assessering van die waas beskermende aktiwiteit van mannoproteïene, vrygestel deur verskillende rasse, tot dusver onderneem nie. In hierdie studie is verskeie gisrasse gekeur vir hul impak op wyn waas-vorming in Chardonnay mos en ook in 'n model druiwesap. Die data wys dat rasse van die spesie Saccharomyces paradoxus besit beter waas beskermende eienskappe as die algemene Saccharomyces cerevisiae wyngisrasse. Verskille in die aard van die proteïene wat vrygestel is deur hierdie twee spesies is ondersoek, en dit is aangedui aangedui dat verskeie mannoproteins vrygestel aan aansienlik hoër vlakke deur S. Paradoxus. Dit is ook aangedui dat sommige van hierdie proteïene wel bydra tot die waas-beskermende aktiwiteit. 'n Verdere verkenning van gis selwand eienskappe het aangedui dat die selwande van waas-beskermende rasse van S. paradoxus hoër vlakke chitien as nie-waas beskermende stamme bevat. Druiwe chitinases is waarskynlik hoofsaaklik verantwoordelik vir wyn waas vorming, en die data toon duidelik dat hierdie ensieme in staat is om te bind aan die gis selwande, en dat die stamme met hoër vlakke chitien in die selwand meer chitinases sal bind. Hierdie bevinding dui daarop dat die waas-beskermende aard van die stamme ten minste gedeeltelik gekoppel is aan die chitien vlakke van die stamme. Die impak van sekere genetiese modifikasies in twee verskillende gisrasse, naamlik die S. cerevisiae ras VIN13 en die S. paradoxus ras RO88, dui verder daarop dat verskeie proteïene dra by tot die beskerming teen wyn waas. Geeneen van die mannoprotein-koderende flokkulasie gene, FLO1, FLO5 en FLO11 het egter 'n impak op hierdie eienskap nie. Verdere studies is nodig om die volle impak van die S. paradoxus rasse op waas beskerming te assesseer. In die besonder, die moontlike gebruik van sulke rasse as 'n inkolasie kultuur of die gebruik van S. paradoxus gis doppe as verheldering agent moet verder ondersoek word.

Natural corks have long been used as wine closures. However, they are associated with causing multiple adverse effects to the wine they are attempting to preserve. Alternative closures such as synthetic corks and screw caps were developed in order to reduce and/or eliminate these problems. However, the major cause of concern regarding these closures is of consumers' acceptance. The effect of how three types of closures (Natural Cork, Synthetic Cork and Screw Cap) affected wine consumers' perceptions of the quality of wine was examined in this study. This project was divided into two experiments. The first experiment determined if frequent wine consumers could detect sensorial differences between the three closure types. The second experiment ascertained if and how regular wine consumers' perceptions were altered based on the type of closure with which the wine samples were bottled. It was determined that the wine consumers could not significantly detect a difference between any of the three closure type samples based only on sensory stimuli. The results from the second experiment found for the Chardonnay samples, the knowledge that the wine samples came from a natural cork or a synthetic cork did not significantly affect the liking, quality or purchase intent scores. However, when the panelists knew that the sample was bottled with a screw cap, they thought it was of lower quality, were less willing to buy a wine like the sample and they lowered the price they were willing to pay. For the Merlot samples, knowledge that the sample came from a natural cork caused the wine consumers to significantly increase both their opinions of the quality of the wine and the amount they were willing to pay for the wine. When they knew that the sample was bottled with a screw cap, they reduced the price they would pay for the wine. / Graduation date: 2005

Thesis (MTech (Agriculture))--Cape Peninsula University of Technology, 2015. / A study was carried out at Bien Donné Experimental Farm, near Paarl in the Western Cape Region (South Africa), to evaluate the performance of solid winery waste compost (WWC) and inorganic fertilizer (N:P:K, 2:3:4 (30) - 5g Zn%) on growth and yield of cabbage (Brassica oleracea var. capitata L.) and Swiss chard (Beta vulgaris subsp. cycla). The experimental plot was fertilized as per treatment with WWC (100% and 400% equivalent recommended fertilizer application using N as reference mineral) and inorganic fertilizer. The experimental design was set up in a Randomized Complete Block Design (RCBD) with 4 treatments (control- without compost and inorganic fertilizers, inorganic fertilizer-2:3:4 (30) - 5g Zn% and LAN (28), WWC application at different application rates were (3485g/plot) (100%) and (13939g/plot) (400%)) replicated four times. Soil analysis showed that the experimental plot is dominated by sandy soil structure. Results of mineral analysis after application of treatments showed a significant (p>0.05) drop in soil pH over time in the untreated control and application of 400% WWC significantly (p<0.05) raised soil pH compared with the control. The application of mineral fertilizer showed significant (p<0.05) increase in soil P compared with the other treatments. However, WWC picked up significant (p<0.05) speed above inorganic fertilizer, thus making P available to the soil than NPK mineral fertilizer. A significant (p<0.05) drop in soil K content by 21% over time on amended soil with inorganic fertilizer treatment was observed. However, the application of WWC at 300 and 400% significantly (p<0.05) raised the soil K by 54.93 and 73.06% respectively. There were no significant differences in soil Ca over time, but high soil Ca concentrations from WWC (100%) were recorded compared to inorganic treatment that showed the lowest soil Ca concentration. There was a slight drop in soil Na over time in control and soil amended with inorganic fertilizer. The effects of the treatment on Mg values were not so prominent, suggesting that concentrations of nutrients are less essential characteristics of the soil or small portion of nutrients were readily available on the soil.

