Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The two white seedless table grape cultivars, Regal Seedless and Thompson Seedless fulfil a very important role in securing foreign income not only for the South African table grape industry, but the South African economy as a whole. These two cultivars, however, are like so many other white table grape cultivars, also prone to browning, especially netlike browning on Regal Seedless and internal browning on Thompson Seedless grapes. This leads to huge financial losses every year, since there is no established way to assess at harvest, during storage or during packaging, whether the grapes will eventually turn brown. In other words, there is no well-known protocol of assessing the browning risk of a particular batch of grapes prior to export. Numerous studies have been undertaken to determine the exact cause of browning and how it should be managed, but to date, no chemical or physical parameter has been firmly associated with the phenomenon. The overall aim of this study was thus to find an alternative way to deal with the problem by investigating the potential of near infrared (NIR) spectroscopy as a fast, non-destructive measurement technique to determine the browning potential of whole white seedless table grapes. A secondary aim was the determination of optimal ripeness of table grapes. In this way harvest maturity and quality indicative parameters namely total soluble solids (TSS), titratable acidity (TA), pH, glucose and fructose, also associated with the browning phenomenon, was quantified using models based on infrared spectra. Three different techniques (a) Fourier transform Near Infrared (FT-NIR), (b) Fourier transform – Mid Infrared (FT-MIR) and (c) Fourier transform – Mid Infrared Attenuated Total Reflectance (FT-MIR ATR) spectroscopy were investigated to determine these parameters. This was done so that a platform of different technologies would be available to the table grape industry. The grapes used in this study were harvested over two years (2008 and 2009) and were sourced from two different commercial vineyards in the Hex River valley, Western Cape, South Africa. Different crop loads (the total amount of bunches on the vines per hectare) were left for Regal Seedless (75 000, 50 000 and 35 000) and for Thompson Seedless (75 000 and 50 000). Three rows were used for Regal Seedless and two rows for Thompson Seedless. Each row had six sections which each represented a repetition for each crop load. In 2008 these cultivars were harvested early at 16°Brix, at optimum ripeness (18°Brix) and late at 20°Brix. In 2009 they were harvested twice at the optimum ripeness level. Berries from harvested bunches were crushed and the juice was used to determine the reference values for the different parameters in the laboratory according to their specific methods. The obtained juice was also scanned on the three different instruments. Different software (OPUS 6.5 for the FT-NIR and FT-MIR ATR instruments and Unscrambler version 9.2 for the FT-MIR instrument) as well as different spectral pre-processing techniques were also evaluated before construction of the models for all the instruments. Partial least squares (PLS) regression was used for the construction of the different calibration models. Different regression statistics, that included the root mean square error for prediction (RMSEP); the coefficient of determination (R2); the residual prediction deviation (RPD) and the bias were used to evaluate the performance of the developed calibration models. Calibration models which are fit for screening purposes were obtained on the FT-NIR and FTMIR ATR instruments for TSS (11.40 - 21.80°Brix) (R2 = 85.92%, RMSEP = 0.71 °Brix RPD = 2.67 and bias = 0.03°Brix), pH (2.94 - 3.9) (R2 = 85.00%, RMSEP = 0.08 RPD = 2.59 and bias = -0.01) and TA (4.3 - 13.1 g/L), (R2 = 90.77%, RMSEP = 0.48 g/L RPD = 3.30 and bias = -0.03 g/L). Models for fructose (46.70 – 176.82 g/L) (R2 = 74.66%, RMSEP = 9.28 g/L RPD = 2.00 and bias = 1.10 g/L) and glucose (20.36 – 386.67 g/L) (R2 = 70.71%, RMSEP = 11.10 g/L RPD = 1.87 and bias = 1.64 g/L) were obtained with the FT-NIR and FT-MIR ATR instruments that were in some instances fit for screening purposes and in some instances unsuitable for quantification purposes. The FT-MIR instrument gave models for all the parameters that were not yet suitable for quantification purposes. Combined spectral ranges used for calibration were often similar for some parameters, namely 12 493 - 5 446.2 for TSS and pH, 6 101.9 - 5 446.2 for TSS, TA and fructose and 4 601.5 - 4 246.7 for pH and fructose on the FT-NIR instrument, 2 993.2 - 2 322.3 for pH, TA and glucose and 1 654.3 - 649.4 for pH and glucose on the FT-MIR ATR instrument and sometimes they were adjacent (3 996.6 - 3 661.2, 3 663.5 - 3 327.7 and 3 327.2 - 2 322.3 for TSS and glucose, 1 988.3 - 1 652.8 and 1 654.3 - 649.4 for TSS, pH and TA. Other times they were overlapping (1 654.3 - 649.4 and 1 318.8 - 649.4) for pH, TA and fructose on the FT-MIR ATR instrument. This is a very good sign for transfer of this technology to a handheld device, where adjacent and/ or overlapping wavenumbers are crucial. Instruments which have to determine different parameters over large spectral ranges are not only impractical, because the instrument has to be big, but because it is also very expensive. Another advantage of implementing especially FT-NIR spectroscopy as a fast, accurate and inexpensive technique for determining harvest maturity and quality parameters is because no sample preparation is necessary and very little waste (few single berries tested) is produced. This is a pre-requisite which is highly recommended in the green era that we are currently living in and will do so for aeons to come. A platform of technologies has now been made available through this study for the determination of the respective parameters in future table grape samples by just taking their spectra on one of the instruments. Indeed something that has not been possible or available for the South African table grape industry before. Berries for the browning experiments were scanned on a FT-NIR instrument immediately after harvest (before cold storage) and again after cold storage. Before cold storage they were scanned on each side of the berry and after cold storage they were scanned twice on a brown spot if browning was present and twice on a clear spot, irrespective of whether browning was present or not. Inspection of the berries for the incidence of browning after cold storage revealed that Regal Seedless had a higher incidence of browning (68% in 2008 and 66% in 2009) than Thompson Seedless (21% in 2008 and 25% in 2009). Regal Seedless was also more prone to external browning, specifically netlike browning, whereas Thompson Seedless was more prone to internal browning, despite the different phenotypes of browning that were present on both. Principal component analysis (PCA) done on the spectra obtained before and after cold storage revealed that NIR can capture the changes related to cold storage with the first principal components explaining almost 100% of the variation in the spectra. Classification models also build using PCA was based on spectra of berries that remained clear before and after cold storage and those that turned brown after cold storage. Classification models of berries based on spectra obtained after cold storage (browning present) had a better total accuracy (94% for training- and 87% for test datasets), than the classification models based on spectra obtained before cold storage (79% for training- and 64% for test datasets). The implication of this is that the current models will be able to classify berries in terms of those which have turned brown already and those that remained clear better after cold storage than before cold storage, which is the critical stage where we want to actually know whether the berries will turn brown or not. The potential, however, to use NIR spectroscopy to detect browning before harvest already on white seedless grapes is still present, since all these models were built using the whole NIR spectrum. No variable selection was thus done and all the different browning phenotypes were also used