Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Non-Saccharomyces yeasts of oenological origin have previously been associated with spoilage or regarded as undesired yeasts in wine. However, these yeasts have recently come under investigation for their positive contribution towards wine aroma especially when used in sequential or co-inoculated fermentations with Saccharomyces cerevisiae. These yeasts are also known to secrete a number of enzymes that could be applicable in wine biotechnology. Amongst these enzymes are aspartic proteases. The secreted proteases from some non-Saccharomyces yeast may play a role in protein haze reduction, as demonstrated by some authors, while simultaneously increasing the assimilable nitrogen content of the wine for the utilization and growth of fermentative microorganisms. Moreover, the proteases may have an indirect effect on wine aroma by liberating amino acids that serve as aroma precursors. Although many screenings have been performed detecting protease activity in non-Saccharomyces yeasts, no attempts have been made to characterize these enzymes. This study set out to isolate and characterize genes encoding extracellular aspartic proteases from non-Saccharomyces yeasts. An enzymatic activity screening of a collection of 308 Saccharomyces and non-Saccharomyces yeasts, isolated from grape must, was performed. The aspartic protease-encoding genes of two non- Saccharomyces yeasts, which showed strong extracellular proteolytic activity on plate assays, were isolated and characterized by in silico analysis. The genes were isolated by employing degenerate and inverse PCR. One gene was isolated from Metschnikowia pulcherrima IWBT Y1123 and named MpAPr1. The other putative gene was isolated from Candida apicola IWBT Y1384 and named CaAPr1. The MpAPr1 gene is 1137 bp long, encoding a 378 amino acid putative protein with a predicted molecular weight of 40.1 kDa. The CaAPr1 putative gene is 1101 bp long and encodes a 367 amino acid putative protein with a predicted molecular weight of 39 kDa. These features are typical of extracellular aspartic proteases. The deduced protein sequences showed less than 40% homology to other yeast extracellular aspartic proteases. By heterologous expression of MpAPr1 in S. cerevisiae, it was confirmed that the gene encodes an extracellular acid protease. The expression of MpAPr1 was shown to be induced in media containing proteins as sole nitrogen source and repressed when a preferred nitrogen source was available. The gene was expressed in the presence of casein, bovine serum albumin (BSA) and grape juice proteins and repressed in the presence of ammonium sulphate. Expression was most induced in the presence of grape juice proteins, which was expected since these proteins are present in the natural habitat of the yeast. A genetic screening confirmed the presence of the MpAPr1 gene in 12 other M. pulcherrima strains isolated from grape juice. The extracellular protease activity of the strains was also visualized on plates. As far as we know, this is the first report on the genetic characterization of secreted aspartic proteases from non-Saccharomyces yeasts isolated from grape must and provides the groundwork for further investigations. / AFRIKAANSE OPSOMMING: Nie-Saccharomyces giste is voorheen met wynbederf geassosieer en hul teenwoordigheid in wyn is ongewens. Hierdie giste is onlangs ondersoek vir hulle positiewe bydrae tot wyn aroma, in veral sekwensiële en ko-inokulerings met Saccharomyces cerevisiae. Sommige van die nie-Saccahromyces giste skei ‘n verskeidenhied ensieme af wat moontlik vir die wynmaker van nut kan wees. Een groep van hierdie ensieme is die aspartiese suurproteases. Soos deur sommige navorsers aangetoon word, kan die proteases die vorming van proteïenwaasverlaging, terwyl dit terselfdertyd die assimilerende stikstofinhoud van die wyn vir die gebruik en groei van fermentasie-mikroörganismes verhoog. Die proteases kan moontlik ook ‘n indirekte uitwerking op die aromaprofiel van die wyn hê deur die vrystelling van aminosure wat as aromavoorlopers dien. Alhoewel baie studies gedoen is wat die ekstrasellulêre teenwoordigheid van proteases bevestig in nie-Saccharomyces giste wat van druiwesap/wyn afkoms is, is daar geen dokumentasie oor die genetiese karakterisering van hierdie ensieme beskikbaar nie. Die doel van hierdie studie was om gene wat aspartiese proteases in nie-Saccharomyces giste enkodeer, te isoleer en gedeeltelik te karakteriseer. ‘n Versameling van 308 Saccharomyces en nie-Saccharomyces giste wat uit druiwe sap geïsoleer is, is gesif vir ensiematiese aktiwiteit deur plaattoetse uit te voer. Twee gene wat aspartiese protease enkodeer, is geïsoleer van twee nie-Saccharomyces giste. Dit hetpositief gedurende die aktiwiteitstoetse getoets en is deur in silico–analise gekarakteriseer. Die gene is deur die uitvoering van gedegenereerde en inverse PKR geïdentifiseer. Een geen is vanaf Metschnikowia pulcherrima IWBT Y1123 geïsoleer en is MpAPr1 genoem, terwyl die ander van Candida apicola IWBT Y1384 geïsoleer en CaAPr1 genoem is. Die MpAPr1-geen is 1137 bp lank en enkodeer ‘n proteïen wat uit 378 aminosure bestaan met ‘n voorspelde molekulêre massa van 40.1 kDa. Daar teenoor is die CaAPr1-geen 1101 bp lank en enkodeer vir ‘n proteïen wat uit 367 aminosure met ‘n molekulêre massa van 39 kDa bestaan. Hierdie eienskappe is kenmerkend van aspartiese protease. Die afgeleide proteïenvolgorde het minder as 40% homologie met ander ekstrasellulêre aspartiese proteases vertoon, wat dui op die nuwigheid van hierdie ensieme. Die MpAPr1-geen is heterologies in S. cerevisiae YHUM272 uitgedruk en dit het bevestig dat die geen inderdaad ‘n ekstrasellulêre aspartiese protease enkodeer. Die MpAPr1-geen is uitgedruk in media wat alleenlik proteïen as stikstofbron bevat het, terwyl dit onderdruk is in gevalle waar ‘n verkose stikstofbron beskikbaar was. Die geen is uitgedruk in die teenwoordigheid van kaseïen, BSA en proteïene afkomstig vanaf druiwesap en in die teenwoordigheid van ammoniumsulfaat onderdruk. Die hoogste uitdrukking was in die teenwoordigheid van druifproteïene. Hierdie proteïene is teenwoordig in die natuurlike habitat van die gis en is dus dalk ‘n bekende stikstofbron vir die gis. ‘n Genetiese sifting het die teenwoordigheid van die MpAPr1-geen in 12 ander M. pulcherrima–rasse, wat ook van wynkundige oorsprong is, bevestig. Die aspartiese protease-aktiwiteit van die 12 rasse is ook op agarplate waargeneem. Na ons wete, is dit die eerste verslag oor die genetiese karakterisering van afgeskeide aspartiese proteases van nie- Saccharomyces giste van wynkundige oorsprong en verskaf die grondslag vir verdere ondersoek.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Yeast strains contend with numerous stresses during winemaking. An inability to perceive and initiate the physiological changes needed to adapt to stress, has been linked to slow or incomplete (residual sugar > 4 g/L) fermentations. Wine yeast strains differ in genotype; this is manifested as differences in their stress tolerance, and fermentation performance. The first goal of this study was to evaluate how the initial sugar (200 or 240 g/L) and nitrogen (50, 100, 250, or 400 mg/L) content, and the fermentation temperature (15°C or 20°C) affected the fermentation performance of 17 commercial wine yeast strains. Fermentation performance was evaluated based on the fermentation kinetics (lag phase, maximum fermentation rate and total weight loss by CO2 evolution), residual sugar content and yeast dry weight. The results demonstrate that the fermentation performances of commercial yeast cultures are significantly and differently affected by initial nitrogen and sugar levels, as well as the fermentation temperature. Additionally, excess nitrogen had a negative impact on the fermentation kinetics and sugar consumption. Nitrogen deficiency is a common cause of slow and incomplete fermentations, as it affects yeast growth and thus fermentation rates. Nitrogen supplements are routinely added at the onset of fermentation, reducing the risk of problematic fermentations. Therefore characterising the fermentative ability of a strain over a range of oenologically relevant conditions, could aid winemakers in selecting a yeast strain capable of fermenting a grape must (of known sugar and nitrogen levels) to completion at the desired fermentation temperature. Investigations on fermentation related stress generally focus on its influence on fermentation rate and sugar consumption. However, from a winemaking perspective, the strain’s ability to produce the desired volatile aroma compounds is equally important. Yet, literature provides little insight into the influence stress has on the volatile aroma profile; this is surprising as wine aroma is closely linked to wine quality and consumer liking. The final goal of this study was to evaluate changes to the volatile aroma profiles produced by five commercial yeast strains, in response to hyperosmotic and temperature stress. The concentrations of the aroma compounds were quantified using a gas chromatograph coupled to a flame ionization detector. The results show that hyperosmotic and temperature stress caused significant changes in the levels of a number of aroma compounds. Furthermore, the changes observed differed among the evaluated strains, as well as for the fermentation stress treatments studied. Future aims should be directed towards the potential application of yeast strain selection as a means to avoid problematic fermentations in grape must; in addition to the further characterisation of the relationship between stress and the resultant volatile aroma profile in wine. / AFRIKAANSE OPSOMMING: Gisrasse moet verskeie stresfaktore afweer tydens die wynmaak proses. Die onvermoë van ‘n