Thesis (Ph. D.)--Washington State University, December 2008. / Title from PDF title page (viewed on Dec. 24, 2008). "Department of History." Includes bibliographical references.

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2009. / Grape juice is transformed into wine through the complex processes of alcoholic and malolactic fermentation that is performed by yeasts, lactic acid bacteria and acetic acid bacteria. However, the microbes involved in these processes do not only take part in ensuring the successful production of wine, but also cause spoilage of the wine if their growth is not controlled. Conventional, culture-dependent methods of microbiology have been used as the main technique in detecting and identifying these spoilage microbes. Cultureindependent techniques of molecular biology have recently become more popular in detecting possible spoilage microbes present in must and wine, since it allows the detection and identification of viable, but non-culturable microbes and are not as timeconsuming as conventional microbiological methods. The aim of this study was to investigate the sustainability of polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis in detecting wine spoilage microbes inoculated into sterile saline solution (SSS) (0.85% (m/v) NaCl) and sterile white wine and red wine as single microbial species and as part of mixed microbial populations. Three methods of DNA isolation from SSS, sterile white wine and sterile red wine inoculated with reference microbial strains were compared in terms of DNA concentration and purity, as well as simplicity of the technique. These three DNA isolation methods were the TZ-method, the proteinase K-method and the phenol extraction method. DNA could not successfully be isolated from red wine using any of the three DNA isolation methods. The TZ-method was the method of choice for the isolation of DNA from inoculated SSS and sterile white wine as this technique gave the best results in terms of simplicity, DNA concentration and purity. PCR and DGGE conditions were optimised for the universal primer pair, HDA1-GC and HDA2, the wine-bacteria specific primer pair, WBAC1-GC and WBAC2, and the yeast specific primer pair, NL1-GC and LS2. DNA from Acetobacter pasteurianus, Lactobacillus plantarum, Pediococcus pentosaceus, Oenococcus oeni, Brettanomyces bruxellensis and Saccharomyces cerevisiae were amplified with the appropriate primers and successfully resolved with DGGE analysis. PCR and DGGE detection limits were successfully determined when 106 cfu.ml-1 of the reference microbes, A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis were separately inoculated into SSS and sterile white wine. It was possible to detect low concentrations (101 cfu.ml-1) with PCR for A. pasteurianus, Lb. plantarum, Grape juice is transformed into wine through the complex processes of alcoholic and malolactic fermentation that is performed by yeasts, lactic acid bacteria and acetic acid bacteria. However, the microbes involved in these processes do not only take part in ensuring the successful production of wine, but also cause spoilage of the wine if their growth is not controlled. Conventional, culture-dependent methods of microbiology have been used as the main technique in detecting and identifying these spoilage microbes. Cultureindependent techniques of molecular biology have recently become more popular in detecting possible spoilage microbes present in must and wine, since it allows the detection and identification of viable, but non-culturable microbes and are not as timeconsuming as conventional microbiological methods. The aim of this study was to investigate the sustainability of polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis in detecting wine spoilage microbes inoculated into sterile saline solution (SSS) (0.85% (m/v) NaCl) and sterile white wine and red wine as single microbial species and as part of mixed microbial populations. Three methods of DNA isolation from SSS, sterile white wine and sterile red wine inoculated with reference microbial strains were compared in terms of DNA concentration and purity, as well as simplicity of the technique. These three DNA isolation methods were the TZ-method, the proteinase K-method and the phenol extraction method. DNA could not successfully be isolated from red wine using any of the three DNA isolation methods. The TZ-method was the method of choice for the isolation of DNA from inoculated SSS and sterile white wine as this technique gave the best results in terms of simplicity, DNA concentration and purity. PCR and DGGE conditions were optimised for the universal primer pair, HDA1-GC and HDA2, the wine-bacteria specific primer pair, WBAC1-GC and WBAC2, and the yeast specific primer