together. Further analysis of the data will thus be based on using variable selection techniques like particle swarm optimization (PSO) to select certain wavelengths strongly associated with the browning phenomenon and only on the main types of browning (netlike on Regal Seedless and internal browning on Thompson Seedless). This study has major implications for the table grape industry, since it is the first time that the possibility to predict browning with other methods than visual inspection, especially before cold storage, is shown. / AFRIKAANSE OPSOMMING: Die twee wit pitlose tafeldruif kultivars, Regal Seedless en Thompson Seedless onderskeidelik, speel 'n baie belangrike rol in die verkryging van buitelandse inkomste, nie net vir die Suid- Afrikaanse tafeldruif industrie nie, maar ook vir die Suid-Afrikaanse ekonomie as 'n geheel. Hierdie twee kultivars is egter, soos baie ander wit kultivars, ook geneig tot verbruining. Dit is veral netagtige verbruining op Regal Seedless en interne verbruining op Thompson Seedless wat pertinent is. Hierdie belangrike kwaliteitsprobleme lei jaarliks tot groot finansiële verliese, aangesien daar huidiglik geen gevestigde prosedure is om voor oes, tydens opberging of tydens verpakking te bepaal of die druiwe uiteindelik gaan verbruin nie. Met ander woorde, daar is geen gevestigde protokol vir die beoordeling van die verbruinings risiko van 'n bepaalde groep druiwe voor dit uitgevoer word nie. Talle studies is alreeds onderneem om vas te stel wat die presiese oorsaak van hierdie verskynsel is en hoe dit bestuur moet word, maar geen enkele aspek wat bestudeer is kon tot op hede, herhaaldelik ge-assosieer word met die presiese oorsaak van verbruining nie. Die oorkoepelende doel van hierdie studie was dus om 'n alternatiewe manier te kry om hierdie probleem aan te spreek. ‘n Ondersoek na die potensiaal van naby infrarooi (NIR) spektroskopie as 'n vinnige en nie-vernietigende metings tegniek om die verbruinings potensiaal van ‘n wit pitlose tafeldruifkorrel wat nog heel is te bepaal, is onderneem. 'n Sekondêre doel was om die bepaling van optimale rypheid van tafeldruiwe te onderosek. Op hierdie manier is oesrypheid, en die kwaliteitsfaktore, naamlik totale oplosbare vastestowwe (TOVS), titreerbare suur (TS), pH, glukose en fruktose, wat ook gekoppel word aan die voorkoms van verbruining, deur middel van infrarooi (IR) spektroskopie modelle gekwantifiseer. Drie verskillende infrarooi metodes naamlik (a) die Fourier transform naby infrarooi (FT-NIR), (b) Fourier transform - Mid Infrarooi (FT-MIR) en (c) Fourier transform - Mid Infrarooi Verswakte Totale Refleksie (FT-MIR VTR) spektroskopie is gebruik om die aspekte te bepaal. Dis gedoen sodat 'n platform van tegnologie beskikbaar sou wees vir die tafeldruif industrie. Die druiwe wat in hierdie studie gebruik is, is oor twee jaar (2008 en 2009) en van twee verskillende kommersiële wingerde in die Hexriviervallei, Wes-Kaap, Suid-Afrika ge-oes. Verskillende oesladings (die totale aantal trosse op die wingerdstokke per hektaar) is vir Regal Seedless (75 000, 50 000 en 35 000) en Thompson Seedless (75 000 en 50 000) gelaat. Daar is drie rye gebruik Regal Seedless en twee vir Thompson Seedless. Elke ry het ses vakkies gehad wat dan verteenwoordigend was van ‘n herhaling vir elke oeslading. In 2008 is hierdie kultivars by vroeë rypwording (16°Brix), by optimale rypheid (18°Brix) en by laat rypheid (20°Brix) geoes. In 2009 is dit twee keer by die optimale rypheidsgraad geoes. Vir die bepaling van oesrypheid, en die kwaliteitsapekte is verskillende sagteware (OPUS 6.5 op die FT-NIR en FT-MIR VTR instrumente en Unscrambler weergawe 9.2 vir die FT-MIR instrument) sowel as verskillende spektrale voor-verwerking tegnieke ëvalueer voor die konstruksie van die kalibrasie modelle op die verskillende instrumente. Parsiële kleinste kwadraat (PKK) regressie is gebruik vir die opstel van kalibrasiemodelle vir die bepaling van laasgenoemde aspekte. Verskillende statistieke gegewens is gebruik om die kalibrasie modelle te evalueer, naamlik die bepalingskoëffisiënt (R2), die vierkantswortelgemiddelde- kwadraat fout vir voorspelling (VGKV), relatiewe voorspellingsafwyking (RVA) en sydigheid. Kalibrasie modelle wat geskik is vir keuring is verkry op die FT-NIR en FT-MIR VTR instrumente vir TOVS (11.40 – 21.80°Brix) (R2 = 85.92%, VGKV = 0.71°Brix, RVA = 2.67 en sydigheid = 0.03°Brix), pH (2.94 – 3.9) (R2 = 85.00%, VGKV = 0.08 g/L, RVA = 2.59 en sydigheid = -0.01 g/L), en TS (4.3 – 13.1 g/L), (R2 = 90.77%, VGKV = 0.48 g/L RVA = 3.30 en sydigheid = -0.03 g/L). Modelle vir fruktose (46.70-176.82 g/L) (R2 = 74.66%, VGKV = 9.28 g/L RVA = 2.00 en sydigheid = 1.10 g/L) en glukose (20.36 – 386.67 g/L) (R2 = 70.71%, VGKV = 11.10 g/L RVA = 1.87 en sydigheid = 1.64 g/L) is verkry met die FT-NIR en FT-MIR VTR instrumente wat in sommige gevalle gepas was vir keuringsdoeleindes en in sommige gevalle nie geskik was vir kwantifiserings doeleindes nie. Die FT-MIR-instrument het modelle vir al die aspekte gegee wat nog nie vir kwantifiserings doeleindes of vir keuringsdoeleindes geskik was nie. Gekombineerde spektrale reekse is gebruik vir die kalibrasies wat dikwels soortgelyk was vir sommige aspekte naamlik 12 493 - 5 446.2 vir TOVS en pH, 6 101.9 - 5 446,2 vir TOVS, TS en fruktose en 4 601.5 - 4 246.7 vir pH en fruktose op die FT-NIR instrument, 2 993.2 - 2 322.3 vir pH, TA en glukose en 1 654.3 – 649.4 vir pH en glukose op die FT-MIR VTR instrument. Andersyds, was dit aangrensend (3 996.6 - 3 661.2, 3 663.5 - 3 327.7 en 3 327.2 - 2 322.3) vir TOVS en glukose, 1 988.3 - 1 652.8, 1 654.3 – 649.4 vir TOVS, pH en TS en ander tye was dit weer oorvleuelend 1 654.3 – 649.4 en 1 318.8 – 649.4 vir pH, TS en fruktose op die FT-MIR VTR instrument. Dit is 'n baie goeie teken vir die oordrag van hierdie tegnologie na ‘n handgedraagde instrument, waar aanliggende en/of oorvleuelende golfnommers noodsaaklik is. Instrumente wat verskillende aspekte oor groot spektrale reekse moet bepaal is nie net onprakties, omdat die instrument groot moet wees nie, maar dit is ook baie duur. Nog 'n voordeel van die implementering van veral FT-NIR spektroskopie as 'n vinnige, akkurate en goedkoop tegniek vir die bepaling van oesrypheid, en die kwaliteit aspekte van druiwe is omdat daar geen monster voorbereiding nodig is nie en baie min afval (paar enkele korrels word gemonster) geproduseer word. 'n Voorvereiste wat sterk aanbeveel kom in die groen era waarin ons tans leef en nog vir eeue van nou af gaan doen. ‘n Platform van tegnologie is nou beskikbaar gestel deur middel van hierdie studie vir die bepaling van die onderskeie aspekte in toekomstige tafeldruif monsters deur net op een van die instrumente hulle spektra te neem. Inderdaad iets wat nie voorheen moontlik of beskikbaar was vir die Suid- Afrikaanse tafeldruif industrie nie. Korrels vir die verbruiningseksperimente is geskandeer direk na oes (voor koelopberging) en weer na koelopberging. Dit was voor koelopberging op elke kant van die korrel skandeer en na koelopberging was dit twee maal skandeer op 'n bruin vlek indien verbruining teenwoordig was en twee keer op 'n helder plek, ongeag of verbruining teenwoordig was of nie. Inspeksie van die korrels vir die voorkoms van verbruining na koelopberging het aan die lig gebring dat Regal Seedless 'n hoër voorkoms van verbruining (68% in 2008 en 66% in 2009) as Thompson Seedless (21% in 2008 en 25% in 2009) gehad het. Regal Seedless was ook meer geneig om eksterne verbruining, spesifiek netagtige verbruining te vertoon, terwyl Thompson Seedless meer geneig was om interne verbruining te vertoon, ten spyte van die verskillende fenotipes van verbruining wat teenwoordig was op beide kultivars. Hoofkomponente analise (HKA) is op die spektra gedoen voor en na koelopberging en naby infrarooi spektroskopie het aan die lig gebring dat die veranderinge wat verband hou met koelopberging met die eerste hoofkomponent (HK) verduidelik kan word met byna 100% van die variasie in die spektra wat daarin vasgevang is. Klassifikasiemodelle is ook deur die gebruik van HKA gebou en was gebaseer op die spektra van korrels wat vekry is voor en na koelopberging asook die wat verkry is nadat korrels verbruin het na koelopberging. Klassifikasiemodelle van