Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Fermentation is a complex process in which raw materials are transformed into high-value products, in this case, grape juice into wine. In this modern and economically competitive society, it is increasingly important to consistently produce wine to definable specifications and styles. Process management throughout the production stage is therefore crucial to achieve effective control over the process and consistent wine quality. Problematic wine fermentations directly impact on cellar productivity and the quality of wine. Anticipating stuck or sluggish fermentations, or simply being able to foresee the progress of a given fermentation, would be extremely useful for an enologist or winemaker, who could then take suitable corrective steps where necessary, and ensure that vinifications conclude successfully. Conventional methods of fermentation monitoring are time consuming, sometimes unreliable, and the information limited to a few parameters only. The current effectiveness of fermentation monitoring in industrial wine production can be much improved. Winemakers currently lack the tools to identify early signs of undesirable fermentation behaviour and to take preventive actions. This study investigated the application of Fourier transform mid infrared (FT-IR) spectroscopy in transmission mode, for the quantitative and qualitative monitoring of alcoholic fermentation during industrial wine production. The major research objectives were firstly to establish a portfolio of quantitative calibration models suitable for quantification of the major quality determining parameters in fermenting must. The second major research objective focused on a pilot study aimed at exploring the use of off-line batch multivariate statistical process control (MSPC) charts for actively fermenting must. This approach used FT-IR spectra only, for the purpose of qualitative monitoring of alcoholic fermentation in industrial wine production. Towards these objectives, a total of 284 industrial-scale, individual, actively fermenting tanks of the seven major white cultivars and blends, and nine major red cultivars, of Namaqua Wines, Vredendal, South Africa, were sampled and analysed with FT-IR spectroscopy and appropriate reference methods during vintages 2007 to 2009. For the quantitative strategy, partial least squares regression (PLS1) calibration models for determination of the classic wine parameters ethanol, pH, volatile acidity (VA), titratable acidity (TA) and the total content of glucose plus fructose, were redeveloped to provide a better fit to local South African samples. New PLS1 models were developed for the must components glucose, fructose and yeast assimilable nitrogen (YAN), all of which are frequently implicated in problem fermentations. The regression statistics, that included the standard error of prediction (SEP), coefficient of determination (R2) and bias, were used to evaluate the performance of the redeveloped calibration models on local South African samples. Ethanol (SEP = 0.15 %v/v, R2 = 0.999, bias = 0.04 %v/v) showed very good prediction and with a residual predictive deviation (RPD) of 30, rendered an excellent model for quantitative purposes in fermenting must. The models for pH (SEP = 0.04, R2 = 0.923, bias = -0.01) and VA (SEP = 0.07 g/L, R2 = 0.894, bias = -0.01 g/L) with RPD values of 4 and 3 respectively, showed that the models were suitable for screening purposes. The calibration model for TA (SEP = 0.35 g/L, R2 = 0.797, bias = -0.004 g/L) with a RPD of 2, proved unsatisfactory for quantification purposes, but reasonable for screening purposes. The calibration model for the total content of glucose plus fructose (SEP = 0.6.19 g/L, R2 = 0.993, bias = 0.02 g/L) with a RPD of 13, showed very good prediction and can be used to quantify total glucose plus fructose content in fermenting must. The newly developed calibration models for glucose (SEP = 4.88 g/L, R2 = 0.985, bias = -0.31 g/L) and fructose (SEP = 4.14 g/L, R2 = 0.989, bias = 0.64 g/L) with RPD values of 8 and 10 respectively, also proved fit for quantification of these important parameters. The new calibration models of ethanol, total glucose plus fructose; and glucose and fructose individually, showed an excellent relation to local South African samples and can be easily implemented by the wider wine industry. Two calibration models were developed to determine YAN in fermenting must by using different reference methods, namely the enzyme-linked spectrophotometric assay and Formol titration method, respectively. The results showed that enzyme-linked assays provided a good quantitative model for white fermenting must (SEP = 14.10 mg/L, R2 = 0.909, bias = -2.55 mg/L, RPD = 6), but the regression statistics for predicting YAN in red fermenting must, were less satisfactory (data not shown). The Formol titration method could be used successfully in both red- and white fermenting must (SEP = 16.37 mg/L, R2 = 0.912, bias = -1.01 mg/L, RPD = 4). A minor, but very important finding was made with respect to the storage of must samples that were taken from tanks, but that could not immediately be analysed with FT-IR spectroscopy or reference values. Principal component analysis (PCA) of frozen samples showed that must samples could be stored frozen for up to 3 months and still be used to expand the calibration sample sets when needed. Therefore, samples can be kept frozen to a later stage if immediate analyses are not possible. For the purpose of the pilot study that focused on the use of FT-IR spectroscopy for qualitative off-line monitoring of alcoholic fermentation, a total of 21 industrial-scale fermentation tanks were monitored at 8- or 12-hourly intervals, from the onset of fermentation to complete consumption of the grape sugars. This part of the work excluded quantitative data, and only used FT-IR spectra. MSPC charts were constructed on the PLS scores of all the FT-IR spectra taken at the various time intervals of the different batches, using time as the y-variable. The primary aim of this research objective was to evaluate if the PLS batch models could be used to discriminate between normal and problem alcoholic fermentations. The models that were constructed clearly showed the variations in patterns over time, between red- and white wine alcoholic fermentations. One Colombar tank that was fermented at very low temperature in order to achieve a specific wine style, was characterised by a fermentation pattern that clearly differed form the rest of the Colombar fermentations. This atypical fermentation was identified by the batch models constructed in this study. PLS batch models over all the Colombar fermentations clearly identified the normal and problem fermentations. The results obtained in this study showed that FT-IR spectroscopy showed great potential for effective quantitative and qualitative monitoring of alcoholic fermentation during industrial wine production. The work done in this project resulted in the development of a portfolio of calibration models for the most important quality determining parameters in fermenting must. The quantitative models were subjected to extensive independent test set validation, and have subsequently been implemented for industrial use at Namaqua Wines. Multivariate batch monitoring models were established that show good discriminatory power to detect problem fermentations. This is a very useful diagnostic tool that can be further developed by monitoring more normal and problem fermentations. Future work in this regard, will focus on further optimisation and expansion of the quantitative and qualitative calibration models and implementation of these in the respective wineries of Namaqua Wines. / AFRIKAANSE OPSOMMING: Fermentasie is ‘n komplekse proses waartydens rou material getransformeer word na produkte van hoë