Thesis (M.Sc. (Chemistry)) -- University of Limpopo, 2022 / Calcium and magnesium are two dominant species that contribute to water hardness. The aim of this study was to develop a poly (vinylidene fluoride-co hexafluoropropylene) (PVDF-HFP) composite membrane for treatment of water hardness. The synthesis of PVDF-HFP composite membranes was confirmed by X ray diffraction (XRD), scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and Fourier transform infrared (FTIR) spectroscopy. The concentrations of the hardness causing agents in both the simulated and real hard water samples were investigated in batch studies wherein parameters such as pH, contact time, temperature, and adsorbent were optimised. The maximum adsorption efficiency of 56 and 45 mg/g (evaluated by Langmuir isotherm) for Ca(II) and Mg(II) ions were obtained. These were achieved at an optimum pH of 7 and adsorption dosage of 0.5 mg/L using the 3% PVDF-HFP/cellulose acetate (CA) and 1% nitrogen doped multiwalled carbon nanotubes (N-MWCNTs)/CA composite membranes respectively. The adsorption kinetics and isotherm models were all consistent with the pseudo-second order and Freundlich isotherm models for all the membranes suggesting that the sorption process met heterogeneous adsorption. Furthermore, the thermodynamic parameters indicated that the adsorption is physical and endothermic in nature. Reusability studies showed that all the PVDF-HFP based membranes can be recycled at least 3 times and for Ca(II) ions an adsorption loss of only 0.35 % was recorded while using a 3% PVDF-HFP/CA composite membrane. These results were further confirmed by XRD, TGA and inductively coupled plasma mass (ICP-MS) spectrometry. Thus, the findings from this study have shown that the PVDF-HFP based membranes could provide valuable material for hardness removal to acceptable level.

Contamination of peanuts by secondary metabolites of certain fungi, namely aflatoxins present a great health hazard when exposed either at low levels for prolonged times (carcinogenic) or at high levels at once (poisonous). It is important to develop an accurate and rapid measurement technique to trace the aflatoxin and/or source fungi presence in peanuts. Thus, current research focused on development of vibrational spectroscopy based methods for detection and separation of contaminated peanut samples. Aflatoxin incidence, as a chemical contaminant in peanut paste samples, was investigated, in terms of spectral characteristics using FTIR-ATR. The effects of spectral pre-processing steps such as mean-centering, smoothing the 1st derivative and normalizing were studied. Logarithmic method was the best normalization technique describing the exponentially distributed spectral data. Spectral windows giving the best correlation with respect to increasing aflatoxin amount led to selection of fat associated spectral bands. Using the multivariate analysis tools, structural contributions of aflatoxins in peanut matrix were detected. The best region was decided as 3028-2752, 1800-1707, 1584-1424, and 1408-1127 cm-1 giving correlation coefficient for calibration (R2C), root mean square error for calibration (RMSEC) and root mean square error for prediction (RMSEP) of 98.6%, 7.66ppb and 19.5ppb, respectively. Applying the constructed partial least squares model, 95% of the samples were correctly classified while the percentage of false negative and false positive identifications were 16% and 0%, respectively. Aspergillus species of section Flavi and the black fungi, A. niger are the most common colonists of peanuts in nature and the majority of the aflatoxin producing strains are from section Flavi. Seed colonization by selected Aspergillus spp. was investigated by following the chemical alterations as a function of fungal growth by means of spectral readouts. FTIR-ATR was utilized to correlate spectral characteristics to mold density, and to separate Aspergillus at section, species and strain levels, threshold mold density values were established. Even far before the organoleptic quality changes became visually observable (~10,000 mold counts), FTIR distinguished the species of same section. Besides, the analogous secondary metabolites produced increased the similarity within the spectra even their spectral contributions were mostly masked by bulk peanut medium; and led to grouping of species producing the same mycotoxins together. Aflatoxigenic and non-aflatoxigenic strains of A. flavus and A. parasiticus were further studied for measurement capability of FTIR-ATR system in discriminating the toxic streams from just moldy and clean samples. Owing to increased similarity within the collected spectral data due to aflatoxin presence, clean samples (having aflatoxin level lower than 20 ppb, n=44), only moldy samples (having aflatoxin level lower than 300 ppb, n=28) and toxic samples (having aflatoxin level between 300-1200 ppb, n=23) were separated into appropriate classes (with a 100% classification accuracy). Photoacoustic spectroscopy (PAS) is a non-invasive technique and offers many advantages over more traditional ATR system, specifically, for in-field measurements. Even though the sample throughput time is longer compared to ATR measurements, intact seeds can be directly loaded into sample compartment for analysis. Compared to ATR, PAS is more sensitive to high moisture in samples, which in our case was not a problem since peanuts have water content less than 10%. The spectral ranges between: 3600-2750, 1800-1480, 1200-900 cm-1 were assigned as the key bands and full separation between Aspergillus spp. infected and healthy peanuts was obtained. However, PAS was not sensitive as ATR either in species level classification of Aspergillus invasion or toxic-moldy level separation. When run for separation of aflatoxigenic versus non-aflatoxigenic batches of samples, 7 out of 54 contaminated samples were misclassified but all healthy peanuts were correctly identified (15 healthy/ 69 total peanut pods). This study explored the possibility of using vibrational spectroscopy as a tool to understand chemical changes in peanuts and peanut products to Aspergillus invasion or aflatoxin contamination. The overall results of current study proved the potential of FTIR, equipped with either ATR or PAS, in identification, quantification and classification at varying levels of mold density and aflatoxin concentration. These results can be used to develop quality control laboratory methods or in field sorting devices. / Ph. D.