korrels wat gebaseer was op spektra na koelopberging (verbruining teenwoordig) het 'n beter algehele akkuraatheid (94% vir opleidingsdata en 87% vir toetsdata), getoon as die klassifikasiemodelle wat gebaseer was op spektra van korrels voor koelopberging (79% vir opleidings data en 64% vir toetsdata). Die implikasie hiervan is dat die huidige modelle in staat sal wees om korrels beter te klassifiseer in terme van diegene wat alreeds verbruin het en die wat nie verbruin het na koelopberging as daardie voor koelopberging, wat juis die kritieke stadium is waar ons wil weet of die korrels wel gaan verbruin of nie. Daar is wel potensiaal wat verder ontgin kan word, aangesien al hierdie modelle gebou is deur gebruik te maak van die hele NIR spektrum. Geen veranderlike seleksie is dus gedoen nie en al die verskillende verbruiningsfenotipes is ook saam gebruik in die opstel van die modelle. Verdere analise van die data sal dus gebaseer word op die gebruik van veranderlike seleksie tegnieke soos deeltjie swerm optimisasie (DSO) wat sekere golflengtes kies wat sterk verband hou met die verbruining verskynsel en slegs die belangrikste tipes van verbruining (netagtig op Regal Seedless en interne verbruining op Thompson Seedless) sal gebruik word. Hierdie studie het 'n baie belangrike implikasie vir die tafeldruifbedryf, want dit is die eerste keer dat die moontlikheid om verbruining te voorspel met ander metodes as visuele inspeksie, veral voor koelopberging, getoon word. / The Postharvest and Innovation Programme, for financing this study

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The relationships between leaf and fruit represent a fundamental concept in perennial plants. This concept allows to understand and to manage, with regard to farming, the balance of a vine, which is important in terms of fruit quality (i.e. fruit composition), mainly when it comes to producing wines of different categories and styles. The understanding of vine structure, physiology and vine functioning ultimately allows for appropriate recommendations to be given with regard to farming procedures. These include the adaptation of the canopy architecture to achieve a certain yield per vine, the determination of an appropriate fruit microclimate as well as the prediction of harvest dates. One of the central notions of vine balance involves the relationship between the source and the sink organs. The definition of source-sink relationships incorporates several concepts, including the ability of a source tissue to produce carbohydrates through photosynthesis, the transport of these carbohydrates to various plant organs-tissues via appropriate transport channels, and the assimilation and storage of the carbohydrates in the sink organs. In past years, a number of simple ratios have been created to incorporate the relationship between source and sink organs and thereby define vine balance in order to aid in practical management decisions (choice of a training system, irrigation, canopy manipulation etc.). However, vine functioning is very complex and cannot be defined accurately by simple, static ratios. More integrated and dynamic physiological indicators of vine balance and functioning are needed in order to understand the complex communication between organs and ultimately improve on farming practices. In order to achieve this, a better grasp of source-sink relationships, including the signalisation between organs and the functioning of the transport tissues is required. A two year experiment was proposed to study the interaction between source and sink organs using a combination of both primary shoot girdling methods and berry classification according to size. Girdling removes the bark and phloem tissue, thereby interrupting carbon import as well as water flow to the bunch to a certain degree. The aim of the study was to demonstrate the complexity of vine functioning by investigating the dynamics of berry sugar and water accumulation (used as physiological indicators) and the influence thereof on berry fresh mass evolution. Furthermore, the use of berry sugar loading was proposed as an improved physiological indicator of vine balance as it is directly linked to source and sink functioning. Sugar production and the dynamics of berry sugar accumulation rely on photosynthesis which in turn is dependent on stomatal conductance and therefore also incorporates the effects of external abiotic factors (temperature, light and water). It furthermore gives a direct indication of sink functioning as it shows the progressive accumulation of sugar throughout the ripening period and the possible consequences on berry volume evolution. A primary shoot which bore two bunches was used to represent a biological replicate. The lower bunches were girdled above and below in order to completely isolate them from any carbohydrate import. These bunches, along with the upper ungirdled bunches and two control bunches from another shoot were sampled. The berries from these bunches were classified according to diameter, thereby providing the unique opportunity to study berries of the same volume/size. Measurements were done to determine the fresh and dry masses of the sampled berries, as well as to analyse the concomitant sugar concentrations. It was found that girdling clearly had an effect on berry sugar dynamics and the method was improved in the second year of the trial. Girdling in interaction with berry classification according to diameter demonstrated that berries from the same size could have different sugar concentrations. It further showed that, to a certain degree, a relationship exists between the first rapid phase of sugar accumulation and the post véraison increase in berry fresh mass, until the plateau of fruit sugar accumulation, which generally occurs around a sugar concentration of 20 Brix. Additionally, and more importantly, it was found that vine functioning and the balance between the source and the sink organs may be controlled to a certain degree. There is a strong degree of compensation within a vine which results from signalling between and within organs. When taking the results of this study into consideration, it becomes clear that the classical ratios used to quantify the complex relationships between the fruit and the leaves may not be completely adequate to do so. The current way of looking at source-sink relationships and thereby determining whether a vine is balanced or not is over-simplified and there are numerous limitations involved in this approach. The vine is far more complex and various aspects must be taken into consideration before any claims can be made concerning source-sink relationships and consequently leaf to fruit balance. / AFRIKAANSE OPSOMMING: Die verhoudings tussen blaar en vrug verteenwoordig ‘n fundamentele konsep in meerjarige plante. Begrip van hierdie konsep maak dit moontlik om in boerdery die balans van ‘n wingerdstok te verstaan en bestuur. Hierdie wingerdbalans is belangrik in terme van vrugkwaliteit (d.w.s. vrugsamestelling), hoofsaaklik met betrekking tot die produksie van wyne van verskillende kategorieë en style. Begrip van die wingerdstok se struktuur, fisiologie en funksionering maak dit moontlik om gepaste aanbevelings te maak rakende boerdery prosedures. Dit sluit in die aanpassing van die lower argitektuur om ‘n sekere opbrengs per wingerdstok te verkry, die vasstel van ‘n geskikte vrug mikroklimaat asook die voorspelling van oesdatums. Een van die sentrale denkwyses rondom wingerdstok funksionering behels die die bron-vragpunt verhouding. Die definisie van bron-vragpunt verhoudings inkorporeer verskeie konsepte, insluitende die vermoë van ‘n bronweefsel om koolhidrate te produseer deur fotosintese, die vervoer van hierdie koolhidrate na verskeie plantorgaan weefsels via die gepaste vervoerkanale asook die opname en berging van hierdie koolhidrate in die vragpunt organe. In die verlede is ‘n aantal eenvoudige verhoudings geskep om die verband tussen die bron en vragpunt organe te beskryf en sodoende die wingerdstokbalans te definieer met die doel om ondersteuning te bied in praktiese bestuursbesluite (die keuse van opleistelsel, besproeiing, lowermanipulasie, ens.). Wingerdstok funksionering is egter baie kompleks en kan nie