waarde, in hierdie geval, druiwesap na wyn. In die huidige ekonomies-kompeterende samelewing, is dit al hoe meer belangrik om volhoubaar wyn te produseer wat voldoen aan definieerbare spesifikasies en style. Goeie prosesbestuur tydens die wynproduksie stadium is baie belangrik om herhaalbaarheid en gehaltebeheer te verseker. Problematiese wynfermentasies het ’n direkte impak op beide kelderproduktiwiteit en wynkwaliteit. Die voorkoming van slepende- of steekfermentasies, of selfs net om probleme te voorsien, sou uiters bruikbaar wees vir ‘n wynkundige of wynmaker, wat dan die toepaslike regstellende stappe kan neem waar nodig, om te verseker dat die wynbereiding suksesvol voltooi word. Konvensionele metodes van monitering van alkoholiese fermentasie is tydrowend, soms onbetroubaar en die inligting beperk tot ‘n paar parameters. Die huidige effektiwiteit van fermentasie monitering in industriële wynproduksie kan heelwat verbeter word. Wynmakers ervaar tans ’n behoete aan tegnologië wat die vroeë tekens van ongunstige fermentasiepatrone kan identifiseer, en hul doeltreffendheid om moontlike regstellende aksies te neem, is dus beperk. Hierdie studie het die toepassing van Fourier transformasie mid-infrarooi (FT-IR) spektroskopie in transmissie, ondersoek met die oog op kwantitatiewe en kwalitatiewe monitering van alkoholiese gisting tydens industriële wynproduksie. Die vernaamste navorsingsdoelwitte was eerstens om ’n portefeulje van kwantitatiewe kalibrasiemodelle te vestig, wat geskik is om die belangrikste kwaliteitsbepalende parameters in gistende mos te kwantifiseer. Die tweede hoofnavorsingsdoelwit was ’n loodsstudie wat ondersoek ingestel het na die opstel van multiveranderlike statistiese proseskontrole grafieke van aktief-gistende mos, met die oog op aflyn-kwalitatiewe monitering van alkoholiese gisting in industriële wynproduksie. Hiervoor is slegs FT-IR spektra gebruik. Vir die doel van hierdie studie is monsters van ’n totaal van 284 individuele, aktief-gistende tenke van die sewe hoof wit kultivars en hul versnydings en nege hoof rooi kultivars van Namaqua Wyne, Vredendal, Suid Afrika, geneem. Al die monsters is met toepaslike chemiese metodes en FT-IR spektroskopie analiseer tydens die parsseisoene van 2007 tot 2009. Vir die kwantitatiewe strategie is parsiële kleinste kwadraat (PKK1) kalibrasiemodelle vir die bepaling van die klassieke wynparameters etanol, pH, vlugtige suur (VS), titreerbare suur (TS) en die totale konsentrasie van glukose plus fruktose herontwikkel, om beter te pas op plaaslike Suid-Afrikaanse monsters. Nuwe PKK1 kalibrasiemodelle is ontwikkel vir die komponente glukose, fruktose en gis-assimileerbare stikstof, aangesien hierdie komponente gereelde aanduidings van probleemgisting is. Die regressiestatistieke het die standaardvoorspellingsfout (SVF), bepalingskoëffisiënt (R2) en sydigheid ingesluit en was gebruik om die prestasie van die herontwikkelde kalibrasiemodelle vir plaaslike Suid-Afrikaanse monsters te evalueer. Etanol (SVF = 0.15 %v/v, R2 = 0.999, sydigheid = 0.04 %v/v) het baie goeie regressiestatistiek getoon en met ‘n relatiewe voorspellingsafwyking (RVA) van 30, was dit ‘n uitstekende model vir kwantifisering in gistende mos. Die modelle vir pH en VS met RVA waardes van 4 en 3 onderskeidelik, is geskik vir semi-kwantitatiewe toepassings. Die kalibrasiemodel vir TS met ‘n RVA waarde van 2, was nie geskik vir akkurate kwantifisering nie, maar wel vir semikwantitatiewe analises. Die kalibrasiemodel vir die totale glukose plus fruktose inhoud in gistende mos, met ‘n RVA waarde van 13, het uitstekende regressiestatistiek gegee en is geskik vir akkurate kwantifiseringsdoeleindes. Die nuut-ontwikkelde kalibrasiemodelle vir glukose en fruktose, met RVA waardes van onderskeidelik 8 en 10, is geskik vir akkurate kwantifisering van hierdie belangrike parameters. Die kalibrasiemodelle vir etanol, totale glukose plus fruktose, en glukose en fruktose afsonderlik, het uitstekende korrelasies getoon met plaaslike Suid-Afrikaanse monsters en is gereed om toepassing te vind in die wyer wynindustrie. Twee kalibrasiemodelle is ontwikkel om gis-assimileerbare stikstof in gistende mos te bepaal, deur gebruik te maak van verskillende verwysingsmetodes van analise; hierdie metodes was ‘n ensiem-gekoppelde spektrofotometriese toets en die Formoltitrasie metode. Resultate het getoon dat goeie regressiestatistiek vir FT-IR spektroskopie-gebaseerde kalibrasiemodelle waar data wat met die ensiem-gekoppelde toetse verkry is, as verwysingwaardes gebruik is, in wit gistende mos (SVP = 14.10 mg/L, R2 = 0.909, sydigheid = -2.55 mg/L, RVA = 6), maar nie in rooi gistende mos nie. Die Formoltitrasie metode as verwysingsmetode, was geskik vir die ontwikkeling van goeie kalibrasiemodelle in beide rooi- en wit gistende mos (SVP = 16.37 mg/L, R2 = 0.912, sydigheid = -1.01 mg/L, RVA = 4). ’n Sekondêre, maar baie belangrike bevinding is gemaak met betrekking tot die stoor van mosmonsters wat geneem is van tenke, maar wat nie dadelik met die verwysingsmetodes en FT-IR spektroskopie analiseer kon word nie. Multiveranderlike hoofkomponentanalise op vars en gevriesde sapmonsters het getoon dat gevriesde monsters gebruik kan word om die kalibrasie datastel uit te brei, wanneer benodig. Dus, sapmonsters kan gevries word tot ’n later stadium as onmiddelike analises nie moontlik is nie. Vir die doel van die tweede navorsingsdoelwit van die studie, naamlik kwalitatiewe af-lyn monitering van alkoholiese fermentasie met FT-IR spektroskopie, is ‘n totaal van 21 industriëlegrootte fermentasietenks ge-monitor deur sapmonsters met 8- tot 12-uurlikse intervalle te trek, vanaf die begin van fermentasie, totdat al die druifsuiker gemetaboliseer is. Vir hierdie deel van die werk is die kwantitatiewe data nie gebruik nie; slegs die FT-IR spektra. Multiveranderlike statistiese proseskontrole grafieke is opgestel op grond van die PKK tellings wat bereken is op al die FT-IR spektra wat gemeet is by die verskillende tydsintervalle. Vir hierdie analise is tyd as y-veranderlike gebruik. Die vernaamste doel van hierdie ondersoek was om te evalueer of die PKK-gebaseerde modelle kon onderskei tussen normale en slepende gistings. Die modelle wat verkry is, het die variasie oor tyd in die fermentasiepatrone tussen wit- en rooiwyn fermentasies tydens alkoholiese gisting, duidelik uitgewys. Een Colombar tenk wat teen baie lae temperatuur gefermenteer is om ‘n spesifieke wynstyl te verkry, se fermentasiepatroon het aansienlik verskil van die ander Colombar tenks wat gemonitor is, en hierdie atipiese patroon is ook deur die kwalitatiewe modelle identifiseer. ‘n PKK model oor al die Colombar fermentasies kon duidelik tussen normale en slepende gistings onderskei. Die resultate wat in hierdie studie verkry is, het getoon dat FT-IR spektroskopie baie goeie potensiaal toon vir die aanwending van kwantitatiewe en kwalitatiewe monitering van alkoholiese fermentasie tydens industriële wynproduksie. Die werk wat in hierdie projek gedoen is, het gelei tot die vestiging van ‘n portefeulje van kalibrasiemodelle vir die belangrikste kwaliteitsbepalende parameters in fermenterende mos. Die kwantitatiewe modelle is baie deeglik getoets met onafhanlike toets datastelle, en daarna is die kalibrasiemodelle geimplementeer vir industriële gebruik by Namaqua Wyne. Multiveranderlike statistiese proseskontrole grafieke wat baseer is op data wat vanaf 21 verskillende fermentasietenks verkry is, het baie goeie potensiaal getoon om probleemfermentasies vroeg te identifiseer. Dié grafieke is ‘n baie nuttige diagnostiese hulpmiddel wat verder ontwikkel kan word om verskillende tipes probleemfermentasies te monitor. Toekomstige navorsing in hierdie konteks, sal toegespits word op die optimisering en uitbreiding van die kwantitatiewe en kwalitatiewe modelle, sowel as toepassing van die tegnieke in die onderskeie kelders van Namaqua Wyne.