wyngis om stres waar te neem en die nodige fisiologiese veranderinge te inisieer om aan te pas by die strestoestande word met slepende of onvolledige fermentasies (met ‘n residuele suiker van meer as 4 g/L) geassosieer. Wyngisrasse verkil in genotipe; wat as groot verskille in die graad van strestoleransie, en dus ook fermentasie sukses geopenbaar word. Die eerste doelwit van hierdie studie was om te evalueer hoe die suiker (200 of 240 g/L) en stikstof (50, 100, 250, of 400 mg/L), asook die fermentasie temperatuur (15°C of 20°C) die fermentasie prestasie van 17 kommersiële wyngiskulture beïnvloed. Die sukses van fermentasie is geëvalueer op grond van fermentasie kinetika (sloerfase, maksimum fermentasiespoed en totale gewigsverlies as CO2 verlies), die residuele suiker inhoud en die gis droë massa. Die resultate demonstreer dat die fermentasie sukses van kommersiële giskulture beduidend en verskillend beïnvloed word deur die aanvangsstikstof en – suikerkonsentrasies, asook die fermentasie temperatuur. Daarbenewens, wanneer stikstof in oormaat teenwoordig is kan dit ‘n negatiewe impak op fermentasietempo en suiker metabolisme hê. Beperkende vlakke van stikstof ‘n algemene oorsaak van slepende of onvolledige fermentasies, aangesien stikstof die groei en gevolglik ook die fermentasiespoed van gis beïnvloed. Stikstofaanvullings word dikwels tot druiwemos toegevoeg aan die begin van gisting, wat die risiko van probleemfermentasies verlaag. Dus kan die karakterisering van die fermentasievermoë van ‘n gisras vir ‘n reeks wynkundig relevante kondisies die wynmaker help om ‘n gisras te selekteer wat in staat is om ‘n druiwemos (waarvan die suiker en stikstofvlakke bekend is) droog te gis by die gewenste temperatuur. Meeste studies wat fermentasieverwante stress ondersoek, fokus op die die invloed daarvan op fermentasietempo en suikerverbruik. Van ‘n wynmaakperspektief is die gis se vermoë om die gewensde vlugtige aroma komponente te produseer egter ewe belangrik as die vermoë om fermentasie te voltooi. Tog verskaf die literatuur min insig tot die invloed van stres op die vlugtige aromaprofiel; wat verbasend is aangesien die aromaprofiel ‘n belangrike faktor is van die waargenome wynkwaliteit en daarom ook verbruikersvoorkeur. Die finale doelwit van hierdie projek was om die veranderinge tot die vlugtige aromaprofiel geproduseer deur vyf kommersiële gisrasse in reaksie op hiperosmotiese stres en temperatuur stres te evalueer. Die konsentrasies van die aromakomponente is gekwantifiseer deur gas chromatografie gekoppel aan vlam‐ioniserende deteksie. Die resultate wys dat hiperosmotiese‐ en temperatuur stres beduidende veranderinge meebring in die vlakke van ‘n aantal aromakomponente. Verder is die waargenome veranderinge ook verskillend vir die geëvalueerde gisrasse, asook vir die verskille stresbehandelings wat ondersoek is. Toekomstige studies behoort gerig te wees op die toepassing van gis seleksie om potensiële probleemfermentasies in druiwemos te voorkom; asook die verdere karakterisering van die verhouding tussen omgewingstresfaktore en die gevolglike vlugtige aromaprofiel in wyn.

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 (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Flocculation can be defined as the asexual aggregation of yeast cells in a liquid environment. This aggregation of cells, also referred to as "floc formation", will in most cases lead to rapid settling or sedimentation. However, in so-called top-fermenting yeast strains, the floes can move to the surface of the liquid growth substrate to form a thin layer, called a "velum", that has been compared to other microbial biofilms. The factors that trigger flocculation can be divided into two groups, physical/chemical (e.g. sugar content, the presence of inorganic salts, organic solvents, ethanol concentration, pH, agitation etc.) and genetic factors (genes that encode for proteins that are either directly or indirectly involved in flocculation). In top-fermenting yeast strains, several physical and chemical factors that trigger the process have been described, including ethanol concentration, the presence of organic solvents, the absence of molecular oxygen and the presence of inorganic salts (Ca2+ and Mg2+). These factors appear to affect the cell hydrophobicity and the cell surface charge. As for genetic factors, no specific genes have thus far been associated with flocculation in top fermenting yeast strains. In bottom-fermenting yeast strains, the physical and chemical factors that affect the process are similar to the ones described for top-fermenting yeast strains, but include, more specifically, the concentration of hexoses in the media (mannose or glucose), which may inhibit the process. Indeed, flocculation in bottom-fermenting yeast strains has been divided into the NewFlo type (inhibited by both mannose and glucose) and the Fl01 type (inhibited by mannose) on the basis of the inhibitory effect of specific sugars. Various genes have been associated with the flocculation of bottom-fermenting yeast strains. Through genetic analysis, the genes have been categorised into dominant genes, semidominant genes and recessive genes. In order to better understand the role of some of the proteins responsible for flocculation in S. cerevisiae, and to create strains whose flocculation properties would correspond to those wanted in the wine and beer industries, three of the dominant flocculation genes, FL01, FL05 and FL011, were placed under the control of the promoters of the stationary phase-induced genes, ADH2 and HSP30. This was achieved by replacing the native promoters of the flocculation genes with the heterologous promoters through homologous recombination. The laboratory strain FY23, which is nonflocculent due to the absence of the transcription factor that is required for flocculation, F108p,was used as a model system. Some of the transformed strains showed high flocculation, especially when the genes were placed under control of the ADH2 promoter. In addition to this, the strains carrying a modified FL011 gene showed increased adhesion to solid agar media and were able to invade the growth substrate. These strains also showed an increased velum-forming ability when grown in media containing only non-fermentable carbon sources. / AFRIKAANSE OPSOMMING: Flokkulasie kan gedefinieër word as die ongeslagtelike aggregasie van gisselle in 'n vloeibare medium. Hierdie aggregasie van selle, kan ook na verwys word as flok formasie, en in meeste gevalle lei dit tot In vinnige sedimentering. In oppervlak-fermenterende giste, beweeg die flokke na die oppervlakte van die vloeibare medium om sodoende 'n flor -lagie te vorm. Hierdie verskynsel was ook al gevind in ander organismes. Verskeie faktore is verantwoordelik vir die effektiwiteit van flokkuklasie. Hierdie faktore kan in twee groepe verdeel word, nl. fisiese en chemiese faktore (byv. suikerkonsentrasie, die teenwoordigheid van anorganiese soute, organiese oplossings, etanol konsentrasie, pH, ens.) en genetiese faktore (gene wat kodeer vir die proteïene wat of direk of indirek betrokke is by flokkulasie). In oppervlak-fermenterende giste is daar al heelwat informasie beskikbaar omtrent fisies en chemiese faktore se effekte op flokkulasie. Van die faktore waarvan heelwat informasie beskikbaar is sluit in, etanol konsentrasie, die teenwoordigheid van organiese oplossings, die afwesigheid van molekulêre suurstof en die teenwoordigheid van anorganiese soute (Ca2+ en Mg2+). Hierdie faktore toon 'n effek of hidrofobisiteit en elektriese lading op die seloppervlakte. Geen genetiese faktore kon tot dusver gekoppel word aan flokkulasie in oppervlak-fermenterende giste nie. Benede-oppervlak fermenterende giste se fisies en chemiese faktore wat effektiwiteit van flokkulasie beïnvloed is dieselfde as die van oppervlak-fermenterende giste, maar sluit in meer spesifiek, die konsentrasie van heksoses in die media (nl. mannose en glukose), wat 'n inhiberende effek het op flokkulasie. Die benede-oppervlak fermenterende giste se flokkulasie kan in twee segmente verdeel word nl. die NewFlo tipe (word geïnhibeer deur die teenwoordigheid van mannose en glukose) en die Flo1-tipe (word geïnhibeer deur slegs die teenwoordigheid van mannose). Verskeie gene was ook al geidentifiseer wat die effektiwiteit van flokkulasie beïnvloed in benede-oppervlak fermenterende giste. Hierdie gene kan in drie kategorieë opverdeel word, nl dominante-, semi-dominante- en ressessiewe flokkulerende gene. Ten orde 'n beter begrip te kry rondom die proteïene verantwoordelik vir die meeste effektiwiteit ten opsigte van flokkulasie in S. cerevisiae, asook om giste te manipuleer om spesifieke flokkulasie eienskappe te toon volgens die belange van die wyn en bierindustrieë, was drie dominante flokkulerende gene, nl. FL01, FL05, en FL011, onder regulering van stationêre fase-geïnduseerde promotors, PADH2 en PHSP30, geplaas. Dit was verkry deur die vervanging van die wilde tipe promotors van die drie gene met die stationêre fase-geïnduseerde promotors deur middel van homoloë rekombinasie. Die laboratorium gisras, FY23, wat 'n nie-flokkulerende gisras is vanweë die afwesigheid van 'n transkripsionele faktor, Flo8p, wat verantwoordelik is vir die aktivering van belangrike gene in flokkulasie, was gebruik as 'n wilde tipe ras. Sommige van die transformante het In hoë mate van flokkulasie getoon, veral wanneer onder die regulering van die PADH2. Tesame met laasgenoemde verskynsel, was daar gevind dat FL011-transformante 'n verhoging in hul vermoeë het om te bind aan die agar en ook om die agar te penetreer. Laasgenoemde gisrasse het ook die vermoë getoon om 'n flor-lagie te vorm bo-op die oppervlakte van die medium, maar slegs wanneer dit in niefermenteerbare koolstofbronbevattende media opgegroei word.