In the electronics industry, several greenhouse gases (GHGs) are used as process gases in manufacturing processes. The organization RF360 as a Qualcomm Inc. subsidiary is using GHGs as input gases in the manufacturing processes dry etching, Chemical Vapor Deposition (CVD) and trimming in the fabrication plant in Munich.The estimation of GHG emissions from the use of process gases under Scope 1 requires a global and comprehensive approach to determine emission sources. This work provides the basis for the GHG emission estimation from process gas use under consideration of the 2019 Refinement to the 2006 IPCC Guidelines for National Greenhouse Gas Inventories. Dry etching and CVD process GHG emissions arecalculated using the Tier 2c method with process specific default emission factors. The process GHG emissions from trimming are characterized under Tier 3a, by determination of site-specific process emission factors. These emission factors are obtained from FTIR measurements in the inline. The measurement results show the input gas NF3 is largely not converted or destroyed in the trimming process. The total GHG emissions resulting from process gas use in the considered processes are determined by emissions of NF3, CF4 and N2O. The implementation and improvement of the approach requires further measurements of site-specific emission factors in the processes and Destruction Removal Efficiencies of the abatement systems.

Doctor of Philosophy / Department of Chemical Engineering / Keith L. Hohn / To characterize surface properties by current techniques, metal oxides typically have to be pre-treated at high temperature to remove surface absorbents. Therefore, a new low temperature method which can provide information on the surface chemistry is desired. In this work, the surface properties of metal oxide samples were studied by tetrakis(dimethylamino)ethylene (TDE) chemiluminescence (CL). This chemiluminescent method was also employed in probing the properties of reversed microemulsions. It was found that the emission intensity vs. reaction time curve (I[subscript]t) of catalyzed TDE CL on MgO was affected by the distributions and types of surface hydroxyl groups. Isolated hydroxyls with lower coordination were found to have higher catalytic reactivity for the emission of TDE CL. Although hydrogen bonded hydroxyls also catalyze the TDE oxidation reaction, the influence on the light emission was negative. Because the properties of surface hydroxyls are associated with specific orientations of adjacent ions, information on surface hydroxyls can provide information about some general surface characteristics of a metal oxide. When characterizing surface hydroxyls on Al[subscript]2O[subscript]3 by TDE CL, it was found that the catalytic reactivity of isolated hydroxyl groups is strongly associated with the stretching frequency of isolated hydroxyl. The stretching frequency of an isolated hydroxyl group is related to the modification of the adjacent ions and the coordination of the isolated hydroxyl. The results showed that the blue-shifts in the stretching frequencies of isolated hydroxyls led to increases in the catalytic reactivity of Al[subscript]2O[subscript]3 surfaces for the emission of TDE CL. TDE CL was further applied in characterizing the surfaces of other metal oxides and chemically grafted Al[subscript]2O[subscript]3. The results indicated that the isolated hydroxyl groups with fewer adjacent ions likely have higher affinity for the binding of grafting agents. Higher emission intensities were obtained from catalyzed TDE CL on metal oxides featuring higher percentages of isolated hydroxyls. The determination of a surfactant’s critical micellar concentration was accomplished by measuring the decay rate of the emission of TDE CL in a reversed microemulsion system. In this study, the CMC values of non-ionic and ionic surfactants were measured in different non-polar solvents.

Thesis (MScEng)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: The simplified Perturbed-Chain Statistical Associating Fluid Theory (sPC-SAFT) is characterised by the dual advantages of decreased computational intensity, while remaining accurate for a variety of systems. Vapour-liquid equilibrium data are used to generate equation of state parameters. However, incorporating monomer fraction data into the parameter regression has long been advocated as a good, or even preferred, practice. Therefore, the monomer fraction data of dilute alcohol-acetone systems were analysed in this study. A small stainless steel sample vessel was constructed with temperature control, manual pressure control and a mechanism for liquid phase analysis via infrared spectroscopy. The performance of the spectrometer was verified by comparison with the ethanol – n-hexane data of von Solms et al. (2007), after which new monomer fraction data were obtained for dilute solutions (between 0.01 and 1.5 mol%) of methanol, ethanol, 1-propanol and 2-propanol in acetone near 23 °C. For dilute alcohol-acetone systems it was found that the propanols had the highest monomer fractions, and methanol the lowest. With increasing alcohol concentration, the monomer fractions decreased exponentially to values of 0.4 and 0.1 for methanol and the other alcohols respectively. The excess availability of hydrogen bond acceptors in the mixtures explains the equivalency observed for ethanol, 1-propanol and 2-propanol. For