MScAgric / ENGLISH ABSTRACT: The Little Karoo region of South Africa stretches from Montagu in the west, through Barrydale on the Langeberg Mountain, towards Ladismith, Calitzdorp, Oudtshoorn and De Rust in the east, with the Swartberg mountain range in the north. The Wine of Origin district of Calitzdorp is a small, demarcated area around Calitzdorp in the Little Karoo, surrounded by the Rooiberg, Swartberg and Kleinberg mountains. With a mean February temperature (MFT) of 23.7ºC and a low annual rainfall of 233 mm, the district of Calitzdorp has a similar climate to that of the Douro Demarcated Region (DDR). The MFT is comparable to the DDR mean July temperature, and it seems that the Douro Superior sub-region to the east of the DDR has a mean July temperature of higher than 25°C. In the Cima Corgo sub-region (in the centre of the DDR), and the Baixo Cargo sub-region, the mean July temperatures are ±25°C and ±22°C respectively. Annual rainfall in the DDR is much higher, with Baixo Cargo recording 1 018 mm, Cima Corgo recording 658 mm and Douro Superior in the east recording only 437 mm. Touriga Naçional is one of the highest quality Portuguese red grape varieties. It produces high-quality port-style wine as well as table wines. Excellent quality Touriga Naçional wines have a dark black/purple colour, good extract, high, elegant tannin content and intense aromas, with typical plum, raisin, wild fruit, mulberry, “fynbos” and cherry aromas. The most suitable terroir for Touriga Naçional in the DDR has been found to be on sites that restrain the natural vigour of the grapevine. Soils with moderate to low water-holding capacity, in association with low rainfall, result in water deficits during the growing season and are considered optimal to restrict growth vigour. A steep, northern middle slope is ideal in the southern hemisphere for high temperatures and sunlight interception. Warm temperatures (25 to 30 °C) during the day and cooler temperatures during the night are optimal for photosynthesis and colour development. In order to study factors affecting the quality of Touriga Naçional in Calitzdorp, two Vitis vinifera L. cv. Touriga Naçional commercial vineyards in the Calitzdorp district were selected. Each vineyard was divided into two separate management blocks based on their empirically determined quality of production. Two crop-reduction treatments, the standard 50% crop reduction (which was considered to be the control) and a further less drastic treatment of 25% crop reduction, were applied. Significant differences were found in viticultural performance between the two adjacent Touriga Naçional management blocks in each vineyard, especially with respect to vigour. The upper management blocks, which provided grapes for reserve-quality port-style wines, experienced a higher water deficit due to the moderate soil water-holding capacity and higher temperatures in comparison to the lower sites. The higher water deficits had a restraining effect on the Touriga Naçional vines, and therefore the upper sites had lower vigour, which contributed to better quality of both the wine and port-style wine, and this could be recognised sensorially. However, it was not reflected in the chemical analytical results. Crop load also appeared to have an effect on the Touriga Naçional grapevines, but this appeared to be dependent on the management block. The 50% crop reduction had a significant positive effect on the sensory analyses, but did not significantly affect the chemical analyses. Calitzdorp terroir has a similar effect on Touriga Naçional compared to the DDR terroir, and that is why Calitzdorp can produce good table and port-style wines from Touriga Naçional. / AFRIKAANSE OPSOMMING: Die Klein Karoo-streek in Suid Afrika strek vanaf Montagu in die weste, deur Barrydale teen die Langeberg, na Ladismith, Calitzdorp, Oudtshoorn en De Rust in die ooste, met die Swartberg in die noorde. Die distrik van Calitzdorp is ‘n klein area rondom Calitzdorp, in die Klein Karoo, wat deur die Rooiberg, Swartberg en Kleinberg omring is. Calitzdorp het ‘n gemiddelde Februarie-temperatuur (GFT) van 23.7°C en ‘n lae jaarlikse reënval van 223 mm, wat soortgelyk is aan die klimaat van die Douro Vallei in Portugal. Die Douro Vallei se gemiddelde Julie-temperatuur (GJT) in vergelyking met die GFT van Calitzdorp is hoër, met temperature van meer as 25°C in die substreek Douro Superior. Vir die substreke Cima Cargo en Baixo Cargo is die GJT ±25°C en ±22°C onderskeidelik. Die jaarlikse reënval is ook hoër by Baixo Cargo, met 1 018 mm, Cima Cargo met 658 mm en Douro Superior met slegs 437 mm. Touriga Naçional is een van die beste Portugese rooi kultivars wat hoëkwaliteit tafel- en portwyne produseer. ‘n Tipiese hoëkwaliteit Touriga Naçional-wyn het ‘n swartpers kleur, hoë ekstrak, hoë elegante tanniene en intense aromatiese geure wat tipiese pruim, rosyne, wilde vrugte, moerbei, fynbos en kersie aromas insluit. Die geskikste terroir vir Touriga Naçional is op swak gronde wat die natuurlike groeikrag van die wingerdstok strem. Gronde met matige tot lae grondwaterhouvermoë tesame met lae reënval veroorsaak ‘n waterstremming in die wingerdstok gedurende die groeiseisoen en word as optimaal beskou omdat dit beheersde groei veroorsaak. In die suidelike halfrond word relatief steil, noordelike middelhange as ideaal beskou vir hoë temperature en maksimale sonligonderskepping. Gepaardgaande hiermee is die interne dreinasie verantwoordelik vir vinniger uitdroging van die grond. Hoë temperature (25 tot 30°C) gedurende die dag en koue nagte is optimaal vir fotosintese en kleurontwikkeling. Twee Vitis vinifera L. cv. Touriga Naçional kommersiële wingerde in die Calitzdorp-distrik is geselekteer en in twee afsonderlike bewerkingsblokke verdeel, gebaseer op kwaliteitsverskille. In elke blok was die verdeling van so ‘n aard dat daar ‘n hoërliggende helfte en ‘n laerliggende helfte was. Twee trosverminderingsbehandelings, nl. 50% (kontrole) en 25%, gebaseer op trosgetalle, is toegepas. By elkeen van die wingerde was daar betekenisvolle groeiverskille tussen die twee aangrensende helftes. Die boonste helftes (of gedeeltes) het minder gegroei a.g.v. ‘n hoër waterstremming sowel as hoër temperatuur as die laer helftes. Dit het geblyk om ‘n positiewe invloed op die kwaliteit van beide die tafel- en portwyne uit te oefen. Troslading het ook ‘n effek op die Touriga Naçional-wingerde gehad, maar dit blyk of dit blok-afhanklik is. Die 50% trosverminderingsbehandeling het ‘n beduidende positiewe verskil in die sensoriese analises gemaak, maar nie ‘n beduidende verskil in die chemiese analises van die wyne nie. Calitzdorp se terroir het ‘n soortgelyke effek op Touriga Naçional as dié van die DDR terroir en daarom kan Calitzdorp soortgelyke goeie tafel- en portwyne van Touriga Naçional produseer.