akkuraat gedefinieer word deur eenvoudige, statiese verhoudings nie. Meer geïntegreerde en dinamiese fisiologiese aanwysers van wingerdstokbalans en funksionering is nodig om die komplekse kommunikasie tussen organe te verstaan en uiteindelik boerdery praktyke te verbeter. Om dit te bereik is ‘n beter begrip van bron-vragpunt verhoudings asook die seinoordrag tussen organe en die werking van die vervoerweefsels nodig. ‘n Twee jaar lange eksperiment is voorgestel om die interaksie tussen bron- en benuttingsorgane te ondersoek deur gebruik te maak van beide die primêre loot ringelering metode en korrel klassifikasie volgens grootte. Ringelering verwyder die bas en floëem weefsel en onderbreek sodoende koolstof invoer sowel as watertoevoer na die tros tot ‘n sekere mate. Die doel van die studie was om die kompleksiteit van wingerdstok funksionering aan te toon deur die dinamika van suiker en water akkumulasie in die korrel te ondersoek asook die invloed daarvan op korrel vars massa ontwikkeling. Verder is die gebruik van korrel suikerlading voorgestel as ‘n beter fisiologiese aanduiding van wingerdstok funksionering aangesien dit direk geassosieer is met bron-vragpunt funksionering. Suikerproduksie en die dinamika van suiker akkumulasie in die korrel berus op fotosintese wat weer afhanklik is van stomatale geleiding en daarom ook die effek van eksterne abiotiese faktore (temperatuur, lig en water) inkorporeer. Dit gee verder ‘n direkte aanduiding van die funksionering van die vragpunt organe omdat dit die progressiewe akkumulasie van suiker gedurende die rypwordingsperiode aantoon, asook die moontlike gevolge op korrelvolume ontwikkeling. ‘n Primêre loot wat twee trosse dra is gebruik om ‘n biologiese herhaling te verteenwoordig. Die laer trosse is bo en onder geringeleer om hulle heeltemal te isoleer van enige koolhidraat invoer. Hierdie trosse, tesame met boonste ongeringeleerde trosse en twee kontrole trosse vanaf ‘n ander loot is gemonster. Die korrels van hierdie trosse is geklassifiseer volgens hulle deursnee, om sodoende die unieke moontlikheid daar te stel om korrels van dieselfde volume/ grootte te bestudeer. Metings is gedoen om die vars en droë massas van die gemonsterde korrels te bepaal, asook om die gepaardgaande suikerkonsentrasies te analiseer. Daar is gevind dat ringelering duidelik ‘n effek gehad het op korrelsuiker dinamika en die metode is verbeter in die tweede jaar van die proef. Ringelering in wisselwerking met korrel klassifikasie volgens korrel deursnee het aangetoon dat korrels met dieselfde grootte verskillende suikerkonsentrasies kon hê. Dit het verder aangedui dat daar, tot ‘n sekere mate, ‘n verhouding bestaan tussen die vinnige fase van suiker akkumulasie en die na-véraison toename in korrel vars massa, totdat die plato in suiker akkumulasie bereik word, gewoonlik rondom ‘n suikerkonsentrasie van 20 Brix. Daarbenewens, en van groter belang, is gevind dat wingerdstok funksionering en die balans tussen die bron en vragpunt organe onder ‘n mate van beheer is. Daar is ‘n sterk mate van kompensasie binne ‘n wingerdstok wat die gevolg is van seinoordrag tussen en binne organe in die wingerdstok. Wanneer die resultate van hierdie studie in aanmerking geneem word, word dit duidelik dat die klassieke verhoudings, wat gebruik word om wingerdstok funksionering en balans mee te bepaal, moontlik nie beduidend betekenisvol is nie. Die wyse waarop bron-vragpunt verhoudings tans beskou word is, tot ‘n mate, ‘n oorvereenvoudiging en daar is heelwat beperkinge betrokke by hierdie benadering. Die wingerd is baie meer kompleks en verskeie aspekte moet in aanmerking geneem word voordat enige bewering gemaak kan word rakende bron-vragpunt verhoudings. / The University of Stellenbosch and Winetech for financial support

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The conversion of sugar into ethanol and carbon dioxide is a process that has been intertwined with human culture and long as civilized man has existed. This fermentation process has been dominated by the micro-organism Saccharomyces cerevisiae and from providing ancient seafaring explorers of a non perishable beverage to equipping bakers with a raising agent to turn flour into bread; this organism with its fermentative potential, has formed an essential part of most societies. In more recent times, many industries still rely on this basic principle. The complexities and efficiencies of the conversion of sugar into its various fermentative byproducts have been studied and optimised extensively to meet the specific demands of industries. Depending on the raw material used as starting point, the major beneficiaries of the useful characteristics have been alcoholic beverage producers (wine, beer, and whiskey amongst others), bakers (bread leavening) and biofuel producers. One of the obstacles in fermentation optimisation is the sugar consumption preferences displayed by the organism used. S. cerevisiae can consume a wide variety of sugars. Depending on the complexities of its structures, it shows a preference for the simpler saccharides. The fermentation of certain more complex sugars is delayed and runs the risk of being left residually after fermentation. Many of the crops utilised in fermentation-based products contain large amounts of starch. During the starch degradation process many different forms of sugars are made available for fermentation. Improved fermentation of starch and its dextrin products would benefit the brewing, whiskey, and biofuel industries. Most strains of Saccharomyces ferment glucose and maltose, and partially ferment maltotriose, but are unable to utilise the larger dextrin products of starch. This utilisation pattern is partly attributed to the ability of yeast cells to transport the aforementioned mono-, di- and trisaccharides into the cytosol. The inefficiency of maltotriose transport has been identified as the main cause for residual maltotriose. The maltotriose transporting efficiency also varies between different Saccharomyces strains. By advancing the understanding of maltotriose transport in yeast, efforts can be made to minimise incomplete fermentation. This aim can be reached by investigating the existing transporters in the yeast cell membrane that show affinity for maltotriose. This study focuses on optimising maltotriose transport through the comparison of the alpha glucoside transporter obtained from different strains of Saccharomyces. Through specific genetic manipulations the areas important for maltotriose transport could be identified and characterised. This study offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer, whiskey, and biofuel industries. / AFRIKAANSE OPSOMMING: Die transformasie van suiker na etanol en koolstof dioksied is so oud soos die beskawing self, en dit is van die vroegste tye af onlosmaaklik met die mens se kultuur verbind. Hierdie fermentasie-proses word gedomineer deur die Saccharomyces cerevisiae mikroorganisme. Hierdie organisme het antieke seevaarders voorsien van ‘n nie-bederfbare drankie en van ouds af aan bakkers ‘n rysmiddel verskaf waarmee meel in brood verander kon word. As gevolg van hierdie fermenteringspotensiaal het hierdie organisme ‘n onmisbare rol in meeste beskawings gespeel. Baie industrieë is steeds op hierdie basiese beginsel gebou. Die kompleksiteite en effektiwiteit van die transformasie van suiker na sy verskeie gefermeenteerde neweprodukte is breedvoerig bestudeer en geoptimiseer om aan die spesifieke behoeftes van verskeie industrieë te voeldoen. Afhangend van die grondstowwe wat as beginpunt gebruik is, is die primêre begunstigdes van die fermentasie proses die alkoholiese drankprodusente (onder andere die wyn-, bier- en whiskey produsente), bakkers en biobrandstofprodusente. Die suikerverbruik-voorkeur van die organisme wat die fermentering fasiliteer is een van die struikelblokke in die optimisering van die proses. S. cerevisiae kan ‘n wye spektrum van suikers verbruik maar dit toon ‘n voorkeur vir die eenvoudiger suikers. Die fermentasie van sekere van die meer komplekse suikers is vertraag en loop die risiko om agtergelaat te word na fermentasie. Vele van die gewasse wat in die gefermenteerde produkte gebruik word bevat groot hoeveelhede stysel. Vele soorte suikers