dilute acetone-alcohol systems it was found that, especially for methanol and ethanol, there was a pronounced trend towards acetone monomer fractions of 1 at infinite dilution. For the acetone – 2-propanol system, a previously unrecorded monomer peak was observed and quantified. Acetone monomer fractions tended to decrease as alcohol chain-length increased, showing that acetone could more easily penetrate the hydrogen bond network of the solvent when the solvent-solvent bonds were weaker. Monomer fraction data were compared to predictions for the sPC-SAFT scheme and parameters combinations published in the literature. The experimental data were accurately modelled using modified association parameters such that the solute associates strongly (εAB≈103 κ≈1), while the solvent parameters were decreased (εAB≈102 κ≈10˗3) to give a weakened solvent association effect. The difficulty for the dilute solute in penetrating the solvent bonding network appeared to be similar to the hydrophobic effect. Two new association schemes were proposed for acetone, assigning a single (N) or two (2N) negative association sites to represent the oxygen valence electron pairs. These schemes showed relative success in modelling acetone as the solvent in the mixture, while not being able to predict acetone monomer fractions when acetone was the solute. For dilute acetone-alcohol systems, the data were best described using the 2B model for acetone, while the best choice of scheme for the alcohol varied from system to system. For dilute alcohol-acetone mixtures it was generally found that a 2B-N model (with modified association parameters) provided the best fit to those experimental data. Accurate modelling below 0.1 mol% was difficult to attain with average errors decreasing to the order of 10% when this area was excluded. In this highly dilute region, not one of the models could describe the rapid change in (monomer fraction) gradient sufficiently while simultaneously offering accurate predictions over the entire experimental range. / AFRIKAANSE OPSOMMING: Die sPC-SAFT of simplified Perturbed-Chain Statistical Associating Fluid Theory toestandsvergelykings word wyd gebruik as gevolg van sy goeie akkuraatheid vir ‘n wye reeks sisteme, ten spyte van verminderde berekeningsintensiteit. Die parameters vir dié toestandsvergelyk word afgelei van faseewewig data, maar monomeer fraksie data word voorgestel vir die verbetering van (veral) die assosiasie parameters. Ten opsigte hiervan, was alkohol-asetoon sisteme bestudeer en hul monomeer fraksies gemeet. ‘n Staal reaktor was ontwerp (met ‘n temperatuurbeheerstelsel sowel as drukbeheer) om vloeistof monsters voor te berei vir analise d.m.v. infrarooi-spektroskopie. Die akkuraatheid van die eksperimentele apparaat is bewys deur nabootsing van etanol – n-heksaan data van von Solms et al. (2007), waarna nuwe monomeer fraksie data gegenereer is vir verdunde mengsels (0.01 tot 1.5 mol%) van metanol, etanol, 1- en 2-propanol met asetoon by 23 °C. Metanol monomeer fraksies het eksponensieël afgeneem na 0.4, terwyl etanol en propanol fraksies afgeneem het na ‘n gemene waarde van ongeveer 0.1. Hierdie tendens word toegeskryf aan ‘n oormaat van toeganklike waterstofbindingontvangers in hierdie mengsels. Vir verdunde asetoon-alkohol sisteme is daar ‘n tendens, (veral vir verdunnings met metanol en etanol) vir die monomer fraksies om te neig na 1 by oneindige verdunning. ‘n Monomeer piek is ook waargeneem vir die asetoon – 2-propanol sisteem. Hierdie piek is nie voorheen gesien in ander studies nie en dit is ook die eerste keer wat sulke data gekwantifiseer is. Daar is bevind dat asetoon monomeer fraksies afneem soos alkohol kettinglengte toeneem. Die gegenereerde monomeer fraksie data word vergelyk met verskeie sPC-SAFT parameterstelle vanuit die literatuur. Oor die algemeen, is die beraamde fraksie veel hoër as die eksperimentele data wanneer die 2B/3B/2C skemas met ‘n nie-assosiërende asetoon molekuul gemodelleer word. Wanneer die 2B parameters van von Solms et al. (2004) gebruik word, toon die beraming ‘n drastiese onderskatting van die data. Om ‘n akkurate beraming van die monomeer fraksie data te kry, moet die assosiasie parameters van die opgeloste stof vermeerder word (met εAB≈103 κ≈1) terwyl die oplosmiddel s’n drasties verswak moet word (met εAB≈102 κ≈10-3). Hierdie patroon kan vergelyk word met die hidrofobiese effek waar die kragte binne die oplosmiddel ‘n netwerk vorm wat die opgeloste stof uitstoot. Twee nuwe assosiasie skemas word ook voorgestel vir asetoon waar onderskeidelik een (N) en twee (2N) negatiewe sones, wat die valenselektroonpare op die suurstofatoom voorstel, aan asetoon geheg word. Hierdie twee skemas het relatiewe sukses getoon in die modellering van verdunde alkohol-asetoon sisteme terwyl dit ‘n swak beskrywing van die verdunde asetoon-alkohol mengsels voorspel het. ‘n Gewysigde 2B asetoon skema gee ‘n goeie beskrywing van die eksperimentele data. In hierdie geval, is die keuse van alkohol skema minder belangrik, terwyl die waardes van die assosiasie parameters verminder moet word. Vir verdunde alkohol-asetoon mengels word daar bevind dat ‘n 2B-N model met nuwe assosiasie parameters die beste passing van die eksperimentele data gee. Daar was ook bevind dat die modelle se akkuraatheid drasties afneem (met fout vermeerdering in die orde van 10%) wanneer die konsentrasie van die opgeloste stof minder as 0.1 mol% is.