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: One of the most striking qualities of wine is its tart, sour taste. The sensory perception of sourness is mainly attributed to the presence of hydrogen ions (protons) at high concentrations. Large amounts of weak carboxylic acids (organic acids) are the main sources of these ions within wine. Once wine enters a person's mouth, the dissociable protons of the weak organic acids within wine are partially neutralized or, in other words, titrated by the saliva secreted inside one's mouth. This explains why the duration and intensity of a wine's sourness is related to its titratable acidity content. The sour taste of wine is usually considered refreshing and it helps balance wine flavour. In fact, wines become watery when its titratable acidity content is too low. After alcoholic fermentation, the titratable acidity of wine will usually be less than that of the grape juice from which was made due to ethanol-induced precipitation of potassium bitartrate crystals and partial consumption of malic acid by fermenting wine yeasts. Occasionally however, increases in titratable acidity are observed during alcoholic fermentation. If wine is produced from grape juice with optimum levels of titratable acidity, unforeseen increases in titratable acidity during alcoholic fermentation can be detrimental to the quality of the final product. Although the net production of malic acid by wine yeasts contributes to increases in titratable acidity seen during grape juice fermentations, the production of succinic acid is regarded as the primary contributor. In fact, succinic acid accounts for approximately 90% of the non-volatile acids produced during fermentation of grape juice. Between 0.5 and 1.5 g/L succinic acid is normally found in wine, but higher concentrations thereof (up to 3.0 g/L) have been detected within certain red wines. Acidity adjustments should preferably be carried out before the onset of alcoholic fermentation to allow better integration of the added compound(s) and to ensure that conditions during fermentation favour the quality and microbial stability of the final product. In doing so unfortunately, winemakers run the risk of ending up with wines that may taste too sour if they are unable to accurately predict and take into consideration the amount of succinic acid produced during alcoholic fermentation. Knowledge with regard to the factors involved in succinic acid's production by fermenting wine yeasts is therefore required in order to manage the titratable acidity of wines more accurately. Ever since Louis Pasteur first noticed succinic acid amongst the by-products of alcoholic fermentation, attempts have been made to determine the metabolic pathways and factors involved in its production by fermenting wine yeasts. Up until now however, it remains unclear why wines sometimes end up with exceptionally high levels of succinic acid. For these reasons it was decided to investigate the possible causes of very high succinic acid concentrations within wine. Due to complexity of grape juice's chemical composition and the problems associated with sterilizing grape juice, fermentation experiments were conducted within a chemically defined grape juice-like medium. Succinic acid production by nine different industrial wine yeast strains was studied under various conditions with regard to the nutrient status of the synthetic grape juice, temperature and availability of molecular oxygen during alcoholic fermentation. The amount of succinic acid produced during alcoholic fermentation was found to depend on the yeast strain, fermentation temperature and chemical composition of the synthetic grape juice. Out of the nine commercial yeast strains selected for this study, strain WE372 produced the largest amount of succinic acid in synthetic grape juice at 28°C. Strain WE372 produced significantly smaller amounts of acetic acid than the other yeast strains of this study and very little acetic acid at 28°C, which indicated that strain WE372 may have less acetaldehyde dehydroganase activity than the other yeast strains of this study under the conditions tested. The effect this has on NAD: NADH balance is the probable cause for its ability to form more glycerol, succinic and malic acid than the other strains. Results from our study show that succinic acid production is influenced primarily by the metabolizable fraction of YAN, which we termed metabolically available nitrogen (MAN). Succinic acid production by fermenting yeasts will be favoured by moderate to high fermentation temperatures (20°C to 28°C) in grape juice with a nicotinic acid and/ or nicotinamide deficiency, high sugar content (200 g/L to 240 g/L), moderate amounts of metabolically available nitrogen (300 ± 50 mg/L MAN), the presence of flavonoids and large supplies of unsaturated long-chain fatty acids. Even higher concentrations of succinic acid were produced when oxygen was made available to fermenting yeasts by aerating the fermenting grape juice. Fermentation temperatures below 18°C, too much metabolizable nitrogen (> 450 mg/L MAN), very high concentrations of fermentable sugar (> 240 g/L), lipid deficiencies and a lack of pantothenic acid, thiamine, biotin or pyridoxine will decrease the amount of succinic acid produced fermenting yeasts. / No Afrikaans summary available.