Thesis (MScAgric.)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Changes in the bacterial population throughout the malting process of two barley cultivars, i.e. Clipper (local cultivar) and Prisma (imported cultivar), malted at Southern Associated Maltsters (SAM), Caledon, South Africa, were studied. Samples were taken from four individual runs of each cultivar at ten different stages, i.e. dry barley before steep, water from the first steep water-stand, barley after draining the first steep, water from the second steep water-stand, barley from the second steep water-stand, barley after draining of the second steep, barley from the first, second and third days of germination in the germination vessels (GV), and malt after kilning. Emphasis was placed on the taxonomy and composition of the lactic acid bacteria (LAB) isolated from the ten different phases. The LAB were identified to species level by using numerical analysis of total soluble cell protein patterns, RAPD-PCR banding patterns and 16S rRNA sequencing. The Gram-negative bacteria were identified to genus level by using the API 20E system and included Citrobacter spp., Enterobacter spp., Pantoea spp., Proteus spp., Seratia spp., Kluyvera spp., Klebsiella spp., Vibrio spp. and Escherichia coli. The number of viable bacteria throughout the malting process of the two cultivars did not differ significantly, although the LAB counts in the barley before steep and on the kilned malt were higher in Prisma than in Clipper. Leuconostoc argentinum, Leuconostoc laetis and Weissella confusa were the most predominant in both cultivars. A few strains of Weissella paramesenteroides, Lactobacillus casei, Lactococcus laetis and Lactobacillus rhamnosus were also isolated. Lb. casei and Lb. rhamnosus were not isolated from the Prisma cultivar, whilst W paramesenteroides and Le. laetis were absent in the Clipper cultivar. Kilned malt of the Clipper cultivar contained predominantly Le. argentinum, whereas the Prisma cultivar contained mainly Le. lactis. The effect of these bacteria on the fermenting ability of the brewer's yeast Saccharomyces cerevisiae SAB 05, was also studied. Fermentations were conducted in wort prepared from Clipper and Prisma malt. Yeast in combination with the different bacteria were used in the fermentation studies. Wort with only yeast was used as control. Emphasis was placed on the effect the bacteria has on the gravity, pH, yeast- and bacterial- counts and the different volatile aroma compounds produced throughout the fermentations. The presence of LAB and Gram-negative bacteria had no effect on the yeast to reduce the gravity of the fermenting wort, whilst the LAB caused a decrease in the pH of the fermentations in both Clipper and Prisma wort. The cell numbers of the Gram-negative bacteria decreased throughout the fermentations, whilst the LAB cell numbers remained constant. Comparisons could be drawn between the volatile aroma compounds produced in the control fermentation and fermentations with yeast and Gram-negative bacteria, yeast and Lactobacillus spp. and yeast and Weissella spp. Leuconostoc spp. had a much greater influence on the aromatic composition of fermented malt, with much more clear variations between Prisma and Clipper. No major differences were recorded in the aroma profiles of Prisma and Clipper malt fermented in the presence and absence of Lactococcus spp. The Gram-negative bacteria had no significant effect on the volatile aroma compounds produced by the yeast, whilst the LAB had a definite effect on aroma composition in both cultivars. The levels of four of the five principle aroma compounds, present in beer, were in the acceptable concentration range on the fmal day of fermentation. The compounds with the highest concentrations were iso-amyl alcohol, acetic acid and acetoin, with acetic acid being present in the highest concentration in all the fermentations. / AFRIKAANSE OPSOMMING: Veranderinge in die bakteriese populasie van die gars kultivars, Clipper (plaaslik) en Prisma (ingevoer), vermout by Southern Associated Maltsters (SAM), Caledon, Suid Afrika, is ondersoek. Monsters is van vier individuele lopies van elke kultivar en tydens tien verskillende fases van die vermoutingsproses geneem. Die tien verskillende stadia het die volgende ingesluit: Droë gars voor benatting, water van die eerste benattingsfase, gars nadat water van die eerste benattingsfase gedreineer is, water van die tweede benattingsfase, gars van die tweede benattingsfase, gars na die dreinering van water in die tweede benattings fase, gars na die eerste, tweede en derde dag van ontkieming binne die ontkiemingstenke, en mout na droging. Klem is geplaas op die taksonomie en samestelling van melksuurbakterieë (MSB) wat tydens die tien verskillende fases geïsoleer is. Die MSB is tot spesievlak geïdentifiseer deur gebruik te maak van numeriese analise van totale oplosbare selproteïen bandpatrone, RAPD-PKR bandpatrone en 16S rRNA volgorde-bepaling. Gram-negatiewe bakterieë is tot op genusvlak geïdentifiseer deur gebruik te maak van die API 20E toetssisteem. Spesies van die genera Citrobacter, Enterobacter, Pantoea, Proteus, Seratia, Kluyvera, Klebsiella, Vibrio asook Escherichia coli is geïdentifiseer. Tydens die vermoutingsproses van die twee kultivars is geen beduidende verskille in die lewensvatbare bakterietellings gevind nie, alhoewel die MSB-tellings in die gars voor benatting en mout na droging in Prisma hoër was as in Clipper. Leuconostoc argentinum, Leuconostoc laetis en Weissella confusa het die meeste voorgekom in beide kultivars. Kleiner hoeveelhede van Weissella paramesenteroides, Lactobacillus casei, Lactococcus laetis en Lactobacillus rhamnosus is ook geïsoleer. Lb. casei en Lb. rhamnosus het nie in die Prisma-kultivar voorgekom nie, terwyl W paramesenteroides en Le. laetis nie in die Clipper-kultivar teenwoordig was nie. Le. argentinum het meestal in die gedroogde mout van die Clipper-kultivar voorgekom, terwyl Le. laetis meestal in die Prisma-kultivar waargeneem is. Die effek van hierdie bakterieë op die fermentasievermoë van die brouersgis Saccharomyces cerevisiae SAB 05 is ook bestudeer. Die fermentasies is in Clipper- en Prisma- wort gedoen. Vir die fermentasiestudies is gis in kombinasie met verskillende bakterieë gebruik. Wort met slegs gis het as kontrole gedien. Klem is geplaas op die effek van die bakterieë op die digtheid, pH, gis- en bakterietellings en die verskillende vlugtige komponente wat tydens die fermentasies geproduseer is. Die teenwoordigheid van MSB en Gram-negatiewe bakterieë het geen effek gehad op die vermoë van die gis om die digtheid van die gefermenteerde wort te verlaag nie. Die MSB het wel 'n verlaging van die pH in beide Clipper- en Prisma- wort teweeggebring. Tydens die fermentasie het die Gramnegatiewe bakterietellings verminder, terwyl die MSB-tellings konstant gebly het. 'n Verband is gevind tussen vlugtige komponente geproduseer in die kontrole-fermentasie en fermentasies met gis en Gram-negatiewe bakterieë, gis en Lactobacillus spp. en gis en Weissella spp. Leuconostoc spp. het groter veskille in die samestelling van die gefermenteerde wort teweeg gebring met duidelike verskille tussen Clipper en Prisma. Die teenwoordigheid van Lactococcus spp. het nie groot verskille in die samestelling van die gefermenteerde wort getoon nie. Op die laaste dag van die fermentasies was die vlakke van vier uit die vyfbelangrikste vlugtige aroma komponente wat in bier voorkom in die kontrole fermentasies in aanvaarbare konsentrasies teenwoordig. Die Gramnegatiewe bakterieë het geen beduidende invloed gehad op die vlugtige aroma komponente wat deur die gis geproduseer is nie, terwyl die MSB 'n besliste effek in die aroma-samestelling van beide die kultivars gehad het. Die komponente met die hoogste konsentrasies was, isoamiel-alkohol, asynsuur en asetoin. Asynsuur was in al die fermentasies in die hoogste konsentrasie teenwoordig.