BACKGROUND:In a globalized word, prevention of infectious diseases is a major challenge. Rapid detection of viable virus particles in water and other environmental samples is essential to public health risk assessment, homeland security and environmental protection. Current virus detection methods, especially assessing viral infectivity, are complex and time-consuming, making point-of-care detection a challenge. Faster, more sensitive, highly specific methods are needed to quantify potentially hazardous viral pathogens and to determine if suspected materials contain viable viral particles. Fourier transform infrared (FTIR) spectroscopy combined with cellular-based sensing, may offer a precise way to detect specific viruses. This approach utilizes infrared light to monitor changes in molecular components of cells by tracking changes in absorbance patterns produced following virus infection. In this work poliovirus (PV1) was used to evaluate the utility of FTIR spectroscopy with cell culture for rapid detection of infective virus particles.RESULTS:Buffalo green monkey kidney (BGMK) cells infected with different virus titers were studied at 1 - 12 hours post-infection (h.p.i.). A partial least squares (PLS) regression method was used to analyze and model cellular responses to different infection titers and times post-infection. The model performs best at 8 h.p.i., resulting in an estimated root mean square error of cross validation (RMSECV) of 17 plaque forming units (PFU)/ml when using low titers of infection of 10 and 100 PFU/ml. Higher titers, from 103 to 106 PFU/ml, could also be reliably detected.CONCLUSIONS:This approach to poliovirus detection and quantification using FTIR spectroscopy and cell culture could potentially be extended to compare biochemical cell responses to infection with different viruses. This virus detection method could feasibly be adapted to an automated scheme for use in areas such as water safety monitoring and medical diagnostics.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The polysaccharides that are present in wine originate from the grapes, the fungi that grow on the grapes and from other microorganisms that come into contact with the must during winemaking. The grape-derived polysaccharides of most concern in winemaking are pectin, glucan and xylan that can be enzymatically degraded by pectinases, glucanases and xylanases, respectively. These are the main structural polysaccharides of the cell wall of the grape cell. Degradation of the cell walls will result in the separation and rupture of the grape cells, and cell wall-bound compounds will be released into the must. Treating the must with pectinase and macerating enzyme preparations can result in an increase in free-flow juice, an improvement in must clarification and filtration, and an increased extraction of phenols and tannins. The tannins that are extracted polymerise with anthocyanins in red wine during ageing, resulting in increased colour intensity and stability. Wine aroma is also influenced by enzyme treatment. The degradation of the cell wall contributes to the release of glycosidically-bound terpene or alcohol precursors from the berries. The hydrolysis of these precursors during fermentation can result in an improvement in aroma. It can thus be seen that it is possible to improve wine quality and processing by supplementing the endogenous enzymes that are present in the fermentation with commercial enzyme preparations. Commercial enzymes are typically crude fungal preparations. The majority of commercial pectinase and glucanase preparations are derived from Aspergillus and Trichoderma, respectively. Since the endogenous polysaccharase activity of Saccharomyces cerevisiae is very limited, the heterologous expression of specific polysaccharase genes in an industrial yeast strain can improve the winemaking process, resulting in a higher quality wine without the addition of expensive commercial enzyme preparations. Since only the desired enzymes are secreted by the recombinant strain, there will be no undesired sideactivities, which can be detrimental to wine quality. Several pectinase-, glucanaseand xylanase-encoding genes, cloned from a variety of organisms, have been expressed successfully in laboratory strains of S. cerevisiae. Attempts have also been made to construct industrial wine yeast strains that express these polysaccharase genes and secrete the encoded enzymes. Fermentation with some of these strains resulted in a decrease in total phenolics and turbidity, an increase in juice extraction, and alterations in the colour and aromatic profile of the resulting wines. In this study, four polysaccharide-degrading, recombinant wine yeast strains were constructed. The endo-β-1,4-xylanase gene, XYN2, and the endo-β-1,4-glucanase gene, end1, were previously cloned from the soft rot fungus Trichoderma reesei and the rumen bacterium Butyrivibrio fibrisolvens, respectively. These genes were subcloned into different expression cassettes which were used to construct the four integration plasmids. The recombinant plasmids contained the following gene cassettes: TEF1P-XYN2-ADH2T (plasmid pDLG29) ADH1P- MFα1S -end1-TRP5T (plasmid pDLG30) ADH1P-MFα1S-end1-TRP5T and ADH2P-XYN2-ADH2T (plasmid pDLG33), ADH1P-MFα1S-end1-TRP5T and YG100PXYN2- ADH2T (plasmid pDLG39). These four plasmids were then separately integrated into the ILV2 locus of the commercial wine yeast strain S. cerevisiae VIN13. Wine was made with the four strains constructed in this study, a pectolytic strain, VIN13[pPPK], a glucanase- and xylanase-secreting strain, VIN13[pEX], an untransformed VIN13 strain, and an untransformed strain with the addition of the commercial enzyme preparation Rapidase EX Colour. Microvinification experiments were carried out on Pinot noir, Ruby Cabernet and Muscat d’Alexandria wines. Fermentation with the polysaccharide-degrading strains resulted in significant improvements in juice extraction, colour intensity and stability, and in alterations in the aromatic profiles of the wines