Thesis (MScAgric)--Stellenbosch University, 1948. / NO ABSTRACT AVAILABLE

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: The varietal character of Sauvignon blanc wine is mostly defined by the balance between tropical and green vegetative flavour nuances. Grape derived methoxypyrazines are the main aroma contributors towards green vegetative flavours. Methoxypyrazines are heat and light sensitive. Due to warm climatic conditions in South Africa, methoxypyrazine levels decrease during grape ripening. The addition of food flavourings to Sauvignon blanc wine, a practice known as spiking, has occurred in the past to improve the green character of the wines. Adding flavourings to wine and selling the wine as natural certified wine is illegal in South Africa. Currently, adulterated Sauvignon blanc wines are identified using gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) methods to quantify methoxypyrazines and compare levels to an established database. Although of high sensitivity, GC-MS and LCMS methods are costly and time consuming, therefore not optimal for routine screening of wines. Hence the need for the development of a fast and cost effective method for routine screening of large amounts of wines to identify adulteration. Small scale vinification practices were used to prepare experimental Sauvignon blanc wine. Flavourings were added to Sauvignon blanc grape juice before fermentation, during the preparation of experimental spiked wines. Control wines, containing no flavouring, were also prepared. Commercial wines were spiked after fermentation and bottling. Each wine was only spiked with a single flavouring. The flavourings added were the juice of homogenised fresh green peppers and commercially available flavourings for wine. The following commercial flavourings were used: green pepper, asparagus, grassy and tropical. The above mentioned wines were analyzed using Fourier transform infrared (FT-IR) spectroscopy, GC-MS, LC-MS and descriptive sensory analysis. The FT-IR techniques used were Fourier transform mid infrared (FT-MIR) transmission, FT-MIR attenuated reflection and Fourier transform near infrared (FT-NIR) reflection spectroscopy. The data was interpreted using the following multivariate statistical techniques: principal component analysis (PCA), partial least squares discrimination (PLS-D) and conformity testing. Multivariate models constructed from FT-MIR and FT-NIR data were able to discriminate between spiked and control wines. Sensory analysis results clearly showed differences between non-spiked wines and spiked wines with 3-isobutyl-2-methoxypyrazine concentrations 10 times higher than naturally occurring in wine. Differences between control and spiked wines with concentrations of 3-isobutyl-2-methoxypyrazine similar to concentrations naturally occurring in wines could not be detected to prove adulteration conducting sensory analysis. However, differences between control and spiked wines with levels of 3-isobutyl-2-methoxypyrazine similar to levels naturally occurring in wines could be detected using FT-IR data in conjunction with multivariate statistics. This study showed that, FT-IR spectroscopy in conjunction with multivariate statistical methods can be a possibility for the screening and identification of wines suspected of adulteration in terms of added flavourings. Descriptive sensory analysis also proved to be a potentially useful tool. However screening and training of potential panel members are time consuming. / AFRIKAANSE OPSOMMING: Die variëteitskarakter van Sauvignon blanc wyn word grotendeels gedefinieer deur die balans tussen tropiese en groen vegetatiewe aromas. Metoksipirasiene is die hoof aroma verbindings wat verantwoordelik is vir groen vegetatiewe aromas. Metoksipirasien is hitte- en ligsensitief. Warm klimaatsomstandighede in Suid-Afrika het tot gevolg dat metoksipirasien konsentrasies daal tydens druif rypwording. Sauvignon blanc wyne is in die verlede vervals deur middel van die byvoeging van vars groen soetrissies om die groen vegetatiewe karaktereienskappe van die wyne te bevorder. Die byvoeging van geurmiddels of plantekstrakte by wyn en verkoop van daardie wyn as gesertifiseerde natuurlike wyn is onwettig in Suid-Afrika. Tans word vervalsde wyne met behulp van gaschromatografie-massaspektrometrie (GC-MS) en vloeistofchromatografie-massaspektrometrie (LC-MS) opgespoor. Kwantifisering van metoksiepirasien konsentrasies in wyne en druiwesappe word vergelyk met konsentrasies in ‘n bestaande databasis. Alhoewel GC-MS en LC-MS hoë sensitiwiteitsmetodes is, is dit duur en tydrowende metodes, dus nie optimaal vir roetine sifting nie. Dus word ‘n koste- en tydseffektiewe roetine metode benodig om vervalsing van wyne op te spoor. Eksperimentele wyne is op klein skaal berei. Geurmiddels is voor fermentasie by die druiwesap gevoeg. Kontrole wyne waarby geen geurmiddels gevoeg is nie, is ook berei. Kommersiële wyne is gegeur na fermentasie en bottelering. Elke wyn is met ‘n enkele geurmiddel gegeur. Gehomogeniseerde vars groen soetrissie asook kommersieel beskikbare geursels vir wyn is gebruik. Die volgende kommersiële geursels is gebruik: groen soetrissie, aspersie, gras en tropiese geursel. Die volgende analitiese tegnieke is gebruik vir analisering van bogenoemde wyne: Fourier transformasie infrarooi (FT-IR) spektroskopie, GC-MS, LC-MS en beskrywende sensoriese analise. Die spesifieke FT-IR tegnieke wat gebruik is, is: Fourier transformasie mid-infrarooi (FT-MIR) transmissie, FT-MIR verswakte weerskaatsing en Fourier transformasie naby-infrarooi (FT-NIR) reflektansie. Die volgende multiveranderlike statistiese tegnieke is gebruik ter interpretasie van data: hoof komponent analise (PCA), parsiële kleinste kwadraat diskriminant analise (PLS-D) en gelykvormigheidstoetsing. Multiveranderlike modelle, bereken met behulp van FT-MIR en FT-NIR data, kon diskrimineer tussen gegeurde en kontrole wyne. Resultate wat verkry is tydens sensoriese analises het duidelike verskille uitgewys tussen gegeurde en kontrole wyne met betrekking tot 3-isobutiel-2- metoksipirasien konsetrasies waar 3-isobutiel-2-metoksipirasien konsentrasies 10 keer hoër was as wat natuurlik voorkom in wyn. Geen beduidende verskille kon waargeneem word in gevalle waar wyne vervals is met laer konsentrasies van geurmiddels deur sensoriese data te ontleed nie. Nietemin, statisitiese verskille tussen kontrole en vervalsde wyne kon waargeneem word vir lae-konsentrasie-geurmiddel vervalsde wyne deur FT-IR data met behulp van multiveranderlike statisitiese metodes te ontleed. Hierdie studie het gewys dat FT-IR in kombinasie met multiveranderlike statistiese tegnieke spesifiek hoof komponent analise (PCA) en parsiële kleinste kwadraat diskriminant analise (PLS-D) asook gelykvormigheidstoetsing bruikbare tegnieke is om te onderskei tussen kontrole (egte natuurlike) en vervalsde wyne ten opsigte van die byvoeging van geurmiddels. Beskrywende sensoriese analise kan ook nuttig gebruik word, alhoewel keuring en opleiding van paneellede tydrowend is.