word gedurende die afbreek van die stysel beskikbaar gestel vir fermentasie. Die brouers-, whiskey- en biobrandstof industrieë sal almal voordeel trek uit die verbeterde fermentasie van stysel en sy gepaardgaande dekstrin produkte. Meeste Saccharomyces gisrasse fermenteer glucose en maltose; maltotriose word gedeeltelik gefermenteer, maar die meer komplekse dekstrien produkte gevind in stysel word nie gefermenteer nie. Hierdie verbruikerspatroon kan gedeeltelik toegeskryf word aan die vermoë van gisselle om die bogenoemde mono-, di- and trisaccharides in die sitosol op te neem. Die oneffektiwiteit van maltotriose transport is identifiseer as die hoofoorsaak van post-fermentatiewe, oortollige maltotriose. Die effektiwiteit van maltotriose transport verskil ook tussen verskillende Saccharomyces rasse. Pogings om onvolledige fermentasie te veminder kan bevorder word deur die kennis rondom maltotriose transport in gis uit te bou. Hierdie oogmerk kan bereik word deur die bestaande transporters in die gissel se membraan wat ‘n affiniteit vir maltotriose toon te ondersoek. Hierdie studie fokus op die optimisering van maltotriose transport deur die vergelyking van die alpha glucoside transporter (AGT1) wat van verskillende Saccharomyces rasse afkomstig is. Die areas wat relevant is tot maltotriose transport kon deur spesifieke genetiese manipulasies identifiseer en gekarakteriseer word. Hierdie studie bevorder die vooruitsig op die ontwikkeling van gisrasse met verbeterde maltose en maltotriose transport vermoëns wat op sy beurt weer kan aanleiding gee tot die verbeterde fermentasie effektiwiteit in die bier, whiskey en biobrandstof industrieë.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The yeast Brettanomyces was isolated from beer in 1904 and associated with wine thereafter. A sporulating form, Dekkera, was discovered later. Brettanomyces bruxellensis produces high levels of volatile phenol off-flavours in wine. Sulphur dioxide (SO2) is the most widely used chemical preservative in wine. Yeasts have several mechanisms to cope with the SO2, namely Ssu1p, a membrane bound SO2 transporter; sulphite reduction, sulphite oxidation and acetaldehyde production. In unfavourable environmental conditions, certain yeasts can enter a viable-but-non-culturable (VBNC) state which is characterised by reduced metabolic rate, inability to reproduce on solid media and a reduction of cell size. VBNC can be triggered by chemical stress such as high SO2 levels. The objectives of this study were to examine the SO2 tolerance of B. bruxellensis and Saccharomyces cerevisiae, to quantify their rates of SO2 accumulation and efflux, determine the effect of SO2 on their energy metabolism and investigate if B. bruxellensis possesses an orthologue to S. cerevisiae SSU1. In this study, the identity of a number of Brettanomyces/Dekkera strains was confirmed using 5.8S rDNA-ITS RFLP analysis and DNA sequencing. Sporulation assays were used to confirm whether these strains belonged to the Dekkera or Brettanomyces genus. A method to accurately quantify SO2 in laboratory conditions was optimised. Molecular SO2 tolerance was tested by spotting fresh yeast cultures on media with SO2 and/or ethanol. Tolerance to SO2 and/or ethanol showed highly strain dependent results with S. cerevisiae showing the highest tolerance levels while B. bruxellensis tolerated SO2 and ethanol poorly but certain strains grew well with only SO2. The SO2 accumulation and efflux rates of 3 S. cerevisiae strains and 3 B. bruxellensis strains were determined. It was shown that the S. cerevisiae strains followed the same trends as previously found in literature whereas B. bruxellensis strains showed similar trends but displayed highly variable strain-dependent results. B. bruxellensis CB63 and S. cerevisiae VIN13 were investigated for their response to SO2 in two different media, TA and SWM, over a 48-hour and 32-day period respectively. Acetic acid, acetaldehyde, D-glucose, D-fructose (only in SWM) and ethanol (only in TA) were regularly monitored over the time course of each experiment. SO2 had the greatest impact on B. bruxellensis with decreased rates of glucose consumption and ethanol production as well as increased acetic acid. Acetaldehyde peaked shortly after SO2 addition with the subsequent restarting of sugar consumption for certain samples. This suggests that sufficient acetaldehyde was produced to bind free SO2 to reduce SO2 stress. Volatile phenols were quantified for day 32 of the SWM experiment. An increase of 4-ethyl guaiacol was correlated to higher molecular SO2 levels. SO2 negatively affected both yeasts energy metabolism, forcing the yeasts metabolism to adapt to ensure survival. In general, SO2 was shown to have a negative impact on all aspects of a yeasts growth and metabolism and that SO2 tolerance is highly strain dependent and a far more complicated characteristic than currently understood. / AFRIKAANSE OPSOMMING: Die gis Brettanomyces is in 1904 uit bier geïsoleer en daarna met wyn geassosieer. 'n sporulerende vorm, Dekkera, is later ontdek. Brettanomyces bruxellensis produseer hoë vlakke van vlugtige fenol afgeure in wyn. Swaweldioksied (SO2) is die mees gebruikte chemiese preserveermiddel in wyn. Giste het verskeie meganismes om SO2 te hanteer, naamlik Ssu1p, 'n membraan-gebonde SO2 transporter, sulfietvermindering, sulfiet-oksidasie en asetaldehiedproduksie. In ongunstige omgewingstoestande kan sekere giste 'n lewensvatbare, maar nie-kultiveerbare (LMNK)-toestand aanneem wat gekenmerk word deur verlaagde metaboliese tempo, onvermoë om voort te plant op soliede media en 'n vermindering van die selgrootte. LMNK kan veroorsaak word deur chemiese stres, soos hoë SO2-vlak. Die doelwitte van hierdie studie was om die SO2 -bestandheid van B. bruxellensis en Saccharomyces cerevisiae te ondersoek, hul spoed van SO2 -opneming/akkumulasie en -uitskeiding te kwantifiseer, die invloed van SO2 op energiemetabolisme te bepaal en te ondersoek of B. bruxellensis oor ‘n soortgelyke geen as die S. cerevisiae SSU1 beskik. In hierdie studie is die identiteit van 'n aantal Brettanomyces/Dekkera-stamme bevestig deur 5.8S rDNA-ITS RFLP-analise en DNA-opeenvolging te gebruik. Sporulasietoetse is gebruik om te bevestig of hierdie stamme aan die genus Dekkera of Brettanomyces behoort. 'n Metode om SO2 onder laboratoriumtoestande akkuraat te kwantifiseer, is geoptimiseer. Molekulêre SO2- bestandheid is getoets deur vars giskulture op media met SO2 en/of etanol te groei. Bestandheid teen SO2 en/of etanol het stam-afhanklike resultate getoon, S. cerevisiae wat die hoogste toleransievlakke getoon het, terwyl B. bruxellensis SO2 en etanol swak tolereer, maar sekere stamme het goed gegroei met slegs SO2. Die SO2-akkumulasie en -uitskeidingtempo van 3 S. cerevisiae-rasse en 3 B. bruxellensis-stamme is bepaal. Daar is gevind dat die S. cerevisiae-rasse dieselfde tendens soos voorheen in die literatuur beskryf, gevolg het, terwyl B. bruxellensis-stamme soortgelyke tendense getoon het,maar hoogs veranderlike stamafhanklike resultate vertoon. B. bruxellensis CB63 en S. cerevisiae VIN13 is ondersoek vir hul reaksie tot SO2 in twee verskillende media, TA en SWM, oor 'n tydperk van 48-uur en 32-dae onderskeidelik. Asynsuur, asetaldehied, D-glukose, D-fruktose (slegs in SWM) en etanol (slegs in TA) is gereeld gemoniteer oor die verloop van elke eksperiment. SO2 het die grootste impak op B. bruxellensis met ‘n verlaagde tempo van glukoseverbruik en etanolproduksie, sowel as verhoogde asynsuur. ‘n Asetaldehiedhoogtepunt is bereik kort na die SO2-byvoeging met die daaropvolgende hervatting van suiker wat vir sekere monsters gebruik is. Dit dui daarop dat voldoende asetaldehied geproduseer is om vry SO2 te bind om SO2-stres te verminder. Vlugtige fenole is op dag 32 van die SWM-eksperiment gekwantifiseer. 'n Toename van 4-etiel-guajakol korreleer met hoër molekulêre SO2-vlakke. SO2 het beide giste se energiemetabolisme negatief beïnvloed, wat die gis dwing om sy metabolisme aan te pas om oorlewing te verseker. Oor die algemeen het SO2 'n negatiewe impak op alle aspekte van giste se groei en metabolisme, en SO2-bestandheid is hoogs stam–afhanklik. Dit is ook 'n baie meer ingewikkelde kenmerk as wat tans verstaan word.