produced. Subject to the approval by the regulatory authorities and eventual consumer acceptance of the use of genetically modified organisms (GMOs) in fermented foods and beverages, it might be required that the GM status of the yeast that is used appears on the label. Currently, there is no robust technique available with which the use of GM yeast can be revealed in a finished wine because the yeast cells and their DNA are removed from or denatured in the wine during filtration and processing. One way with which the undeclared use of a GM yeast in winemaking could be exposed would be to compare the chemical profile of a suspect wine with that of non-GM wine. In order to explore this concept further, a secondary aim of this study was to investigate whether Fourier Transformation Infra Red (FT-IR) spectroscopy coupled with multivariate data analysis could distinguish between wines fermented with transgenic and non-transgenic yeast strains, or between wines fermented with different transgenic strains. The results showed that this method could be used to classify wines fermented with different yeast strains if fermentation with the strain resulted in a unique chemical profile in the resulting wine. This was a preliminary study and these findings were summarised as an addendum to the thesis. / AFRIKAANSE OPSOMMING: Die polisakkariede wat in wyn teenwoordig is, is afkomstig van die druiwe, die swamme wat op die druiwe groei en vanaf ander mikroörganismes wat tydens die wynmaakproses met die mos in aanraking kom. Die belangrikste druifpolisakkariede in wynbereiding is pektien, glukaan en xilaan, wat onderskeidelik deur pektinases, glukanases en xilanases afgebreek kan word. Hierdie is die vernaamste strukturele polisakkariede van ‘n druifsel se selwand. Die afbreking van die selwande veroorsaak dat die druifselle skei en skeur, met die gevolg dat die selwandgebonde verbindings in die mos vrygelaat word. Die behandeling van die mos met pektinase en versappingsensiempreparate kan tot ʼn toename in vry-afloopsap lei, sowel as ʼn verbetering in mosverheldering en -filtrasie en ʼn verhoogde ekstraksie van fenole en tanniene. Die tanniene wat geëkstraheer word, polimeriseer in rooiwyn tydens veroudering, en dit lei tot verhoogde kleurintensiteit en -stabiliteit. Wynaroma word ook deur ensiembehandeling beïnvloed. Die afbreking van die druifselwand dra by tot die vrylating van glikosidiesgebonde terpeen- en alkoholvoorlopers uit die korrels. Die hidrolise van hierdie voorlopers tydens gisting kan lei tot ʼn verbetering van die aroma. Dit is dus duidelik dat dit moontlik is om wynkwaliteit en wynbereiding te verbeter deur die endogene ensieme wat in die gisting teenwoordig is met kommersiële ensiempreparate te supplementeer. Kommersiële ensiempreparate is tipies ongesuiwerde swampreparate. Die meerderheid kommersiële pektinase- en glukanasepreparate word onderskeidelik vanaf Aspergillus en Trichoderma verkry. Aangesien die endogene polisakkaraseaktiwiteit van Saccharomyces cerevisiae baie beperk is, kan die heteroloë uitdrukking van spesifieke polisakkarase-gene in ʼn industriële gisras die wynbereidingsproses verbeter en lei tot ʼn hoër kwaliteit wyn sonder die byvoeging van duur kommersiële ensiempreparate. Omdat die verkose ensieme deur die rekombinante ras uitgeskei word, sal daar geen ongewenste newe-effekte teenwoordig wees wat ʼn nadelige effek op wynkwaliteit kan hê nie. Verskeie mikrobiese gene wat vir pektinases, glukanases en xilanases kodeer, is reeds voorheen uit ‘n wye verskeidenheid van organismes gekloneer en suksesvol in laboratoriumrasse van S. cerevisiae uitgedruk. Pogings is ook aangewend om industriële wyngisrasse te konstrueer wat hierdie polisakkarasegene uitdruk en hul enkodeerde ensieme uitskei. Gisting met sommige van hierdie rekombinante gisrasse het gelei tot ʼn afname in totale fenoliese verbindings en troebelheid, ʼn verhoging in sapekstraksie, en veranderings in die kleur en aromatiese profiel van die gevolglike wyne. In hierdie studie is vier polisakkaried-afbrekende, rekombinante wyngisrasse gekonstrueer. Die endo-β-1,4-xilanasegeen, XYN2, en die endo-β-1,4- glukanasegeen, end1, is voorheen reeds onderskeidelik vanaf die sagte vrotswam, Trichoderma reesei, en die rumenbakterium, Butyrivibrio fibrisolvens, gekloneer. Hierdie gene is in vier integrasieplasmiede in verskillende ekspressiekassette gesubkloneer. Die plasmiede het die volgende geenkassette bevat: TEF1P-XYN2- ADH2T (plasmied pDLG29) ADH1P- MFα1S -end1-TRP5T (plasmied pDLG30) ADH1PMFα1S- end1-TRP5T and ADH2P-XYN2-ADH2T (plasmied pDLG33), ADH1P-MFα1S end1-TRP5T and YG100P-XYN2-ADH2T (plasmied pDLG39). Hierdie vier plasmiede is toe afsonderlik in die ILV2-lokus van die kommersiële wyngisras, S. cerevisiae VIN 13, geïntegreer. Wyn is met hierdie vier gekonstrueerde gisrasse gemaak, die pektolitiese gisras, VIN13[pPPK], die glukanase- en xilanase-afskeidende gisras, VIN13[pEX], die ongetransformeerde VIN13-ras, en met ʼn ongetransformeerde VIN13 gis waarby die kommersiële ensiempreparaat, Rapidase EX Colour, bygevoeg is. Mikro-wynbereidingseksperimente is op Pinot noir-, Ruby Cabernet- en Muscat D’Alexandria wyne uitgevoer. Gisting met die polisakkaried-afbrekende gisrasse het gelei tot ʼn noemenswaardige verbetering in sapekstraksie, kleurintensiteit en kleurstabiliteit, asook in veranderinge in die aromatiese profiele van die geproduseerde wyne. Indien die gebruik van geneties gemodifiseerde organismes (GMOs) in gefermenteerde voedsel en drank deur die reguleringsowerhede goedgekeur en uiteindelik deur die verbruiker aanvaar sou word, sou dit vereis kon word dat die GMstatus van die wyngisgis op die etiket van die wynbottel aangebring word. Verpligte etikettering van GM-wyn sal metodes vereis waarmee die ‘nalentskap’ van GMgisselle in die finale produk geïdentifiseer en gemoniteer kan word. Tans is daar geen robuuste tegnieke beskikbaar waarmee die gebruik van GM-giste openbaar kan word nie, aangesien die gisselle en hul DNA tydens filtrasie en prosessering verwyder word. Een wyse waarop die onverklaarde gebruik van ‘n GM-gis in wynbereiding blootgestel sou kno word, is om die chemiese profiel van die verdagte wyn met dié van ‘n nie-GM-wyn te vergelyk. Ten einde hierdie konsep verder te ondersoek was ‘n sekondêre doelwit van hierdie studie om te bepaal of FT-IR (Fourier-transformasie-infrarooi) spektroskopie tesame met meervariante dataanalise gebruik kan word om te onderskei tussen wyne wat met transgeniese en nietransgeniese gisrasse gegis is, of tussen wyne wat met verskillende transgeniese rasse gegis is. Die resultate het aangedui dat hierdie metode gebruik kan word om wyne wat met verskillende gisrasse gegis is, te klassifiseer indien die betrokke gisras ʼn unieke chemiese profiel in die uiteindelike wyn veroorsaak het. Dit was egter ʼn voorlopige ondersoek en is as ʼn byvoegsel tot die tesis geskryf.