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Hen egg white lysozyme (HEWL; muramidase; EC 3:2:1:17) is an enzyme present in high concentrations in chicken (Gallus gallus) egg whites. It hydrolyses the link between N-acetylmuramic acid and N-acetylglucosamine in Gram positive bacterial cell walls, resulting in cell death. It is thus active against lactic acid bacteria (LAB), which may be present in grape juices and musts. These bacteria are responsible for malolactic fermentation of wines although many species, particularly of the genera Lactobacillus and Pediococcus, are considered spoilage organisms. The growth of LAB is therefore closely monitored and controlled during winemaking. The most common means of control is growth inhibition by chemical treatment (usually with SO2). Lysozyme is a commonly used wine processing aid, complementing the antimicrobial activity of SO2 . It allows for lower doses of SO2 to be used, thus improving the wholesomeness of wine. The OIV (Organisation Internationale de la Vigne et du Vin) approved its use in quantities up to 500 mg per liter of wine in 1997. This study evaluated the effect of different secretion signals on the secretion of lysozyme by the haploid auxotroph Saccharomyces cerevisiae strain FY23. Secretion by an industrial strain (VIN13) transformed with a single copy of the HEWL gene with the MF-a secretion signal under the control of the PGK1 (phosphoglycerate kinase 1) prompter and terminator was also evaluated. In the case of FY23 four secretion signals were used, namely the native lysozyme signal and the S. cerevisiae mating factor-a signal as well as mutants of these signals. These mutants incorporated two additional arginines at the N-terminus of the signals immediately downstream of the terminal methionine. The effect of these mutations was to increase the positive charge of the secretion signal N-terminals. The secretion signal-lysozyme fusions were placed under the regulation of the S. cerevisae PGK1 gene’s promoter and terminator. The resulting expression cassettes were cloned into integrating vectors YIpLac211 and pDMPOF1b and episomal vector pHVX2. These were used to transform FY23 and VIN13. FY23 as well as VIN13 transformants were evaluated in an artificial medium designed to reflect the nutrient content of grape juice, with particular attention being paid to assiminable nitrogen. Three hexose concentrations were tested in order to determine the effect thereof on lysozyme secretion titer. Lysozyme secreted under all tested growth conditions was found to be too low for detection by either enzymatic assay or HPLC-FLD. For this reason secreted lysozyme was isolated and concentrated 10x by means of cation-exchange. Subsequently, lysozyme concentrations in the concentrates was determined by means of the aforementioned techniques. SDS-PAGE analysis of lysozyme concentrates was also performed. No significant differences were found between native or MF-a secretion signals and their mutated counterparts in terms of secretion titer or proteolytic maturation. Lysozyme secreted with the MF-a signal was found to be misprocessed in all cases, with both an authentically processed and a larger form, in which the secretion signal was not completely removed, being present. Lysozyme secreted with the native signal appeared to be correctly processed in all cases. Secretion titer from high copy number episomal FY23 tranformants was similar to that of integrants containing a single copy of the gene. Sugar concentration affected lysozyme production, with higher quantities of the enzyme being secreted when higher initial sugar concentrations were used. Lysozyme titers were extremely low (< 0:25 mg/L) with all expression cassettes under all the tested conditions with both FY23 and VIN13. In the case of the VIN13’s a lower final biomass was found for the secretor strain tested in comparrison to the VIN13 wild-type. / AFRIKAANSE OPSOMMING: Hoendereierwitlisosiem (HEWL; muramidase, EG 3:2:1:17) is ´n ensiem teenwoordig in hoë konsentrasies in hoender (Gallus gallus) eierwitte. Dit hidroliseer die binding tussen N-asetielmuramiensuur en N-asetielglukosamien in Gram positiewe bakteriese selwande, wat tot seldood lei. Dit is dus aktief teen melksuurbakterieë (MSB), wat in druiwesap en mos teenwoordig kan wees. Hierdie bakterieë is verantwoordelik vir appelmelksuurgisting van wyne, hoewel baie spesies, veral van die genera Lactobacillus en Pediococcus, ook as bederforganismes beskou word. Die groei van MSB word dus noukeurig tydens wynbereiding gemoniteer en beheer. Die algemeenste wyse van beheer is groei-inhibisie deur chemiese behandeling (gewoonlik SO2). Lisosiem is ´n algemeen gebruikte wyntoevoegingsmiddel en vul die antimikrobiese aktiwiteit van SO2 aan. Met lisosiem kan ´n laer dosis van SO2 gebruik word, wat lei tot ´n verbetering van die heilsaamheid van die wyn. Die OIV (Organisasie Internationale de la Vigne et du Vin) het die gebruik daarvan goedgekeur tot en met 500 mg per liter wyn vanaf 1997. Hierdie studie het die effek van verskillende sekresieseine op die uitskeiding van lisosiem deur die haploïede ouksotrofe Saccharomyces cerevisiae stam, FY23, geëvalueer. Uitskeiding deur ´n industriële stam (VIN13), wat getransformeer is met ´n enkelkopie van die HEWL-gene met die MF-a sekresiesein onder die beheer van die PGK1 (Fosfogliseraat kinase 1) promotor en termineerder, is ook geëvalueer. In die geval van FY23 is vier sekresieseine gebruik, naamlik die inheemse lisosiemsein, S. cerevisiae MF- a sein, asook mutante van hierdie seine. Hierdie mutante het twee bykomende arginienresidu’s by die N-terminus van die seine direk stroom-af van die terminale metionien. Die effek van hierdie mutasies was om die positiewe lading van die uitskeidingsein N-terminale te verhoog. Die gevolglike uitdrukkingskassette is in die integrasievektor YIpLac211 en pDMPOF1b, en die episomale vektor pHVX2, gekloneer. Dit is gebruik om VIN13 en FY23 te transformeer. FY23, sowel as VIN13-transformante, is geëvalueer in ´n kunsmatige medium wat ontwerp is om die voedingsinhoud van druiwesap te weerspieël, met besondere aandag aan assimileerbare stikstof. Drie heksose konsentrasies is getoets om te bepaal wat die uitwerking daarvan op die lisosiemsekresietiter is. Onder alle groeitoestande was die isosiem wat uitgeskei is, te laag om deur ensimatiese toetse of HPLC-FLD bepaal te word. Om hierdie rede is uitgeskeide lisosiem geïsoleer en 10x gekonsentreer deur middel van katioon-uitruiling. Daarna is lisosiemkonsentrasies bepaal deur middel van bogenoemde tegnieke. SDS-PAGE-ontleding van lisosiemkonsentraat is ook uitgevoer. In terme van sekresietiter of proteolitiese maturasie, is geen beduidende verskille gevind tussen inheemse of MF-a sekresieseine en hul gemuteerde eweknieë nie. Lisosiem wat deur die MF-a sein uitgeskei is, is in alle gevalle foutief geprosesseer, met ´n teenwoordigheid van beide die regte produk en ´n groter produk, waarin die uitskeidingsein nie heeltemal verwyder word nie. Lisosiem wat met die inheemse sein uitgeskei is, blyk in alle gevalle korrek verwerk te wees. Sekresietiter van ´n aantal hoë-kopie episomale FY23-transformante was soortgelyk aan dié van integrante met ´n enkelkopie van die geen. Suikerkonsentrasie beïnvloed lisosiemproduksie, met ´n hoër hoeveelheid van die ensiem wat uitgeskei word wanneer die aanvanklike suiker in hoër konsentrasies gebruik is. Lisosiemtiters was baie laag (< 0:25 mg/L), met al die kassette onder al die getoetste toestande vir beide FY23 en VIN13. In die geval van die VIN13’s, is ´n laer finale biomassa vir die uitskeidingstam in vergelyking met die VIN13 wilde-tipe gevind.