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Grapevine is constantly under attack from a wide variety of pathogens including viruses, bacteria and fungi. In order to ensure survival, the grapevine has developed a vast array of defense mechanisms to combat invading organisms. A key element of this disease resistance is the production of phytoalexins, of which resveratrol is the primary component. The synthesis of resveratrol, together with other structural and biochemical defense mechanisms equips the plant to combat a number of pathogens resulting in the production of healthy grapes for the vinification of top quality wine. As part of the active disease response resveratrol is synthesised de novo in the berry skin at the site of infection, on recognition of the pathogen. Here it is able to limit the damage caused by the pathogen as well as preventing it from spreading. This gives the plant the opportunity to initiate its systemic acquired resistance thereby protecting the rest of the plant and preventing secondary infections. The fermentation of red wine on the grape skins allows for the extraction of resveratrol from the skin into the wine. Red wines therefore have a significantly higher concentration of resveratrol than white varieties, which contain little or no resveratrol at all. It is for this reason that the moderate consumption of wine, in particular red wine, is synonymous with a healthy lifestyle. The antioxidant and anti-inflammatory activities of resveratrol are important contributors to the cardiovascular benefits derived from the consumption of red wine. It now seems, however, that significant cardiovascular protection is derived from the synergistic action of resveratrol, the polyphenols and the alcohol in wine. With the wholesomeness of any food or beverage being of extreme importance, the aim of this project was to manipulate wine yeast to produce resveratrol during fermentation. This required the introduction of an entire metabolic pathway, by integrating plant genes into the yeast. Resveratrol synthase utilises three malonyl-CoA and one pcoumaroyl- CoA molecules to produce one molecule of resveratrol, Saccharomyces cerevisiae produces malonyl-CoA but no p-coumaroyl-CoA. Therefore, the following genes were obtained to enable yeast to produce p-coumaroyl-CoA: PAL, encoding phenylalanine ammonia-lyase to convert phenylalanine into cinnamic acid; C4H, encoding cinnamate-4- hydroxlyase to convert cinnamic acid into p-coumaric acid; and 4CL9 or 4CL216 encoding CoA-ligases to convert the p-coumaric acid into p-coumaroyl-CoA. To attain high-level expression, the genes were subcloned under the control of the phosphoglycerate kinase gene (PGK1) promoter and terminator. Due to integration problems with these expression cassettes and the fact that the yeast was able to consume p-coumaric acid, the 4CL9, 4CL216 and Vst1 (encoding resveratrol synthase) genes were subcloned under the control of the alcohol dehydrogenase (ADH2) and PGK1 promoters into episomal plasmids, respectively. A laboratory yeast strain containing both the Vst1 and 4CL9, or the Vst1 and 4CL216 genes was evaluated for its ability to utilise p-coumaric acid and produce resveratrol. Northem analysis confirmed that the Vst1, 4CL9 and 4CL216 genes were transcribed and over-expressed compared to the control strain. The transformants expressing the CoA-ligase genes utilised the p-coumaric acid faster than the control, although it was not possible to determine whether p-coumaroyl-CoA was produced. No resveratrol was produced under the assay conditions used. The results indicated that the yeast is unable to produce active resveratrol synthase, which is required to catalyse the final reaction in the production of resveratrol. Posttranslational modification, such as overglycosylation and disulphide formation, of the heterologous protein in yeast has been indicated as the possible reason for the lack of enzyme activity. This introduces an exciting area of research for the development of biotechnological tools with the ability to increase the production of active heterologous proteins in yeast. / AFRIKAANSE OPSOMMING: Wingerde word voortdurend deur 'n groot verskeidenheid patogene, insluitende virusse, bakteriee en swamme, aangeval. Ten einde oorlewing te verseker, het die wingerdstok In wye reeks verdedigingsmeganismes ontwikkel om weerstand te bied teen indringerorganismes. 'n Belangrike faktor in hierdie weerstand teen siektes is die produksie van fitoaleksiene, waarvan resveratrol die hoofkomponent is. Oeur die sintese van resveratrol, asook ander strukturele en biochemiese verdedigingsmeganismes, word die plant toegerus om weerstand te kan bied teen In hele aantal patogene ten einde gesonde druiwe te produseer wat gebruik kan word vir die vinifikasie van topgehalte wyn. As deel van die aktiewe reaksie teen siektes, word resveratrol de novo in die dop van die korrel by die plek van infeksie gesintetiseer sodra 'n patogeen herken word. Hier kan dit die skade deur die patogeen veroorsaak, beperk en verhoed dat dit versprei. Oit gee aan die plant die geleentheid om sy sistemies-verworwe weerstand te inisieer, en daardeur die res van die plant te beskerm, sowel as sekondere infeksies te verhoed. Die fermentasie van rooiwyn op die druifdoppe maak voorsiening vir die ekstraksie van resveratrol uit die dop na die wyn. Die konsentrasie van resveratrol in rooiwyn is dus beduidend hoer as in die wit varietelte, wat geen of baie min resveratrol bevat. Oit is dan juis die rede waarom die matige inname van wyn, veral rooi wyn, gesien word as In integrale deel van 'n gesonde leefwyse. Resveratrol se aktiwiteit as antioksidant en antiinflammatoriese middel lewer In belangrike bydrae tot die kardiovaskulere voordele wat verkry word uit die inname van rooiwyn. Oit blyk egter nou dat die beduidende kardiovaskulere beskerming gesetel is in die sinergistiese werking van resve ratro I, die polifenole en die alkohol in wyn. Aangesien die heilsaamheid van enige voedsel of drank van die uiterste belang is, was dit die doel van hierdie projek om wyngis te manipuleer ten einde tydens die fermentasieproses resveratrol te produseer. Hiervoor moes 'n volledige metaboliese pad daargestel word deur plantgene in die gis te inkorporeer. Resveratrol-sintase maak gebruik van drie maloniel-KoA-molekules en een p-kumarotel-Kos-molekule om een molekule resveratrol te produseer. Saccharomyces cerevisiae produseer maloniel-KoA, maar nie p-kumaroiel-Kcs, nie. Oie volgende gene is dus aangewend om die gis in staat te stel om p-kumarolel-Koe, te produseer: PAL, wat fenielalanien-ammoniak-liase enkodeer om fenielalanien om te sit na kaneelsuur; C4H, wat sinnamaat-4-hidroksliase enkodeer om kaneelsuur om te sit na p-kumaarsuur; en 4CL9 of 4CL216 wat KoA-ligases enkodeer om p-kumaarsuur om te sit na p-kumarolel-Kos, Om hoevlak-uitdrukking te verkry, is die gene gesubkloneer onder beheer van die fosfogliseraat-kinase-geen(PGK1)- promotor en -terminator. As gevolg van integrasieprobleme met hierdie uitdrukkingskassette en die feit dat die gis die p-kumaarsuur kon verteer, is die 4CL9-, 4CL216- en Vst1- (wat resveratrol-sintase enkodeer) gene na episomale plasmiede gesubkloneer onder beheer van die alkohol-dehidrogenase(ADH2)- en PGK1-promotors onderskeidelik. 'n Laboratorium-gisstam wat 6f beide die Vst1-geen en die 4CL9-geen, 6f die Vst1-geen en die 4CL216-geen bevat het, is geevalueer vir die verrnoe om pkumaarsuur te benut en resveratrol te produseer. Noordelike klad analises het bevestig dat die Vst1-, 4CL9- en 4CL216-gene getranskribeer en ooruitgedruk was in vergelyking met die kontrole-stam. Die transformante wat die KoA-ligases uitgedruk het, het die pkumaarsuur vinniger benut as wat die kontrole dit gedoen het, alhoewel dit nie moontlik was om vas te stel of o-kurnarotel-Kos, geproduseer is nie. Met die essai-kondisies wat gebruik is, is geen resveratroI geproduseer nie. Die resultate het daarop gedui dat die gis nie daartoe in staat is om aktiewe resveratrol-sintase, wat nodig is vir die katalise van die finale reaksie in die produksie van resveratrol, te produseer nie. Naomsettingsmodifikasies van die heteroloe protelen in die gis, soos oor-glikosilasie en disulfiedvorming, is aangewys as die moontlike rede vir die gebrek aan ensiemaktiwiteit. Dit stel In opwindende veld vir verdere navorsing voor, naamlik die ontwikkeling van biotegnologiese middele met die vermoe om die produksie van aktiewe heteroloe protelene in gis te verhoog.