Thesis (MSc)--Stellenbosch University, 2012 / ENGLISH ABSTRACT: The aroma and flavour of wine are important aspects that form the basis for consumers’ organoleptic experience of wine. Therefore, an understanding of the chemical composition of wine aroma is of major importance, to establish possible links between wine chemistry, sensory attributes and consumer preference for a product. For this purpose analytical chemistry and multivariate techniques are indispensable tools for the metabolic profiling of wine. Chenin blanc is one of the most important South African export white wine varieties. However, despite its importance, very limited profiling of Chenin blanc aroma compounds has been done and information is restricted to isolated and dated reports on a few chemical compounds only. Therefore, the overall aim of this study was to obtain an in-depth view of the volatile chemical profile of this cultivar. The first task was to perform targeted volatile metabolic profiling of the three dry and offdry Chenin blanc styles, fresh and fruity, rich and ripe unwooded and rich and ripe wooded. To this end, a new, simple and robust liquid-liquid extraction technique using dichloromethane was developed and validated for extraction of analytes prior to gas chromatography flame ionization detection (GC-FID) analysis, to quantify 57 analytes in one rapid analytical procedure. This method was applied to profile 48 Chenin blanc wines. Very successful discrimination between the three styles, using the quantified volatile compounds, was obtained with two multivariate methods. These were partial least squares regression-discriminant analysis, (PLS-DA), as well linear-DA, using best subset selection for identifying the most important variables. According to the classification models, a higher content of maturation derived, malolactic fermentation derived and wood derived compounds were predominantly characteristic of the wooded wines. Higher content of some terpenes and ethyl esters were predominantly associated with the rich and ripe unwooded style Chenin blanc wines, while the fresh and fruity style were generally characterized by high levels of acetate esters. Secondly, untargeted analysis of 21 wines was done with gas chromatography mass spectrometry (GC-MS). Mathematical chromatography, using PARAllel FACtor analysis (PARAFAC and PARAFAC2), was applied to the GC-MS data for resolution of the complex chromatographic results by multi-way modeling, and to derive unbiased multivariate classification models of the three styles. This approach provided excellent style differentiation, without the arduous task of analysis of numerous standards and setting up of calibration curves, required by the targeted approach described above. Additionally, the data generated during this study will form part of the current South African wine aroma database, which does not contain any data regarding Chenin blanc at present. / AFRIKAANSE OPSOMMING: Die aroma en geur van wyn is belangrike aspekte aangesien dit die basis vorm van die wynverbruiker se organoleptiese ervaring van die produk. Derhalwe, is ‘n deeglike kennis van die chemiese samestelling van wynaroma baie belangrik, ten einde die korrelasies tussen wynchemie, sensoriese eienskappe en verbruikersvoorkeure te bepaal. Vir hierdie doel, is die insameling van analitiese chemiese data, tesame met multi-veranderlike tegnieke om relevante inligting uit die data te onttrek, onmisbaar vir die metaboliese profilering van wyn. Chenin blanc is huidiglik een van Suid-Afrika se belangrikste uitvoer wit wynvariëteite. Ten spyte hiervan, is daar tot hede egter baie min profilering van Chenin blanc se aromakomponente gedoen en die beskikbare inligting is beperk tot geïsoleerde en verouderde navorsingsbevindinge. In die lig van bogenoemde, is die oorkoepelende motivering vir hierdie studie dus om die vlugtige chemiese komponente se profiel in Chenin blanc wyn, in diepte te bepaal. Die eerste taak was die geteikende bepaling van die metaboliese profiel van drie droë of halfdroë Chenin blanc wynstyle, nl. vars en vrugtig, ryk en ryp ongehout, asook ryk en ryp gehout. Om dit te bereik, is ‘n nuwe eenvoudige en robuuste vloeistof-vloeistof ekstraksieprosedure met dichlorometaan ontwikkel, wat analise met gaschromatografie – vlamionisasie deteksie (GC-FID) voorafgaan, om die konsentrasies van 57 komponente in een vinnige analise te bepaal. Hierdie metode is gebruik om 48 Chenin blanc wyne te profileer. Deur gebruik te maak van multi-veranderlike data analitiese tegnieke, is die gekwantifiseerde vlugtige komponente data gebruik in diskriminant analise. Baie suksesvolle onderskeid tussen die drie style, is verkry deur gebruik te maak van twee multi-veranderlike metodes, naamlik: parsiële kleinste kwadrate regressie, diskriminant analise, asook liniêre diskriminant analise. Vir laasgenoemde analise, is die seleksie van die mees belangrike veranderlikes met beste sub-groep regressie bepaal. Volgens hierdie klassifikasie modelle is ‘n hoër inhoud van veroudering-, appelmelksuurgisting- en houtverwante aroma komponente baie kenmerkend in die houtbehandelde wyne. Hoër vlakke van sommige terpene en etiel esters was kenmerkend met betrekking tot ryk en ryp ongehoute Chenin blanc style, terwyl die vars en vrugtige style meer gekenmerk was met hoë vlakke van asetaat esters. Tweedens is ‘n seleksie van 21 wyne geanaliseer deur gebruik te maak van gaschromatografie – massa spektrometrie (GC-MS) in ‘n ongeteikende metaboliese profileringsbenadering. Wiskundige chromatografiese metodes, spesifiek, parallelle faktor analise (PARAllel FACtor analysis (PARAFAC and PARAFAC2)), was voorts gebruik om komplekse chromatogramme te prosesseer met wiskundige, multi-vlak modellering. Met hierdie nie-selektiewe benadering, is ook suksesvolle klassifikasiemodelle gegenereer vir die diskriminasie tussen die drie verskillende Chenin blanc style. Die voordeel van die ongeteikende GC-MS analise, gekoppel met die data hanterings- en prosesseringsprotokols in hierdie studie gebruik, is dat die arbeidsintensiewe taak om kalibrasiekurwes op te stel vir elke individuele komponent, soos wat vereis word in die geteikende benadering, nie nodig is nie. Die data wat ingewin is gedurende hierdie studie, sal ook bygevoeg word tot ‘n bestaande Suid-Afrikaanse aroma databasis, wat tans geen data aangaande Chenin blanc wyn bevat nie.