Thesis (MScAgric)--Stellenbosch University, 1974. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Phenolic compounds are important quality indicators of a red wine, as they can contribute to the colour of a young red wine, colour stability during ageing as well as astringency, bitterness, body and overall mouthfeel properties. Wine composition is commonly influenced by winemaking and viticultural practices. In South Africa it often happens that vines are excessively vigorous, resulting in canopies that are too dense, which in turn could have a negative effect on the quantity and quality of the grapes produced. Viticultural practices such as judicious canopy management and irrigation are designed to control vine vigour and yield, thus improving fruit ripening and colour development. Artificial shading and water deficit have been reported to have an influence on the sensory properties of red wine as well as on the flavonoid composition. These effects are dependent on a number of factors, however, including the season, cultivar, light intensity, and the extent and timing of water deficit. There is limited research on the possible interactive effects of grapevine water deficits and canopy manipulation on grape and wine flavonoid composition in Shiraz, as well as the relationships between berry and wine composition. We thus investigated the effect of canopy reduction in combination with water deficit on the phenolic and colour composition of Shiraz grapes at different levels of ripeness, and in their corresponding wines after alcoholic and malolactic fermentation as well as after six months’ ageing. This study found that it is possible to improve the phenolic composition of grapes and wine by shoot removal, and some of the tendencies in the wines were also observed after the ageing period. If the shoot removal is not performed at a very early stage, sunburn damage can occur and this will result in berries with a lower mass and volume at harvest due to excessive exposure without the berry having adapted to the imposed conditions. Harvesting at different ripeness levels also affected the chemical and phenolic composition of the grapes and resulting wines. The water deficit effect on most phenolic parameters measured in the grapes and wine was not as prominent as that of the canopy manipulation treatment. This study improved our understanding of how an improvement in the canopy microclimate of Shiraz could be reflected in the phenolic composition of wines, along with a potentially important effect of harvesting date. On this basis it may be possible to attain a specific wine style. Harvesting at a ripe stage, for example, could result in the production of wines with higher colour density and astringency, while unripe grapes could result in wines with higher levels of perceivable fresh berry attributes. In particular, canopy reduction could increase the astringency and body of wines made from grapes subjected to water deficit. / AFRIKAANSE OPSOMMING: Fenoliese verbindings is belangrike kwaliteitsparameters van rooiwyn, aangesien dit kan bydra tot die kleur van ‘n rooiwyn, kleurstabiliteit tydens veroudering sowel as frankheid, bitterigheid en mondgevoel. ‘n Wyn se fenoliese samestelling word algemeen bepaal deur wynmaak- en wingerdkundige praktyke. In Suid-Afrika gebeur dit gereeld dat wingerde uitermatig groeikragtig is, wat lei tot te digte lower wat sodoende ‘n negatiewe effek op kwantiteit en kwaliteit van die druiwe wat geproduseer word, het. Wingerdkundige praktyke soos oordeelkundige lowerbestuur en besproeiing is ontwerp om wingerdstokke se groeikrag en opbrengs te beheer, en sodoende vrugrypwording en kleurontwikkeling te verbeter. Kunsmatige beskaduwing en waterstres is gerapporteer om ‘n invloed te hê op die sensoriese eienskappe van rooiwyn sowel as op die flavonoïedsamestelling. Hierdie effekte is egter afhanklik van ‘n verskeidenheid faktore, insluitende die seisoen, kultivar, ligintensiteit en die mate en tyd van waterstres toegepas. Daar is beperkte navorsing op die moontlike interaktiewe effekte van waterstres en lowermanipulasie op die druif en wyn flavonoïedsamestelling in Shiraz, sowel as die verhoudings tussen druif en wyn samestelling. Ons het dus die effekte van lowerbestuur in kombinasie met waterstres op die fenoliese en kleursamestelling van Shiraz druiwe by verskillende rypheidsvlakke ondersoek, asook in hul ooreenstemmende wyne na alkoholieseen appelmelksuurfermentasie sowel as na ses maande veroudering. Hierdie studie het gevind dat dit moontlik is om die fenoliese samestelling van druiwe en wyn deur lootverwydering te verbeter, en sommige van die tendense is ook waargeneem in die wyn na die verouderingsperiode. Indien lootverwydering nie toegepas word by ‘n baie vroeë stadium nie, kan sonbrand voorkom en dit kan lei tot korrels met ‘n laer massa en volume by oes as gevolg van oormatige blootstelling sonder dat die korrel aangepas het by die spesifieke kondisies. Oes by verskillende rypheidsvlakke affekteer ook die chemiese en fenoliese samestelling van die druiwe en ooreenstemmende wyne. Die waterstreseffek op meeste van die fenoliese parameters gemeet in druiwe en wyn was nie so prominent soos dié van die lowermanipulasie behandeling nie. Hierdie studie het ons begrip verbeter van hoe ‘n verbetering van die lower mikroklimaat van Shiraz gereflekteer kan word op die fenoliese samestelling van die wyn, saam met ‘n potensiële belangrike effek van oesdatum. Op grond van hierdie basis is dit dus moontlik om ‘n spesifieke wynstyl te verkry. Oes by ‘n ryp stadium, byvoorbeeld, kan die produksie van wyn met ‘n hoër kleurdigtheid en frankheid tot gevolg hê, terwyl onryp druiwe wyne met hoër vlakke van waarneembare vars bessiekenmerke tot gevolg kan hê. Verlaging van lowerdigtheid kan veral die frankheid en mondgevoel van wyne gemaak van druiwe blootgestel aan waterstres, verbeter.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Wine fermentation is a continuously changing biological process whereby the raw product, grape juice is transformed into a high value product wine. In an ideal situation the fermentation kinetics of batch fermentations should follow the same trend over time. This however is not the case in industrial wine fermentations where significant batch-to-batch variation is present. The time trajectories of fermentation processes are therefore often unpredictable in absolute terms. The monitoring of substrate (sugar) and product concentrations (ethanol) as well as other quality parameters during a wine fermentation, is therefore of extreme importance to ensure effective control and management of wine fermentation processes. Conventional methods for fermentation monitoring are however costly, time consuming and often unreliable. For these reasons the modern wine industry requires rapid, reliable, non-destructive monitoring techniques which would meet the criteria of providing critical real-time process information that is displayed in easily interpretable graphical format, in order to ensure the highest quality and continuous consistency throughout all the stages of a process. This research study in particular, addressed the current need for alternative fermentation monitoring strategies that meet these criteria, by evaluating the potential use of spectroscopy as an analytical technique for fermentation monitoring. The overall objective of this study was to use chemometric modelling of information obtained by Fourier transform mid-infrared (FT-MIR) and near-infrared (FT-NIR) spectroscopy, to quantitatively and qualitatively monitor both alcoholic (AF) and malolactic fermentation (MLF) processes. Towards this objective 11 batch fermentations elaborated with Oenococcus oeni and Lactobacillus plantarum strains in respectively a co-inoculation and sequential inoculation scenario, were sampled and analysed at regular time intervals with FT-MIR and FT-NIR spectroscopy and enzymatic reference methods during 2011. Samples were also analysed by gas chromatography flame ionisation detection (GC-FID) and mass spectrometry (GC-MS) at two critical stages during fermentation, namely 50% completion of MLF and 100% completion of MLF, in order to obtain a profile of the evolution of the aroma compounds associated with each inoculation scenario. Three clearly defined research objectives were set for this project. The first objective involved the expansion of the existing quantitative platform for fermentation monitoring. Towards the outcomes of this objective, partial least squares (PLS) calibration models for prediction of malic acid and lactic acid in fermenting