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2006. / The production of premium quality wine is dependant on excellent management of the total wine production value chain. To achieve this we need rapid and reliable analytical tools. Over the last decade Fourier transform infrared (FT-IR) spectroscopy has made a significant contribution to wine research and in the last five years South African institutions have also exploited the use of this technology not only for research, but also in industrial cellars. The FT-IR apparatus is equipped with global calibrations and therefore we first investigated the validity of these for South African conditions. To achieve this new calibration sets for pH, titratable acidity and °Brix were made and compared to the global calibrations with statistical methods. Results obtained between the °Brix calibrations displayed high correlation and the global calibration can therefore be implemented. However, the new TA calibration was more accurate than the global calibration. Results were inconclusive for the new pH calibration sample set and therefore needs to be enlarged before it can be validated as the possibility of being more accurate exists. It was concluded that FT-IR spectroscopy in the simultaneous measurement for °Brix, pH and TA in grape must showed potential for accurate analysis and quality control purposes in an industrial cellar. Rapid analysis of these parameters will lead to higher throughput of grape must samples in the laboratory as well as adhering to good laboratory practices by validation. The importance of correct sample preparation in the laboratory was illustrated when using FT-IR spectroscopy for one-step analysis and adjustments to global calibrations. Results obtained showed that grape parameters such as °Brix, nitrogen content were not influenced by the two sample preparation methods (hand pressed vs. homogenised), but pH, TA, colour index, anthocyanins and polyphenols were influenced. Important key factors were identified in the quality control chain from vineyard to the cellar. Numerous grape loads had an increase in microbial populations after harvesting the vineyard and transport to the weighbridge. Transport is critical especially for the vineyards in the Lutzville area (had the highest yeast population), which are situated the furthest from the cellar. Sauvignon blanc had the highest acetic acid bacteria and lactic acid bacteria populations compared to the other cultivars. Gluconic acid, glycerol and arabitol was highly correlated to each other. High populations of acetic acid bacteria and lactic acid bacteria also had high levels of gluconic acid and 2,3-butanediol in the grape juice. Meso-inositol differed significantly between the vineyard and weighbridge and it had a high standard deviation compared to the mean value of all the samples between the vineyard and weighbridge. Temperature of grape loads delivered to the cellar ranged from 14 to 36ºC, which had a major impact on the grape quality and the resultant wine. It can be concluded for this study that management of the total value chain is of critical importance to ensure that A-grade grapes results in good quality wine that merits the effort of the grape producer.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2006. / The assessments of grape and wine quality are complex issues and the wine industry needs more objective analysis of grape and wine quality. The standard quality assessment protocol for grading grapes at most wine cellars in South Africa is based on viticultural practices and the determination of chemical parameters such as ºBrix, pH and titratable acidity (TA). Grape juice indices calculated by formulae such as ºBrix/pH, TA/pH, ºBrix/TA, ºBrix x (pH)2 have been used in the past but these approaches have had limited success. It was shown that the total anthocyanins and total phenolics of red grapes correlate with wine quality and provide additional objective measures of grape quality. Most methods for the quantification of total anthocyanins and total phenolics are complex and time consuming and therefore not easily implemented in the routine laboratory environment. Fourier transform infrared spectroscopy (FT-IR) is widely used in South African laboratories for the routine quantification of wine and grape parameters but the commercial calibration models supplied for the quantification of grape total anthocyanins and phenolics are not satisfactory. The focus of this study was to develop new FT-IR calibration models for the quantification of total anthocyanins and phenolics of grapes and to use the generated data during a preliminary evaluation of the implementation of these parameters as part of the grape quality control protocol at a commercial winery in South Africa. The potential of Fourier transform infrared spectroscopy (FT-IR) for the rapid quantification of total anthocyanins and total phenolics in red grapes was investigated and evaluated for prediction accuracy with independent validation sets. The design of calibration sets aimed at capturing most of the variation due to vintage (2004 and 2005), cultivar (Cabernet Sauvignon, Merlot, Pinotage, and Shiraz) and sugar concentration. Best prediction