Thesis (MSc)--Stellenbosch University, 2007. / ENGLISH ABSTRACT: With ever increasing pressure on wine producers to lower the financial costs involved in winemaking to be able to compete in the market, all while maintaining a high level of wine quality, the focus on maintaining control over all aspects of the winemaking process are greatly emphasized. Malolactic fermentation (MLF) is one of the important processes in red wine production. The advantages of this process, when performed successfully, is widely known and accepted. One way to gain control over MLF is the use of MLF starter cultures. Starter cultures usually consist of Oenococcus oeni that has been isolated from grapes or wines and is in most cases available in a freeze-dried form ready for direct inoculation into the wine when MLF is desired. Starter cultures are induced into wine and usually ensure the immediate onset as well as a fast and clean execution of the process. Starter cultures used in South Africa are in most cases isolated from cooler viticultural regions in the Northern hemisphere. The constitution of wines from cooler viticultural regions, differ from those in South Africa, which has a warm climate. The most important difference is the acid content of the wines which is lower in South African must/wines and results into a higher pH. The three most important changes that develop in wine during MLF are a decrease in acidity due to the conversion of malic acid to the less harsh lactic acid, enhanced flavour and aroma of wine and an increase in the microbiological stability of wine. The decrease in acidity is very important for wines produced for grapes grown in cool viticulture regions. In South Africa though, the climate is warm and higher pH’s are present in the musts and wines and the de-acidification due to MLF is not the main aim but rather the microbiological stabilisation. One of the compounds that could be produced by lactic acid bacteria (LAB) is biogenic amines (BA’s). These compounds can be hazardous to human health. This thesis focussed on the performance of MLF starter cultures in high pH South African red wines. The first objective of the study was to stretch MLF starter cultures in high pH red wines of South Africa. Stretching means to use less than the prescribed dosage or the re-use of starter cultures. The difference in MLF rate, the influence of the natural occurring LAB and the levels of biogenic amines formed during MLF were determined for the different stretching treatments. The results showed that different rates in malic acid degradation were experienced between the treatments, but in all cases MLF fermentation was completed. Biogenic amines were formed at various levels and the influence of the natural occurring LAB also played a role. The second objective of the study was the evaluation of the effect of a wine isolated LAB (Lactobacillus) and an acetic acid bacteria (AAB), inoculated with a MLF starter culture had on MLF at different wine pH’s. It was found that especially in the case where the Lactobacillus was inoculated in combination with the MLF starter culture a possible stimulatory effect was experienced with regards to malic acid degradation rate. Biogenic amine concentration was measured at the end of MLF and it was found that no histamine and tyramine were formed in any of the treatments, while the putrescine and cadaverine levels were found to be at approximately similar levels for the different treatments. The third objective was to evaluate the possible influence of commercial tannin additions and a pectolytic enzyme on rate of MLF and phenolic composition of high pH red wine. The commercial tannins had possible inhibitory as well as stimulatory effects on the rate of malic acid degradation especially during the initial stages of MLF, with the highest dosage having the significant effect. The BA results showed difference in the levels produced due to tannin additions as well as strain differences could exist. The phenolic content showed a decrease in colour density, total red pigments, total phenolics and anthocyanins between AF and MLF. The fourth objective was to evaluate inoculation time of MLF starter cultures. The results showed that the fastest AF/MLF time was with simultaneous inoculation of the yeast and MLF starter cultures. It was also for this treatment where no histamine or tyramine was detected at the end of MLF compared to the other inoculation strategies (before the end of AF and after AF). This study generated a large amount of novel data which made a valuable contribution with regards to MLF in high pH red wines of South Africa. / AFRIKAANSE OPSOMMING: Die druk om wyne van hoë gehalte teen lae insetkoste te lewer om deel te bly van ’n kompeterende mark, plaas die fokus weer sterk op onder andere die beheer van alle aspekte van die wynmaak proses. Appelmelksuurgisting (AMG) is een van die belangrikste prosesse van rooiwyn produksie. Die voordele van AMG, in die geval van die suksesvolle implementering daarvan is vandag bekend en word geredelik aanvaar. Een van die metodes om beheer te verkry oor the proses van AMG is deur die gebruik van AMG aanvangskulture. AMG aanvangskulture bestaan uit Oenococcus oeni wat geïsoleer word vanaf druiwe of mos/wyn en is in meeste gevalle beskikbaar in ’n gevries-droogte vorm wat direk in wyn geïnokuleer kan word. Aanvangskulture word in wyn geïnduseer om die onverpose aanvang van AMG te bewerkstellig asook om ’n vinnige en skoon deurvoering van die proses te verseker. Die aanvangskulture wat in Suid-Afrika vir hierdie doeleinde gebruik word is in meeste van die gevalle verkry uit koue wingerdbou gebiede in die Noordelike Halfrond. Die samestelling van druiwe van koue wingerdbou gebiede en dié van Suid-Afrikaanse warm wingerdbou gebiede verskil. Die belangrikste verskil word ervaar in die suur inhoud, wat laer is in Suid-Afrikaanse druiwe en dus lei tot ‘n hoër pH inhoud. Die drie mees belangrikste veranderinge wat gedurende AMG in wyn plaasvind is die vermindering van die suur, as gevolg van die omskakeling van appelsuur na melksuur, die verbetering van die aroma en geur van wyn en die verbeterde mikrobiologiese stabiliteit. Die afname in suur is veral belangrik in wyne van koue wingerbou gebiede omdat die suur-inhoud daarvan soveel hoër is. In Suid-Afrika kan hierdie verlaging in suur egter lei tot ’n verdere verhoging in die pH wat plat wyne en uiteindelik ’n verlaging in die kwaliteit van wyn tot gevolg kan hê. Biogene amiene (BA) is verbinding wat melksuurbakterieë (MSB) kan vorm gedurende AMG en kan ernstige implikasies hê vir die mens se gesondheid. Hierdie tesis fokus op die evaluering van AMG aanvangskulture in hoë pH rooi wyne van Suid-Afrika. Die eerste doelwit gedurende hierdie studie was om AMG kulture te rek en die invloed daarvan in hoë pH rooiwyn te evalueer ten opsigte van the tempo van AMG, die rol van die natuurlike MSB te bestudeer asook om die vlak van biogene amiene te bepaal vir die verskillende behandelings. Die resultate het aan die lig gebring dat die rek van kulture verskille in die tempo van appelsuur afbraak tot gevolg het, maar dat AMG in alle gevalle wel suksesvol deurgevoer kon word. Die BA’e wat gevorm is, was teenwoordig in verskillende hoeveelhede. Die tweede doelwit was om die effekt van die gesamentlike inokulasie van ’n wyn geisoleerde MSB (Lactobacillus) asook ’n asynsuurbakterie (ASB) met ’n kommersiële AMG aanvangskultuur op AMG te evalueer. Hierdie eksperiment is uitgevoer by verskillende pH’s. Daar is gevind dat veral in die kombinasie inokulasie met die Lactobacillus, die tempo van appelsuur afbraak moontlik gestimuleer was. Geen histamien of tiramien is tydens AMG gevorm in hierdie eksperiment gevorm nie, terwyl putresien en kadaverien teenwoordig was teen ongeveer gelyke vlakke vir die behandelings. Die derde doelwit was om die moontlike invloed van kommersiële tannien toevoegings en die toevoeging van ’n pektolitiese ensiem te evalueer ten opsigte van AMG tempo die fenoliese samestelling van rooiwyn te bestudeer. Verskillende kommersiële tanniene het ’n moontlike sowel as inhiberende uitwerking gehad, veral gedurende die aanvanklike stadium AMG. Die grootste verskille is waargeneem in die behandelings waar die hoogste dosisse tannien bygevoeg is. Die BA resultate toon dat verkillende vlakke geproduseer was en dat hierdie verskille onstaan het as gevolg van verskille in tannien dosisse sowel as aanvangskulture. Die fenoliese inhoud het ’n afname in kleur intensiteit, totale rooi pigmente, totale fenole en antosianiene getoon vir die periode vanaf AF tot die einde van AMG. Die vierde doelwit was om the tyd van inokulasie van AMG aanvangskulture te bestudeer. Die resultate het getoon dat die vinningste tydperk van AF/AMG was ondervind in die geval waar die gis aanvangskulture gelyktydig met die AMG aanvangskulture geïnokuleer was. Geen histamine en tyramine het ook in hierdie behandeling ontwikkel nie, terwyl daar wel vlakke teenwoordig was in die ander behandelings (inokulasie net voor die einde van AF en na afloop van AF). Tydens hierdie studie is ’n groot hoeveelheid nuwe data geskep wat ‘n groot bydrae ten opsigte van AMG in hoë pH rooi wyne vanaf Suid-Afrika kan lewer.