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The production of wine is a complex process, which involves the conversion of sugar in grape must to ethanol, carbon dioxide and other byproducts. The principal organism in winemaking is yeast, of which Saccharomyces cerevisiae is the most important due to its ability to survive winemaking conditions, its GRAS (Generally Regarded As Safe) status and the favourable flavours it imparts during the winemaking process. However, due to the demands of the consumer and the emergence of sophisticated wine markets, a demand is developing for specialised yeast strains with enhanced and new oenological properties. For these reasons, research into the contribution of wine yeast to the aroma bouquet as well the influence of wine or brandy maturation in wood on the aroma bouquet is important for consumer demands to be met. The fruity aroma of wine is associated with esters, which are produced during the alcoholic fermentation by yeast. Important acetate esters in wine and brandy are ethyl acetate, which has a fruity, solvent-like aroma, and isoamyl acetate, which has a banana-like aroma. These esters are produced through the action of acetyltransferases (AATases), which catalyse the reaction between a higher alcohol and acyl Coenzyme A. Esters are mainly a product of alcoholic fermentation. However, their concentration changes during wood maturation and it has been found that the concentration of acetate esters can increase during the maturation period. In this study, the aim was to investigate the influence of AATase I and AATase II, which are encoded by the ATF1 and ATF2 genes respectively, on the aroma bouquet of wine and brandy. Therefore, the first objective of this study was to clone the ATF2 gene from a commercial wine yeast strain and to overexpress this gene in a commercial wine yeast strain and in a wine yeast strain that already has the A TF1 gene overexpressed. Disruption cassettes were also designed in order to disrupt the ATF1 and ATF2 genes in a commercial wine yeast strain. The resultant recombinant wine yeast strains were used for the production of wine and brandy. GC analyses and tasting trials were conducted to determine the effect of the overexpression or disruption of these genes on the aroma bouquet of wine. The results obtained indicated that there are differences in the aroma bouquet of wine and brandy when changes are made in gene expression. The results indicated that the A TF1 gene plays a large role in the production of ethyl and isoamyl acetate. When this gene was overexpressed, the level of ethyl acetate was 5.6-fold more than that of the control and the level of isoamyl acetate was 3.5-fold higher than that of the control. However, no increase in ethyl acetate or isoamyl acetate was observed when the A TF2 gene was overexpressed. An increase in 2-phenylethyl acetate and diethyl succinate was observed in brandy, although there was a decrease in total ester concentration. A decrease in acetic acid was also observed in the brandy produced, which could be an indication of ester production. Similarly, no increase in ethyl acetate or isoamyl acetate was observed in the wine or brandy produced when both the ATF1 and ATF2 genes were overexpressed in a single yeast. Once again, a marked decrease was observed in acetic acid concentration in both the wine and brandy. In conclusion, it is clear that changes in gene expression can change the aroma profile of wine or brandy. However, the role of the ATF2 gene still remains unclear and further studies are needed to clarify its role in yeast. Future studies involving the effect of wood maturation on ester concentration will also be of importance, so that the winemaker or distiller can make a product that suits the ever-changing market. / AFRIKAANSE OPSOMMING: Die produksie van wyn is 'n komplekse proses wat die omskakeling van die suiker in mos tot etanol, koolstofdioksied en ander byprodukte tot gevolg het. Die hooforganisme betrokke in die wynmaakproses is gis, waarvan Saccharomyces cerevisiae as een van die belangrikste geag word as gevolg van die vermoë daarvan om onder die wynfermentasietoestande te kan oorleef, die "GRAS"-status (Generally Regarded As Safe) daarvan en die invloed wat dit op die aroma van die uiteindelike produk het weens die werking daarvan gedurende alkoholiese fermentasie. Die behoefte aan wyn met nuwe, verbeterde eienskappe het die vraag na meer gespesialiseerde gisrasse deur beide die verbruiker en nuwe wynmarkte gedurende die afgelope paar jaar drasties laat toeneem. Dit is om dié redes dat navorsing oor die bydrae van wyngis en houtveroudering tot die aroma van beide wyn en brandewyn so belangrik geag word. Die vrugtige aroma van wyn word geassosieer met die esters wat gedurende die alkoholiese fermentasie deur gis gevorm word. Die belangrikste asetaatesters in wyn en brandewyn is etielasetaat, wat vir 'n oplosmiddelagtige, vrugtige aroma bekend is, en isoamielasetaat, wat 'n piesangaroma veroorsaak. Die esters word geproduseer deur die werking van asetieltransferases (AATases), wat as katalis in die reaksie tussen 'n hoër alkohol en asetiel-Ko-ensiem A optree. Alhoewel esters hoofsaaklik 'n produk van alkoholiese fermentasie is, wissel die konsentrasie daarvan gedurende houtveroudering. Daar is gevind dat die konsentrasie van die asetaatesters gedurende die verouderingsproses kan verhoog. Die studie het ten doelom die invloed van AATase I en AATase II, wat onderskeidelik deur die ATF1- en ATF2-gene geënkodeer word, op die aroma van wyn en brandewyn te ondersoek. Die eerste doelwit van die studie was vervolgens om die ATF2-geen vanaf 'n kommersiële wyngisras te kloneer en dit daarna te ooruitdruk in 'n kommersiële wyngisras, asook die geen te ooruitdruk in 'n kommersiële wyngisras wat reeds die ATF1-geen ooruitdruk. Disrupsiekassette is ook vir die disrupsie van die ATF1- en ATF2-gene in 'n kommersiële wyngisras ontwerp. Die rekombinante wyngisrasse wat gedurnde die studie gemaak is, is vir die produksie van wyn en brandewyn gebruik. Gas chromatografise-ontledings en sensoriese evaluerings is ook op die wyn en brandewyn uitgevoer. Die resultate van die studie het bewys dat daar wel veranderings plaasvind wanneer 'n verandering in geenuitdrukking gemaak is. Die resultate het weereens bevestig dat die ATF1-geen 'n belangrike rol in die produksie van etiel- en isoamielasetaat speel. Wanneer die ATF1-geen ooruitgedruk is, is die etielasetaatproduksie 5.6 keer meer en die isoamielasetaatproduksie 3.5 keer meer as in die kontrole. Die ooruitdrukking van die ATF2-geen het geen verhoging in etielasetaat of isoamielasetaat of in totale esters in die wyn getoon nie, alhoewel die ras 2.7 keer meer diëtielsuksinaat geproduseer het. In die brandewyn wat geproduseer is met die gisras waarin ATF2 ooruitgedruk is, was daar wel 'n verlaging in die asynsuur, wat 'n aanduiding van estervorming kan wees, alhoewel die totale esters wat geproduseer is minder was as in die kontrole. 'n Verhoging in diëtielsuksinaat en 2-fenielasetaat is ook gevind. Daar is geen verhoging in etiel- of isoamielasetaat getoon wanneer die ATF1- en ATF2-geen saam ooruitgedruk is nie. Die ras het minder totale sure in wyn en brandewyn geproduseer en ook geen verhoging in totale esters getoon nie. Uit die resultate is dit duidelik dat veranderings in geenuitdrukking 'n verandering in die aromaprofiel van wyn en brandewyn kan veroorsaak. Die rol van dié A TF2-geen is nog steeds onduidelik en verdere studies sal moet plaasvind om die rol van die geen te verduidelik. Studies wat konsentreer op die invloed van houtveroudering op esterkonsentrasie is ook belangrik vir die toekoms, want dit sal die wyn- of brandewynmaker meer beheer oor die uiteindelike produk gee en daardeur die wyn- of brandewynmaker help om 'n produk te vervaardig wat sy mark bevredig.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The presence and growth of lactic acid bacteria (LAB) in wine and their influence on wine quality has received much attention in recent years. Lactic acid bacteria are responsible for conducting malolactic fermentation (MLF) in wine. The benefits associated with malolactic fermentation in terms of deacidification of wine and the contribution to wine flavour and complexity have also recently been the topic of research. It is impossible to describe malolactic fermentation as distinctly desirable or undesirable in terms of its influence on the final quality of wine. The benefits and disadvantages are dependent upon viticultural region, grape variety, wine composition, winemaking techniques and the style and objectives of the winemaker. Brandy production is a multi-stage process in which base wine production, distillation technique and wood maturation all have a large influence on the final chemical profile and organoleptic quality of the brandy. The volatile composition of the base wine, which basically undergoes a concentration process during the subsequent double distillation phase, is critical in determining the aroma and flavour quality of the final brandy product. Thus, the brandy is only as good as the base wine it is distilled from. The aims of this study were to determine