accuracies were obtained for calibration sets using grapes from a single vintage or cultivar or approximately the same sugar concentration. The highest prediction accuracies were obtained for total anthocyanins calibration sets of grapes with sugar concentrations ≥ 23.5ºBrix (SEP = 0.13 mg/g; R2 validation set = 0.77) and for total phenolics calibration sets of grapes with sugar concentrations < 23.5ºBrix (SEP = 0.13 OD280/g; R2 validation set = 0.74). Strong correlations were found between the spectral data and the total anthocyanins (SEP = 0.12 mg/g; R2 validation set = 0.84) and total phenolics concentration data (SEP = 0.10 OD280/g; R2 validation set = 0.76) for 2005 Merlot calibration sets indicating that the FT-IR spectra captured most of the variation. Overall the RPD (ratio of the standard deviation of the reference data to the standard error of prediction) values of all calibration models were below 3 indicating that calibration models are fit for screening purposes. Spectroscopic absorbance at 280 nm is not specific enough for the quantification of total phenolics and the use of an alternative reference method such as high performance liquid chromatography (HPLC) will be considered in the future. Principal component analysis (PCA) revealed that the major sources of variation in the FT-IR spectra of grapes could be ascribed to vintage and grape sugar concentration and this had an effect on the accuracy of the analytical data generated when using FT-IR spectroscopy. This report is the first to our knowledge where FT-IR has been used for the quantification of total anthocyanins and phenolics of grapes. The evaluation of the reference laboratory protocol for the quantification of total anthocyanins and total phenolics in grapes were evaluated in Chapter 4 and emphasized the importance of meticulous laboratory practices to obtain reliable reference data for calibration purposes. This large scale investigation of the total anthocyanins and phenolics concentrations in grapes is the first of its kind in South Africa and a quantitative database containing analytical data of the anthocyanins and total phenolics concentrations of 692 grape samples representing a wide range of grape maturities of Vitis vinifera cultivars Cabernet Sauvignon, Merlot, Pinotage and Shiraz from the 2004 and 2005 vintages was established based on the reference values. The data were used in a preliminary investigation of the implementation of total anthocyanins and total phenolics concentrations as part of grape quality classification at a commercial South African winery (Chapter 5). The results showed that the total anthocyanins and total phenolics concentration in grapes increased with increasing grape maturity (measured as sugar concentration). ANOVA and post-hoc analysis (Bonferroni testing) revealed significant differences between the total anthocyanins and total phenolics concentrations of the four Vitis vinifera cultivars investigated. Grapes harvested earlier in the season had significantly higher (p≤0.05) total anthocyanins and total phenolics concentrations than grapes harvested later in the season. This implies that grapes harvested earlier in the harvest season could produce wines with higher quality. Grapes from regularly irrigated vineyards had lower total anthocyanins and total phenolics concentrations compared to dryland vineyards. The current grape grading system in use at the industrial cellar did not correlate well with the total anthocyanins and total phenolics concentrations of grapes which highlighted the need for the inclusion of more objective measures during grape grading. The information captured in the database can be used as a starting point to establish profiles of the typical anthocyanins and total phenolics of South African grapes and data from more vintages should be included and continually updated. These findings highlight the important contribution of the results obtained in this preliminary study for the incorporation of total anthocyanins and phenolics concentrations as objective parameters of grape quality. Finally multivariate data analysis of the FT-IR spectra revealed important information regarding factors (both physical and chemical) that contribute to the variation of the spectra. The main variation between the 2004 and 2005 samples can probably be interpreted in terms of the water content of the samples.

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The WineScan FT120 is widely used in wine laboratories across South Africa. The WineScan FT120 uses Fourier transform infrared (FT-IR) spectroscopy with multivariate data analysis to correlate spectra with chemical compositional data. Ready-to-use, commercially available calibration models for a FT-IR spectroscopy instrument are an advantage for unskilled users and routine analysis. Introducing spirit products to this technology introduced new interferences, which necessitated vastly different calibrations models to compensate for the changes. Accuracy, precision and ruggedness of the reference methods validated during method validation, verified the suitability of the reference methods used to quantify the parameters in question before calibration model building was attempted. Various principal component analysis (PCA) were performed prior to the calibration step with the aim to identify outliers and inspect groupings. PCA models could identify samples with atypical spectra and differentiate between product types. Two tactics regarding data sets for calibration set-up was experimented with, all the products together and calibration models per product. Partial least squares (PLS) regression was used to establish the calibration models for ethanol, density, obscuration and colour. With all the calibration models, the calibration models based on the product specific data sets, achieved better predicting statistics. The best performing ethanol calibration models achieved Residual mean square error of prediction (RMSEP) = 0.038 to 0.106 %v/v and showed significant improvement on previously reported