must and wines elaborated in our study, were established, based on the MIR and NIR spectra. The models showed excellent predictive abilities in independent test set validation. This outcome made a significant contribution to our existing PLS calibration capacity, particularly towards monitoring of MLF. Quantitative data obtained with the PLS models were also used to graphically project the rate of AF in the different batches, by non-linear fitted regression plots that easily visualised the overall patterns of sugar and ethanol metabolism in the different fermentations. The second research objective involved the qualitative monitoring of fermentations. This approach used FT-MIR and FT-NIR spectra together with chemometrics to identify trends between the different fermentation treatments. Principal component analysis (PCA) clearly projected the time trend from the onset of fermentation, through AF and MLF. No unique bacterial trend was however observed with spectroscopy. These results illustrate the potential of these techniques to be used for modelling of fermentations in industrial situations, through providing critical information about the evolution of the process. Furthermore, these techniques provide tools for identifying problematic and deviating fermentations. A spectral conformity test based on simple calculations of the standard deviation between the absorbance at each recorded wavenumber in the spectra, further confirmed identification of the critical fermentation stages. This technique by-passes the need for spectral interpretation and is a very useful addition, particularly from the industry perspective, to the portfolio of methods established in this study. The third research objective adressed the need to evaluate the possibility to discriminate between the different process stages and LAB treatments using univariate (ANOVA) and multivariate chemometric techniques such as PCA, PLS discriminant analysis (PLS-DA) and Soft Independent Modelling of Class Analogy (SIMCA) for possible future interpretative and classification purposes. The exploratory tool of PCA was used to investigate the similarities and differences between the chemical footprints of the different treatments. PCA showed clear differentiation between the two process stages using chemical quantified data. Differentiation between the LAB treatments was visible with PCA, showing a more prominent separation at 50% completion of MLF. Furthermore the ability of spectroscopy for potential classification was shown using PLS-DA and SIMCA. This profiling study can be seen as a preliminary study setting the ground work for further in-depth research into the profiling of different LAB treatments and inoculation strategies. / AFRIKAANSE OPSOMMING: Wyngisting is ‘n voortdurend veranderende biologiese proses waarvolgens die rou produk, druiwesap, in ‘n produk van hoë waarde, naamlik wyn, verander word. Onder ideale omstandighede sou die gistingskinetika van lotgistings dieselfde tendens oor tyd volg. Dit is egter nie die geval in industriële wyngistings nie, waar noemenswaardige wisseling van lot tot lot teenwoordig is. Die tydsbaan van gistingsprosesse is dus in baie gevalle in absolute terme onvoorspelbaar. Die monitering van substraat- (suiker) en produkkonsentrasies (etanol), sowel as ander kwaliteitsparameters tydens ‘n wyngisting, is van die uiterste belang om doeltreffende beheer en bestuur van wyngistingsprosesse te verseker. Konvensionele metodes vir die monitering van gisting is egter duur, tydrowend en ook soms onbetroubaar. Om hierdie redes vereis die moderne wynbedryf vinnige, betroubare en nie-destruktiewe moniteringstegnieke wat aan die kriteria sou voldoen vir die verskaffing van kritiese, intydse prosesinligting wat in ‘n maklik interpreteerbare grafiese formaat vertoon word om die hoogste kwaliteit en voortdurende konsekwentheid tydens al die stadia van ‘n proses te verseker. Hierdie navorsingstudie het in besonder die huidige behoefte aan alternatiewe gistingsmoniteringstrategieë wat aan hierdie kriteria voldoen, aangespreek deur die potensiële gebruik van spektroskopie as ‘n analitiese tegniek vir die monitering van gisting te evalueer. Die oorhoofse doelwit van hierdie studie was die chemometriese modellering van inligting wat met behulp van Fourier transform middel-infrarooi (FT-MIR) en naby-infrarooi (FT-NIR) spektroskopie verkry is om die prosesse van alkoholiese en appelmelksuurgisting (AMG) kwantitatief en kwalitatief te monitor. Ten einde hierdie doelwit te bereik, is 11 lotgistings met Oenococcus oeni en Lactobacillus plantarum rasse uitgevoer in scenario’s van ‘n gesamentlike inokulasie en opeenvolgende inokulasie onderskeidelik. Monsters van hierdie gistings is met gereelde tydintervalle in 2011 geneem en analises is met FT-MIR en FT-NIR spektroskopie en ensiematiese verwysingsmetodes gedoen. Monsters is ook met gaschromatografiese vlam ionisasie-opsporing (GC-FID) en massaspektrometrie (GC-MS) op twee kritiese stadia tydens gisting geanaliseer, naamlik toe AMG 50% en 100% voltooid was, om ‘n profiel te verkry van die evolusie van die aromaverbindings wat met elke inokulasie-scenario verband hou. Drie duidelik gedefinieerde navorsingsdoelwitte is vir hierdie studie bepaal. Die eerste doelwit het die uitbreiding van die bestaande kwantitatiewe platform vir gistingsmonitering behels. Hiervoor is gedeeltelike kleinstekwadraat [partial least squares (PLS)] kalibrasiemodelle vir die voorspelling van appelsuur en melksuur in die gistende mos en wyne in ons studie op die basis van MIR- en NIR-spektra bepaal. Die modelle het in onafhanklike geldigheidsbepaling van die toetsstel uitstekende voorspellingsvermoëns getoon. Hierdie uitkoms het ‘n noemenswaardige bydrae gemaak tot ons bestaande PLS kalibrasiekapasiteit, veral om AMG te monitor. Die kwantitatiewe data wat met die PLS-modelle verkry is, is ook gebruik om die tempo van alkoholiese gisting in die verskillende lotte grafies uit te beeld deur middel van kromlynige passing van regressiepersele, waarmee dit maklik was om die algehele patrone van suiker- en etanolmetabolisme in die verskillende gistings te visualiseer. Die tweede navorsingsdoelwit het die kwalitatiewe monitering van gistings behels. Hierdie benadering het FT-MIR en FT-NIR spektra tesame met chemometrie gebruik om tendense tussen die verskillende gistingsbehandelings te identifiseer. Hoofkomponentanalise [principal component analysis (PCA)] het duidelik die tydtendens vanaf die aanvang van gisting, deur alkoholiese gisting en AMG, geprojekteer. Geen unieke bakteriese tendens is egter met spektroskopie waargeneem nie. Hierdie uitslae illustreer die potensiaal van hierdie tegnieke om vir die modellering van gistings in industriële situasies gebruik te word deur kritiese inligting oor die evolusie van die proses te verskaf. Verder verskaf hierdie tegnieke gereedskap vir die identifikasie van problematiese en afwykende gistings. ‘n Spektrale gelykvormigheidstoets gebaseer op eenvoudige berekeninge van die standaardafwyking tussen die absorbansie by elke aangetekende golfgetal in die spektra het ook die identifikasie van die kritiese gistingstadia bevestig. Hierdie tegniek omloop die noodsaak vir spektrale interpretasie en is ‘n baie nuttige byvoeging tot die portefeulje van metodes wat in hierdie studie bepaal is, veral vanuit ‘n bedryfsperspektief. Die derde navorsingsdoelwit het die behoefte aangespreek om tussen die verskillende prosesstadia en melksuurbakterie-behandelings te onderskei deur gebruik te maak van eenvariant- (ANOVA) en meervariant- chemometriese tegnieke soos PCA, PLSdiskriminantanalise en sagte onafhanklike modellering van klasanalogie [soft independent modelling of class analogy (SIMCA)] vir moontlike toekomstige verklarende en klassifikasiedoeleindes. Die ondersoekende gereedskap van PCA is gebruik om ooreenkomste en verskille tussen die chemiese voetspore van die verskillende behandelings te ondersoek. PCA het duidelike differensiasie tussen die twee prosesstadia getoon op grond van chemies gekwantifiseerde data. Differensiasie tussen die melksuurbakterie-behandelings was met PCA sigbaar, met ‘n meer prominente skeiding teen 50% voltooide AMG. Verder is die vermoë van spektroskopie vir potensiële klassifikasie met PLS-diskriminantanalise en SIMCA getoon. Die profielsamestelling in hierdie studie kan beskou word as ‘n voorlopige studie vir verdere diepgaande navorsing oor die profielsamestelling van verskillende melksuurbakteriebehandelings en inokulasiestrategieë. / The National Research Foundation and Winetech

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