pair, NL1-GC and LS2. DNA from Acetobacter pasteurianus, Lactobacillus plantarum, Pediococcus pentosaceus, Oenococcus oeni, Brettanomyces bruxellensis and Saccharomyces cerevisiae were amplified with the appropriate primers and successfully resolved with DGGE analysis. PCR and DGGE detection limits were successfully determined when 106 cfu.ml-1 of the reference microbes, A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis were separately inoculated into SSS and sterile white wine. It was possible to detect low concentrations (101 cfu.ml-1) with PCR for A. pasteurianus, Lb. plantarum, iv Pd. pentosaceus, and B. bruxellensis in SSS when amplified with the HDA1-GC and HDA2 primer pair. A PCR detection limit of 102 cfu.ml-1 was determined in sterile white wine for Pd. pentosaceus and 103 cfu.ml-1 for B. bruxellensis using this primer pair. The results obtained from the PCR amplification with the WBAC1-GC and WBAC2 primer pair compared well with the results of the HDA1-GC and HDA2 primer pair. The results from the DGGE detection limits indicated that it was possible to detect lower concentrations (101 – 102 cfu.ml-1) of A. pasteurianus, Lb. plantarum and Pd. pentosaceus with the HDA1-GC and HDA2 primer pair than the WBAC-GC and WBAC2 primer pair (102 – 104 cfu.ml-1). Lower detection limits were also determined for B. bruxellensis amplified with the HDA1-GC and HDA2 primer pair (103 – 104 cfu.ml-1) than with the NL1-GC and LS2 primer pair (105 cfu.ml-1). PCR and DGGE detection limits for the inoculation of A. pasteurianus, Lb. plantarum and B. bruxellensis at an inoculum of 108 cfu.ml-1 as part of mixed populations in SSS and sterile white wine compared well with the results obtained from the reference microbes inoculated as single microbial species. PCR detection limits of 101 cfu.ml-1 were determined for all three reference microbes inoculated as part of mixed populations when amplified with the HDA1-GC and HDA2 and the WBAC1-GC and WBAC2 primer pairs. It was observed that similar or higher DGGE detection limits were obtained for the reference microbes inoculated in sterile white wine (101 – 107 cfu.ml-1) than when inoculated into SSS (101 – 105 cfu.ml-1). PCR-based DGGE analysis proved to be a technique that could be used successfully with the universal, wine-bacteria and yeast specific primer pairs for the detection of A. pasteurianus, Lb. plantarum, Pd. pentosaceus and B. bruxellensis. The culture-independent technique makes the early detection of possible spoilage microbes at low concentrations in wine possible.

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.

Thesis (MSc Food Sc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: In this study an industry-selected and diverse range of South African red wines were analysed for sensory and chemical attributes, as well as degree of liking using a target group of black South African consumers. Segments of consumers that differed in degree of liking were then tested for their response to intrinsic (sensory) and extrinsic (non-sensory) cues. The selection of wines included eighteen dry and natural sweet red wines, representing low-end inexpensive wines together with high-end, top quality wines. Sensory profiles for all samples were established using Quantitative descriptive analysis (QDA). The results revealed that cultivar specific dry red wines associated with a wide range of sensory descriptors such as woody, vegetative and fruity, while the sweet red wines associated with the fruity and sweet-associated attributes. Chemically there was a significant variation between wines regarding the alcohol and sugar content. Gas chromatography with flame ionisation detection (GC-FID) indicated the major volatile constituents present in the wine, i.e. esters, alcohols and fatty acids. When investigating the association between the chemical and sensory data, it was revealed that the red blends were driven by the presence of alcohols and esters, and sensory descriptors such as high roast oak, coffee and mixed spice, whereas the red cultivar wines were mostly driven by fatty acids and esters and the sensory descriptors, green bean and asparagus. The sweet red blends were closely associated with acids and the sensory descriptors sweet-associated and floral. Degree of liking of a subset of 18 wines was investigated based on the preferences of black consumers from the Western Cape area, South Africa. These consumers predominantly preferred the sweet red wines with high sugar content, in a blind tasting session. Purchase intent was also evaluated by viewing actual photographs of packaging formats of the respective wines and the results indicated that the consumers preferred the well-known cultivar wines with a perception of value and style. Cluster