the effect of lactic acid bacteria and spontaneous malolactic fermentation on the quality of brandy base wine and the resulting distillate, and to determine which LAB species had been responsible for the occurrence of spontaneous MLF. This study showed that LAB are present at high numbers and are able to conduct spontaneous MLF of brandy base wines. It was shown that the incidence of spontaneous MLF varied from year to year. In 1998, 50% of the commercially produced base wines had undergone partial MLF prior to distillation. In 1999 and 2000 respectively, 34% and 45% of the commercial base wines had undergone partial MLF prior to distillation. The occurrence of spontaneous MLF had an influence on the chemical composition and the sensory quality of the base wine and distillate. There was an increase in the concentrations of ethyl lactate, acetic acid and diethyl succinate in samples that had undergone MLF. There was also a decrease in the concentrations of esters, such as iso-amyl acetate, ethyl acetate, ethyl caproate, hexyl acetate and 2-phenethyl acetate in these same samples. Sensory evaluation of the base wines and distillates demonstrated that samples that had undergone MLF differed significantly from samples that had not undergone MLF. It was also shown that distillates that had not undergone MLF had a slightly better aroma profile than those that had. Sweet aromas, like chocolate and caramel, as well as negative aromas, like chemical or solvent, were more prominent in brandy distillates that had undergone MLF. Herbaceous and fruity aromas were more intense in distillates not having undergone MLF. Fifty-four strains, all Gram-positive and catalase negative, were isolated at different stages of brandy production. Seven strains were isolated from the grape juice, 15 strains were isolated from the base wine, 20 strains were isolated during MLF and 12 strains were isolated from the base wine after MLF had been completed. Based on C02 production from glucose and gluconate, 17 strains were classified as facultatively heterofermentative and 37 strains as obligately heterofermentative. Fifteen of the 37 obligately heterofermentative strains were rod-shaped and were regarded as lactobacilli. The remaining 22 strains were oval or cocci-bacilli shaped. The isolates were identified to species level by using numerical analysis of the total soluble cell protein patterns, 16S rRNAsequencing and polymerase chain reaction (PCR) with species-specific primers. The facultative heterofermentative lactobacilli were identified as Lactobacillus paracasei and Lactobacillus p/antarum. The fifteen obligately heterofermentative lactobacilli were identified as members of the species Lactobacillus brevis, Lactobacillus verrniforme, Lactobacillus buchneri and Lactobacillus hi/gardii. The 22 obligate heterofermentative isolates, with a coccoid morphology, could be grouped into two clusters and were identified as Oenococcus oeni. O. oeni was the species responsible for the occurrence of spontaneous MLF in most of the commercial base wines. Lb. brevis, Lb. hi/gardii and Lb. paracasei were also isolated from commercial base wines that had undergone spontaneous MLF. In nine out of 14 experimental base wine samples that had undergone spontaneous MLF, O. oeni was again the predominant species. Lb. brevis, Lb. hi/gardii and Lb. paracasei were identified in the remaining experimental base wine samples. This is the first report of the presence of Lb. perecese! and Lb. vermiforme in brandy base wine. It was shown that the occurrence of spontaneous MLF had a negative effect on the quality of brandy base wine, but that was shown to be due to the different species and strains performing MLF. In the non-preferred distillate samples, Lactobacillus spp. had performed MLF or had developed after or during MLF. / AFRIKAANSE OPSOMMING: Die teenwoordigheid en die vermoë van melksuurbakterieë (MSB) om in wyn te groei, is 'n onderwerp wat al heelwat nagevors is. Melksuurbakterieë is verantwoordelik vir die uitvoering van appelmelksuurgisting (AMG) in wyn. Die voordele verbonde aan appelmelksuurgisting, ten opsigte van die verlaging van die totale suurinhoud en die bydrae tot die verbeterde geur en kompleksiteit van die wyn, is ook al goed bestudeer. Wat die invloed op die finale wynkwaliteit betref, is dit byna onmoontlik om AMG as uitsluitlik gewens óf ongewens te beskou. Die voordele en nadele van AMG is afhanklik van verskeie faktore, nl. wingerdkundige streek, druifkultivar, wynsamestelling, wynmaakpraktyke, asook die styl en doelwitte van die wynmaker. Die produksie van brandewyn is 'n multistapproses waarin die bereidingsmetode van die basiswyn, die distillasietegniek en houtveroudering 'n groot invloed op die finale kwaliteit en chemiese samestelling van die brandewyn het. Die vlugtige verbindings van die basiswyn, wat tydens die dubbele distillasieproses gekonsentreer word, is van wesenlike belang in die bepaling van die aroma en geur van die finale brandewynproduk. Brandewyn is dus inderdaad net so goed soos die basiswyn waarvan dit gestook is. Die doelwitte van hierdie studie was om te bepaal wat die invloed van MSB en die voorkoms van spontane AMG op die kwaliteit van die basiswyn en die distillaat is, asook om die MSB wat vir die voorkoms van spontane AMG verantwoordelik was, te identifiseer. Hierdie studie het bewys dat MSB in hoë getalle teenwoordig was en dat dit in staat is om die spontane AMG van basiswyne uit te voer. Daar is bewys dat die voorkoms van spontaneAMG moontlik van jaar tot jaar kan verskil. In 1998 het 50%, in 1999 het 34% en in 2000 45% van die kommersieel-geproduseerde basiswyn gedeeltelike AMG spontaan voor distillasie ondergaan. Daar is ook gevind dat spontane AMG 'n invloed op die chemiese samestelling en sensoriese kwaliteit van die basiswyn en die distillaat gehad het. Daar was 'n toename in die konsentrasies van etiellaktaat, asynsuur en diëtielsuksinaat in monsters wat spontane AMG ondergaan het. In dieselfde monsters was daar ook 'n afname in die konsentrasies van iso-amielasetaat, etielasetaat, etielkaproaat, heksielasetaat en 2-fenielasetaat. Sensoriese evaluering van die basiswyne en distillate het getoon dat daar betekenisvolle verskille was tussen die monsters wat AMG ondergaan het en dié wat nie AMG ondergaan het nie. Daar is bewys dat die distillate wat nie AMG ondergaan het nie, 'n beter aromaprofiel gehad het as dié wat AMG ondergaan het. Soet geure, soos sjokolade en karamel, en negatiewe geure, soos "chemies" en "oplosmiddel", was prominent in distillate wat AMG ondergaan het. Kruidagtige en vrugtige geure was meer intensief in distillate wat nie AMG ondergaan het nie. Vier-en-vyftig bakteriese rasse, almal Gram-positief en katalase-negatief, is gedurende die verskillende stadia van brandewynproduksie geïsoleer. Sewe rasse is uit druiwesap, 15 rasse gedurende die alkoholiese fermentasie, 20 rasse gedurende AMG en 12 rasse na voltooiing van AMG geïsoleer. Op die basis van koolstofdioksied (C02)-produksie vanaf glukose en glukonaat is 17 rasse as fakultatief heterofermentatief en 37 rasse as obligaat heterofermentatief geklassifiseer. Vyftien van die 37 obligaat-heterofermentatiewe rasse was staafvormig en is as lactobacilli geïdentifiseer. Die oorblywende 22 het ovaal of kokkus-bacillusvormige selmorfologie getoon. Identifikasie tot op spesievlak is gedoen deur van numeriese analise van die totale oplosbare selproteïenprofiele, 16S-rRNAvolgordebepalings en spesie-spesifieke inleiers vir die polimerasekettingreaksie (PKR) gebruik te maak. Die fakultatief-heterofermentatiewe rasse is as Lactobacillus paracasei en Lactobacillus p/antarum geklassifiseer. Die 15 obligaat heterofermentatiewe stafies is as Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus hi/gardii en Lactobacillus vermiforme geïdentifiseer. Die 22 ovaal, obligaat heterofermentatiewe isolate kon in twee groepe ingedeel word en is as Oenococcus oeni geïdentifiseer. Daar is bevind dat O. oeni-isolate vir die voorkoms van spontane AMG in die meeste van die kommersiêle basiswyne verantwoordelik was. Lb. brevis, Lb. hi/gardii en Lb. paracasei is ook uit kommersiêle basiswyne wat spontane AMG ondergaan het, geïsoleer. In nege uit 14 van die eksperimentele basiswyne wat spontane AMG ondergaan het, was O. oeni die dominante spesie. In die oorblywende eksperimentele wyne is Lb. brevis, Lb. hi/gardii en Lb. paracasei aangetref. Hierdie is die eerste vermelding van die teenwoordigheid van Lb. paracasei and Lb. vermiforrne in brandewynbasiswyn. Daar is gevind dat die voorkoms van spontane AMG "n negatiewe invloed op brandewynkwaliteit het, maar dit is as gevolg van die verskeidenheid van MSB-spesies en rasse wat voorkom. In die distillate wat deur die proepaneel afgekeur is, het Lactobacillus spesies die AMG deurgevoer, of het dit tydens of na AMG ontwikkel.