prediction errors by Lachenmeier (2007). The results for the density calibration showed a similar trend, with the product specific calibration models outperforming the calibration model when all samples were included into one calibration model. This study produced novel results for quantification of obscuration (RMSEP = 0.10 and 0.09 in blended brandies and potstill brandies, respectively) and colour (RMSEP < 2.286 gold units) of brandies and whiskies. The correlation coefficients (R²) between true and predicted values, for the four parameters tested, indicated good to excellent precision (0.8 < R² < 1.0). Minimising the variation between the samples of the data set, gave more accurate regression statistics, but this resulted in a lower residual predictive deviation (RPD) value (< 5) that indicated models were not suitable for quantification. Adding more samples per product will add more variability into a data set per product, increase the SD and result in an increase in the RPD. The results pave the way for the development of calibration models for the quantification of other parameters for specific products. Following the groupings of product types, further classifications of brandy brands were investigated. PCA plots showed clear separation between potstill brandies and blended brandies and some degree of clustering between some of the blended brands was observed. Classification of brandies were investigated using the Soft Independent Modeling of Class Analogy (SIMCA) approach resulting in a total correct classification rates between 81.25% and 100% for the various brandy brands. These preliminary results were very promising and highlight the potential of using FT-IR spectroscopy and multivariate classification techniques as a tool for rapid quality control and authentication of brandy brands. Using this work as base for further classification projects, this could be of great benefit to the alcoholic beverage industry of South Africa. Future work will involve the development of a database comprised of more products guaranteed authentic to expand the discriminating options. The results suggest FT-IR spectroscopy could be useful in authentication studies. / AFRIKAANSE OPSOMMING: Die WineScan FT120 is ‘n algemeen gebruikte instrument regoor Suid-Afrika. Die WineScan FT120 gebruik Fourier-transformasie-infrarooi (FT-IR) spektroskopie tesame met multiveranderlike statistiese metodes om spektra te korreleer met chemiese samestellingsdata. Die kommersieël beskikbare kalibrasiemodelle vir die FT-IR spektroskopie-instrument is ‘n voordeel vir onbedrewe gebruikers en roetine ontleding. Blootstelling van spiritusprodukte aan die tegnologie, het nuwe hindernisse bekend gestel en dus is verskillende kalibrasiemodelle genoodsaak om hiervoor te kompenseer. Akkuraatheid, presiesheid en ruheid van die verwysingsmetodes is geëvalueer tydens metodevalidasie. Die verwysingsmetodes is geskik verklaar vir die konstruksie van die kalibrasiemodel met geverifieërde akkurate verwysingsresultate. Verskeie multiveranderlike hoofkomponentanalise (MVK) was uitgevoer voor die kalibrasiestap met die doel om uitskieters te identifiseer en groeperings te inspekteer. MVK modelle kon monsters met atipiese spektra identifiseer en onderskei tussen verskillende produk tipes. Twee taktieke aangaande datastelsamestelling is getoets tydens kalibrasiemodel-opstelling, al die produkte saam en kalibrasiemodelle per produk soos met die MVK aangedui. Parsiële kleinste kwadraat (PKK)- regressie is gebruik vir die opstel van die kalibrasiemodelle vir etanol, digtheid, obskurasie en kleur. Met al die kalibrasiemodelle het die produk spesifieke kalibrasiemodelle beter regressiestatistiek gelewer. Die beste presterende etanol kalibrasiemodelle het ‘n standaardvoorspellingsfout (SVF) = 0.038 tot 0.106 %v/v bereik en het ‘n beduidende verbetering getoon op vorige gerapporteerde studies op spiritusprodukte (Lachenmeier, 2007). Die resultate vir die digtheidskalibrasiemodelle het ‘n eenderse tendens getoon soos die etanol, met die produk spesifieke kalibrasiemodelle wat beter presteer het. Hierdie studie was eerste in sy soort met die kalibrasiemodel vir obskurasie (SVF = 0.10 en 0.09 in gemengde brandewyne en potketel brandewyne, onderskeidelik) en kleur (SVF < 2.286 goud eenhede) van brandewyne en whiskies. Die bepalingskoëffisiënt (R²) vir die vier parameters, dui op goeie tot uitstekende presiesheid (0.8 < R² < 1.0). Vermindering van die variasie tussen die monsters in die datastel, het meer akkurate regressiestatistiek teweeg gebring, maar ‘n laer relatiewe voorspellingsafwyking (RVA) waarde (<5) tot gevolg gehad wat aan dui dat hierdie modelle nie geskik is vir sifting of kwantifisering nie. Die byvoeging van meer monsters per produk sal meer verskeidenheid in die datastel per produk bring, wat dan die standaardafwyking sal laat toeneem en uiteindelik die RVA laat toeneem. Die resultate het die fondasie gelê vir die ontwikkeling van kalibrasiemodelle vir die kwantifisering van ander parameters vir spesifieke produkte. As opvolg tot die groeperings van die produk tipe, waargeneem in die MVK modelle, was klassifikasie van brandewyn handelsmerke ondersoek. MVK modelle het duidelike skeiding gewys tussen potketel en gemengde brandewyne en tot ‘n sekere mate groepering tussen handelsmerke. Klassifikasie van brandewyne was ondersoek met behulp van the Soft Independent Modeling of Class Analogy (SIMCA) met die resultaat van ‘n totale korrekte klassifikasiekoers van tussen 81.25% en 100% vir die verskeie brandewyn handelsmerke. Hierdie voorlopige resultate toon belowend en beklemtoon die potensiaal van FT-IR spektroskopie en chemometrics tegnieke as toerusting vir die vinnige kwaliteitskontrole en egtheid van brandewyn handelsmerke studies. Met hierdie werk as basis vir verdere klassifikasie projekte, kan dit ‘n groot aanwins wees tot die alkoholiese drank industrie van Suid-Afrika. Toekomstige werk sal insluit die ontwikkeling van ‘n databasis saamgestel met meer gewaarborgde egte produkte om die klassifikasie uit te brei.