analysis was furthermore performed to ascertain whether these consumers differed in their degree of liking of the intrinsic character of the respective wines. Four different clusters of consumers were identified: 1) Consumers preferring both dry and sweet red wines equally, 2) Consumers who strongly favoured sweet red wines and moderately liked dry red wines, 3) Consumers who strongly favoured sweet red wines with little preference for dry red wines; and 4) Consumers preferring dry red wines. Consumers were also probed on their general opinions or perceptions on the extrinsic character of the wines, and thus factors that influence the purchasing process. It was found that black consumers who don‟t consume wine often, preferred wines that they are familiar with, while consumers that drink wine more frequently enjoy to broaden their horizons by experimenting with more expensive wine brands. Extrinsic or non-sensory cues such as alcohol content, label, vintage, price and cultivar were found to be the most important considered factors when purchasing red wines, while awards and type of closure were regarded as the least important. It was also found that the discerning consumers, who purchase high-end wines, took more of the latter aspects into consideration, whereas consumers who purchase low-end wines considered a limited number of the non-sensory cues. / AFRIKAANSE OPSOMMING: In hierdie studie is 'n diverse reeks industrie-geselekteerde, Suid-Afrikaanse rooiwyne geanaliseer vir hul sensoriese en chemiese eienskappe. Verbruikersvoorkeur van die wyne is getoets, asook tot watter mate verbruikersvoorkeure beïnvloed word deur intrinsieke (sensoriese) en ekstrinsieke (nie-sensoriese) faktore. Die reeks van agtien wyne het bestaan uit droë en soet rooi wyne, wat op hul beurt verder verdeel kan word in goedkoper, kwaliteit wyne en duurder, ultra-premium wyne. Die sensoriese profiel van al die wyne is bepaal deur beskrywende sensoriese analise. Resultate het getoon dat die kultivar-spesifieke droë rooiwyne geassosieer word met 'n wye reeks sensoriese eienskappe soos houtagtig, kruidagtig en vrugtig, terwyl die soet rooiwyne beskryf is as vrugtige en soet-geassosieerd. In terme van die chemiese analises was daar betekenisvolle verskille betreffende die alkohol- en suikerinhoud van die wyne. Gas chromatografie gekoppel met vlam-ioniserende deteksie (GC-FID) het die mees vlugtige verbindings teenwoordig in die wyn aangedui, naamlik esters, alkohole en vetsure. Met die korrelasie van die chemiese en sensoriese data is gevind dat die droë versnitwyne gedryf word deur die teenwoordigheid van alkohole en esters, asook sensoriese eienskappe soos gehout, koffie, en gemengde spesery, terwyl die kultivar-spesieke wyne weer meestal gedryf word deur vetsure en esters en sensoriese eienskappe soos groenboontjie en aspersie. Die soet rooiwyne het chemies geassosieer met sure en sensoriese terme soos soet-geassosieerd en blomagtig. Die aanvaarbaarheid van 'n kleiner groepering wyne is bepaal deur gebruik te maak van swart verbruikers in die Wes-Kaap area, Suid-Afrika. Die verbruikers het in 'n blinde proesessie onderskeie wyne se wynverpakking besigtig en aangedui of hulle die wyne sou koop. Hierdie resultate het getoon dat die verbruikers bekende kultivarwyne verkies wat 'n persepsie van waarde en styl geïllustreer het. Segmentasie tegnieke is op die data uitgevoer ten einde te bepaal of verbruikers in groepe verdeel kan word, wat betref hul voorkeur van die sensoriese of intrinsieke eienskappe van die wyne. Vier verskillende groepe is geïdentifiseer, nl. verbruikers wat 1) droë en soet rooiwyne ewe veel verkies; 2) soet rooiwyne en tot 'n mate ook droë rooiwyne verkies; 3) soet rooiwyne en tot 'n mindere mate droë rooiwyne verkies; en laastens 4) slegs droë rooiwyne verkies. Verbruikers se algemene opinies en persepsies betreffende die ekstrinsieke eienskappe van die wyne is ook ondersoek, met ander woorde faktore wat die aankoop van wyne beïnvloed. Daar is gevind dat swart verbruikers wat nie gereeld wyn drink, bekende handelsmerke verkies, terwyl verbruikers wat gereeld wyn drink, daarvan hou om hul horisonne te verbreed en te eksperimenteer met 'n verskeidenheid handelsmerke. Ekstrinsieke of nie-sensoriese aspekte soos, alkohol-inhoud, etiket, oesjaar, prys en kultivar is die belangrikste faktore wat in ag geneem word wanneer rooiwyne gekoop word, terwyl wyntoekennings en die feit dat die wyn met kurke gebotteleer word, nie as belangrik beskou word nie. Daar is ook gevind dat die meer ingeligte verbruiker, wat hoë kwaliteit wyne koop, meer van die bogenoemde aspekte in ag neem tydens die aankoopproses, terwyl die verbruiker wat meer geneig is om goedkoper wyne te koop, slegs 'n paar ekstrinsieke faktore in ag neem.