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Plant carotenoid cleavage dioxygenases (CCD) are a family of enzymes that catalyse the oxidative cleavage of carotenoids and/or apocarotenoids. Carotenoids are synthesised in plastids (primarily chloroplasts and chromoplasts), where they are involved in light-harvesting and protecting the photosynthetic apparatus from photo-oxidation. The carotenoid-derived apocarotenoids fulfil a number of roles in plants such as phytohormones, pollinator attractants and flavour and aroma compounds. Due to the floral and fruity characteristics that apocarotenoids contribute to wine, these C13 compounds have received interest in grapevine (Vitis vinifera L.). The CCD gene family in Arabidopsis consists of nine members, all encoding for enzymes that catalyse the cleavage of carotenoids. The enzymes in this family include 9-cis-epoxydioxygenases (NCEDs) and four classes of CCD. NCEDs and CCD7 and CCD8 are involved with plant hormone synthesis, e.g. abscisic acid (ABA) through cleavage by NCED and strigolactone (SL) through the sequential cleavage of carotenoids by CCD7 and CCD8, respectively. SLs are a fairly new class of plant hormone which are involved in several aspects of plant growth and development. The most extensively characterised role of SLs is their involvement in the inhibition of shoot-branching. CCD1 and CCD4 cleave a variety of carotenoids to form pigments and aroma compounds. For example, CCD1 forms β-ionone and β-damascenone, which are important varietal flavours of wine, and CCD4 is involved in synthesis of the pigment and aroma compounds of saffron and annatto. CCD1 enzymes symmetrically cleave the 9,10 (9’,10’) double bonds of multiple carotenoids to produce a C14 dialdehyde and two C13 products. Additional CCD1 cleavage activity at 5,6 (5’,6’) double bonds of lycopene has been reported. Previous studies have shown that CCD1 isolated from V. vinifera (VvCCD1) was able to cleave multiple carotenoid substrates in vitro, namely zeaxanthin, lutein and β-carotene at 9,10 (9’,10’) double bonds and both the 5,6 (5’,6’) and 9,10 (9’,10’) double bonds of lycopene. None of the other VvCCDs, except VvCCD4a have been isolated (but no functionality was illustrated) and characterised yet. CCD4 enzymes also cleave carotenoids at the 9,10 (9’,10’) double bond positions. The presence of plastid-target peptides implies that the CCD4 enzymes have continuous access to carotenoids. Therefore it is suggested that CCD4s are responsible for carotenoid maintenance, where CCD1s contribute towards volatile production. To test this hypothesis VvCCD1, VvCCD4a and VvCCD4b were isolated from V. vinifera (cv Pinotage) cDNA and cloned into a pTWIN1 protein expression vector. Substrate specificity of each VvCCD was tested by co-transforming a carotenoid accumulating E. coli strain with a CCD expression vector. Carotenoids synthesized by the bacteria were identified and quantified by UPLC-analysis, while the concentration of the apocarotenoids, were measured in the headspace of the bacterial cultures using HS-SPME-GC-MS. Several optimisations were done to minimize the natural degradation of the carotenoids; to ensure that the apocarotenoid formation is predominantly due to the enzymatic cleavage by the VvCCDs and not due to oxidation or other non-enzymatic degradation. The HS-SPME-GC-MS analysis indicated that all isoforms cleaved phytoene, lycopene and ε-carotene. Additionally VvCCD1 cleaved a carotenoid involved in photosynthesis, namely β-carotene, while VvCCD4a cleaves neurosporene and VvCCD4b cleaves neurosporene and ζ-carotene, carotenoids not involved in photosynthesis. This study has illustrated that VvCCD1 cleave carotenoids necessary for photosynthesis and VvCCD4s cleave carotenoids which were not present in berry tissue, suggesting their role in carotenoid maintenance. Therefore in planta substrates for CCD1 could possibly be C27 apocarotenoids generated from enzymatic cleavage through CCD4 (role in carotenoid maintenance), CCD7 and/or photo-oxidation, which are then transported from the plastid to the cytosol or possibly C40 carotenoids that are released during senescence or when the plastid membrane is damaged, thus releasing important aroma compounds. Thus the identification of the in vivo substrates has contributed to the understanding the in planta functions of these enzymes / AFRIKAANSE OPSOMMING: Die plant ensiemfamilie van karotenoïedsplitsingdioksigenases (CCDs) kataliseer die oksidatiewe splitsing van karotenoïede en/of apokarotenoïede. Karotenoïede word in plastiede (primêr chloroplaste en chromoplaste) sintetiseer en is betrokke by lig-absorpsie en die beskerming van die fotosintetiese apparaat teen foto-oksidasie. Die apokarotenïede afkomstig van karotenoïede dien onder meer as planthormone, geur- en aromakomponente en om bestuiwers aan te lok. Aangesien apokarotenoïede bydra tot die vrug- en blomgeure van wyn is die C13-verbindings binne wingerd (Vitis vinifera L.) van belang. Al nege lede van die CCD geenfamilie in Arabidopsis kodeer karotenoïedsplitsingsensieme. Die ensiemfamilie sluit 9-sis-epoksidioksigenases (NCEDs), en vier klasse CCD in. NCEDs en CCD7 en 8 is betrokke by die sintese van planthormone, naamlik absissiensuur (ABA) deur NCED en strigolaktone (SL) deur die opeenvolgende aksie van onderskeidelik CCD7 en CCD8. SLe is redelik onlangs as planthormone indentifiseer en is betrokke by ‘n verskeie aspekte van die groei en ontwikkeling van plante. Die rol van SL in inhibisie van vertakking is die beste gekarakteriseerde van hierdie aspekte. CCD1 en CCD4 splits ‘n verskeidenheid karotenoïede om pigmente en aromakomponente te vorm. CCD1 vorm byvoorbeeld β-jonoon en β-damasenoon, beide belangrike kultivar-spesifieke wyngeure. CCD4 vorm weer die pigment en aromakomponente van saffraan en annatto. Die CCD1 ensieme splits die 9,10 (9’,10’) dubbelbindingsetels van verskeie karotenoïede simmetries en vorm een C14-dialdehied en twee C13-produkte. Daar is voorheen melding gemaak van verdere splitsing deur CCD1 by die 5,6 (5’,6’) dubbelbindingsetels van likopeen. Vroeër is getoon dat die CCD1 isovorm wat uit V. vinifera geïsoleer is, naamlik VvCCD1, in vitro seaxantin, luteïen en β-karoteen by die 9,10 (9’,10’) dubbelbindingsetels kon splits, en likopeen by beide die 9,10 (9’,10’) en 5,6 (5’,6’) dubbelbindingsetels. Geen ander VvCCDs is al isoleer en funksioneel gekarakteriseer. VvCCD4a is isoleer, maar geen funksie is bepaal nie. CCD4 ensieme splits ook die 9,10 (9’,10’) dubbelbindingsetels van karotenoïede. Aangesien CCD4 ensieme ‘n plastied-bestemmingspeptied besit behoort dié ensieme konstant toegang tot karotenoïede te hê, wat dui op hul rol in die handhawing van die karotenoïedbalans, terwyl CCD1-ensieme bydra tot die sintese van vlugtige verbindings. Om hierdie hipotese te toets is VvCCD1, VvCCD4a en VvCCD4b uit V. vinifera (kv Pinotage) kDNS isoleer in binne ‘n pTWIN1 proteïenuitdrukkingsvektor kloneer. Die substraatspesifisiteit van elke VvCCD is getoets deur ‘n karotenoïedakkumulerende E. coil stam te transvormeer met ‘n CCD-uitdrukkingsvektor. UPLC-analise is gebruik om karotenoïede wat deur die bakterium sintetiseer is te kwantifiseer en identifiseer, terwyl die apokarotenoïedinhoud en -konsentrasie van die boruimte van die bakteriële kultuur met HS-SPME-GC-MS bepaal is. Verskeie aspekte van die proses is optimaliseer om natuurlike afbreking van karotenoïede te minimeer. Daardeur is verseker dat die apokarotenoïedvorming primêr vanweë die ensiematiese splitsing deur VvCCDs plaasvind en nie deur oksidasie of ander nie-ensiematiese afbreking. Die HS-SPME-GC-MS metings het aangedui dat al drie isovorme fitoëen, likopeen en ε-karoteen kan splits. VvCCD1 kan daarby β-karoteen splits, terwyl VvCCD4a neurosporeen, en VvCCD4b neurosporeen en ζ-karoteen kan splits, beide karotene wat nie betrokke is by fotosintese nie. Dié studie toon dat VvCCD1 die karotenoïede splits wat benodig word vir fotosintese, terwyl beide VvCCD4 isovorme karotenoïede splits wat nie in druiwekorrels gevind word nie. Dit dui op hulle rol in die handhawing van karotenoïedpoele. Die in planta substrate vir CCD1 mag dus die C27-apokarotenoïede wees wat deur CCD4 (as deel van karotenoïedhandhawing), CCD7 en/of foto-oksidasie gevorm word en na die sitosol vervoer word, of moontlik die C40-karotenoïede wat tydens veroudering óf wanner die plastiedmembraan beskadig is in die sitosol vrygestel word. Die identifisering van die in vivo substrate het dus bygedra to die begrip van die in planta funksies van die ensieme.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: During wine fermentation, yeasts release extracellular enzymes that significantly impact wine properties. While the extracellular proteins of Saccharomyces cerevisiae have been characterised, those of non-Saccharomyces yeasts remain largely unknown. Most of these enzymes break down sugar polymers or catalyse the liberation of glycosidically-bound molecules. Another category of enzymes of oenological interest is represented by acid proteases that are able to prevent or reduce protein haze, as reported in literature, while simultaneously increasing the assimilable nitrogen content of wine. The liberation of amino acids from peptides and proteins that serve as aroma precursors may also have an indirect effect on wine aroma. In a recent study performed at the Institute for Wine Biotechnology (IWBT), the sequences of two aspartic proteases were retrieved from non-Saccharomyces yeast species isolated from South African wines. The genes, MpAPr1 and CaAPr1, were isolated from two non-Saccharomyces species, Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384, respectively. However, no further characterization was undertaken. This study aimed to clone these two genes into a recombinant bacterial host for expression and purify the corresponding enzymes as a first step toward characterizing their kinetic properties. Considering that some non-Saccharomyces species have been shown to produce more than one acid protease, an additional aim was to identify novel acid proteases within M. pulcherrima IWBT Y1123. Cloning of the genes and transformation of the expression vectors into E. coli were achieved. Optimal conditions for induced expression were established following extensive optimization. Furthermore, while native extraction of the recombinant proteins was unsuccessful, denaturing conditions allowed their recovery, suggesting that the recombinant proteins are encapsulated into inclusion bodies. Recombinant MpAPr1 was purified by using a nickel based column system and mass fingerprinting of the purified enzyme (MpAPr1) confirmed its identity. Purification was followed by refolding experiments, but yielded poor recovery of active enzymes. Unfortunately, recombinant expression of CaAPr1 could not be observed for reasons yet to be elucidated that may include the large sequence dissimilarities between CaAPr1 and MpAPr1. Finally, Southern blot analysis on the genomes of M. pulcherrima IWBT Y1123 and C. apicola IWBT Y1384 revealed that both possess at least one additional protease other than those previously described. Further analysis of the extracellular proteome of M. pulcherrima IWBT Y1123 also confirmed the presence of at least one enzyme able to hydrolyze BSA at a low pH. Unfortunately, mass fingerprinting performed on the entire extracellular proteome and on small groups of proteins thereof did not allow the identification of these enzymes. / AFRIKAANSE OPSOMMING: Gedurende fermentasie van druiwe sap skei gis ekstrasellulêre ensieme af wat ‘n aanmerklike impak op wyn eienskappe het. Terwyl die ekstrasellulêre proteïene vanaf Saccharomyces cerevisiae al gekarakteriseer is, bly die van nie-Saccharomyces spesies grootliks onbekend. Meeste van hierdie ensieme breek suiker polimere af of kataliseer die vrystelling van glikosiediese verbonde molekules. ‘n Ander klas van ensieme wat van belang is vir oenologie word voorgestel deur proteases wat in staat is daartoe om proteïenewaas te verminder, soos voorheen geraporteer is in literatuur, terwyl dit terselfde tyd die assimileerbare stikstof inhoud kan vermeerder. Die vrystelling van aminosure vanaf peptiede en/of proteïene wat as aroma voorlopers dien mag ook ‘n indirekte effek op die wyn se aroma profiel hê. In ‘n onlangse studie wat uitgevoer is by die Instituut vir Wynbiotegnologie (IWBT) was die volgordes van twee aspartiese proteases bepaal vanaf twee nie-Saccharomyces gis spesies wat geisoleer was uit Suid-Afrikaanse wyne. Die gene MpAPr1 en CaAPr1, was afsonderlik geisoleer vanuit twee nie- Saccharomyces giste, Metschnikowia pulcherrima IWBT Y1123 en Candida apicola IWBT Y1384. Egter was daar geen verder karakterisering van hierdie ensieme nie. Die doel van hierdie studie is om die bogenoemde gene in ‘n rekombinante bakteriese gasheer te kloneer vir uitdrukking en suiwering as ‘n eerste stap tot karakterisering van hul kinetiese eienskappe. Om in ag te neem dat sommige nie-Saccharomyces spesies meer as een protease produseer was ‘n aditionele mikpunt om vir nuwe suur proteases te soek binne M. pulcherrima IWBT Y1123. Klonering van hierdie gene en transformasie van die uitdrukkings vektore in E. coli was suksesvol. Optimale kondisies vir die induksie van ekspressie was bevestig na omvattende optimalisering. Verder, terwyl inheemse ekstraksie van die rekombinante proteïene onsuksesvol was, het denatureerende kondisies toegelaat vir suksesvolle ekstraksie, wat voorgestel het dat die rekombinante proteïene geinkapsileer word in inklusie liggame. Rekombinante MpAPr1 was gesuiwer deur gebruik te maak van ‘n niekel gebaseerde kolom sisteem en massa petied fingerafdrukke van die gesuiwerde ensiem (MpAPr1) het die identiteit bevestig. Suiwering was gevolg deur hervouing eksperimente, maar het swak opbrengste gelewer van die aktiewe ensiem. Ongelukkig kon die rekombinante ekspressie van CaAPr1 nie gevisualiseer word nie vir redes wat nog bevestig moet word, maar wat mag behels dat daar groot volgorde veskille tussen MpAPr1 en CaAPr1 kan wees. Uiteindelik was Southern blot hibridiseering analises uitgevoer op die genome van albei M. pulcherrima IWBT Y1123 en C. apicola IWBT Y1384 wat voorgestel het dat albei ten minste een addisionele protease, anders as die wat voorheen geidentifiseer was, bevat. Verder analiese van die ekstrasellulêre proteoom van M. pulcherrima IWBT Y1123 het ook die teenwoordigheid van ten minste een ensiem bevestig wat die vermoë het om BSA te hidroliseer by ‘n lae pH. Ongelukkig het massa peptied vingerafdrukbepaling wat uitgevoer was op die hele ekstrasellulêre proteoom en op klein groepe protein nie identifikasie van hierdie ensieme bevestig nie.