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Producers of South African (SA) dry and semi-dry Chenin blanc table wines are currently experimenting with winemaking techniques to modulate the flavours of the predominantly fruity styles of this genre. An important stage during wine style development is sensory profiling paired with consumer acceptance testing, before wine is produced on industrial scale. With those aforementioned goals in mind, this study was conducted in partnership with two commercial SA wine cellars. The main focus of the study was an investigation into the treatment effects of two winemaking techniques, respectively grape skin contact and natural fermentation, on the sensory profiles of experimentally produced Chenin blanc wines. Results obtained with descriptive sensory analysis (DA) of the wines were compared to those obtained by two rapid sensory profiling methods, namely projective mapping (PM) and frequency of attribute citation (FC). A consumer preference study was also done on the wines. In order to understand the treatment effects better, the dominant non-Saccharomyces yeasts that were present during the natural fermentations were identified using polymerase chain reaction (PCR), while the major volatile chemical compounds were identified with chromatography and mass spectrometry. The sensory and chemical profiles of the naturally fermented wines were significantly different from those of the inoculated wines. PCR analysis identified some of the yeasts present during alcoholic fermentation. In comparison to the inoculated fermented wines, the naturally fermented wines were generally perceived to have more intense and riper tropical fruity aromas, with enhanced sweetness and reduced intensities of sourness, bitterness and astringency. The wines fermented on the skins (FoS) had lower ester and volatile thiol concentrations than the control wines (with no skin contact) and limited skin contact wines. Sensory attributes linked to the FoS wines included aromas of dried fruit, dried grass and a vegetative character, with an increased sour and bitter taste and astringent mouthfeel. In contrast, the wines that were produced with limited skin contact (12 hours) retained their tropical fruity aromas better than the FoS wines. Limited skin contact seemed to have had a less harsh effect on the taste and mouthfeel than the FoS wines. A consumer study was done to establish a Generation Y consumer group’s (18-35 years) preference for the different treated wines. Overall, the naturally fermented wines, which were described as having a strong tropical fruit character, were preferred. The FoS wines were generally disliked by the consumers. Results obtained from the three sensory analysis methods, respectively DA, PM and FC, were similar, thereby confirming the suitability of the rapid methods PM and FC, to extract qualitative information from the sensory profiling of white wine. The results of this study made a significant contribution towards validation of rapid sensory methods for wine evaluation, which are particularly valuable in the context of sustainability and technology transfer to research and industry alike. The knowledge gained on the chemical profiles of SA Chenin blanc is novel and this is one of the first reports on the volatile thiol content of SA Chenin blanc wine. / AFRIKAANSE OPSOMMING: Produsente van Suid-Afrikaanse (SA) droë en semi-droë Chenin blanc tafelwyne eksperimenteer tans met wynmaaktegnieke om die oorwegend vrugtige style van hierdie genre te varieer. ‘n Belangrike fase van wynstylontwikkeling is sensoriese profilering van die produkte met gepaardgaande verbruikersaanvaardingstoetse, voordat die wyne op industriële skaal geproduseer word. Met dié bogenoemde doelwitte in gedagte, was hierdie studie in vennootskap met twee kommersiële SA wynkelders gedoen. Die hooffokus van die studie was om die invloed van twee wynmaaktegnieke, onderskeidelik druifdopkontak en natuurlike fermentasie, op die sensoriese profiele van eksperimenteel geproduseerde Chenin blanc wyne te ondersoek. Resultate wat met beskrywende sensoriese analise (DA) van die wyne verkry is, is vergelyk met dié wat deur twee vinniger sensoriese profileringsmetodes metodes, naamlik projektiewe kartering (PM) en frekwensie van kenmerkaanhaling (FC) verkry is. 'n Verbruikervoorkeurstudie is ook op die wyne gedoen. Ten einde die behandelingseffekte beter te verstaan, is die dominante nie-Saccharomyces giste wat teenwoordig was tydens die natuurlike fermentasie geïdentifiseer met behulp van die polimerasekettingreaksie (PCR), terwyl die vernaamste vlugtige chemiese verbindings geïdentifiseer is met chromatografie en massaspektrometrie. Die sensoriese en chemiese profiele van die natuurlike gefermenteerde wyne was beduidend anders as dié van die geiinokuleerde wyne. PCR analise het sommige giste wat teenwoordig was tydens alkoholies fermentasie geïdentifiseer. In vergelyking met die geiinokuleerde wyne, het die natuurlik gefermenteerde wyne intenser en ryper tropiese vrugtige aromas getoon, met ‘n verhoogde perspesie van soetheid en laer intensiteite van suurheid, bitterheid en vrankheid. Die wyne wat berei is deur fermentasie op die druifdoppe (FoS) het laer konsentrasies van esters en vlugtige swawelverbinding gehad, as die kontrole wyne (geen dopkontak) en beperkte dopkontak wyne. Sensoriese eienskappe gekoppel aan die FoS wyne was geasssosieer met gedroogde vrugte, gedroogde gras en 'n vegetatiewe aroma, met gepaardgaande verhoogde suur en bitter smaakpersepsies en ‘n frank mondgevoel. In teenstelling, het die wyne wat geproduseer is met 'n beperkte dopkontak (12 uur) hul tropiese vrugtige aroma beter behou as die FoS wyne. Beperkte dopkontak het ook ‘n minder negatiewe uitwerking op die smaakpersepies van bitterheid en vrankheid gehad. ‘n Verbruikerstudie is gedoen om 'n groep jong Generasie Y verbruikers (18-35 jaar) se voorkeure vir die verskillende behandelings te bepaal. Oor die algemeen is die natuurlike gefermenteerde wyne, wat beskryf is met 'n sterk tropiese vrugtekarakter verkies, terwyl die groep nie van die FoS wyne gehou het nie. Resultate wat verkry is met drie sensoriese analise metodes, onderskeidelik DA, PM en FC, was soortgelyk en die geskiktheid van die vinniger metodes, PM en FC, om kwalitatiewe inligting van witwyn se sensoriese eienskappe te verkry, is bevestig. Die resultate verkry met hierdie studie maak ‘n beduidende bydrae tot die validering van vinniger sensoriese metodes vir wynevaluering, wat veral waardevol is in die konteks van volhoubaarheid en tegnologieoordrag na navorsing en die industrie. Die nuwe kennis wat in hierdie studie gegenereer is met die chemiese profilering van SA Chenin blanc wyn is baie waardevol en hierdie is een van die eerste narvorsingsprojekte oor die inhoud van vlugtige swawelverbings in SA Chenin blanc wyn.

Dissertation (PhD(Agric)) -- Stellenbosch University, 2005. / ENGLISH ABSTRACT: Due to increased consumer demand for products labelled by origin, and the requirement that these labels are a guarantee of both quality and product character, there is an increasing global focus on delimitation of denominations of origin. The integrity of denominations of origin and their defensibility can be ensured through the use of terroirs as a basis for delimitation. The aims of this study were to establish the dominant environmental criteria that affect the viticultural behaviour and wine character of two important cultivars (Cabernet Sauvignon and Sauvignon blanc) in the Stellenbosch Wine of Origin District, to use an appropriate methodology to identify viticultural terroirs in this district based on these criteria and with the use of a geographic information system, and finally to use these viticultural terroirs to identify denominations of origin within the same area. A terroir can be defined as a grouping of homogenous environmental units, or natural terroir units, based on the typicality of the products obtained. Identification and characterisation of terroirs depends on knowledge of environmental parameters, the functioning of the grapevine and the characteristics of the final product. Field studies, resulting in point data, are necessary to investigate the functioning of the grapevine but in order for this information to be of use within zoning studies it must be placed in a spatial context. As a first phase in data acquisition, the Stellenbosch Wine of Origin District was characterised and natural terroir units were identified using existing digital data and a geographic information system. A natural terroir unit (NTU) can be defined as a unit of land that is characterised by relatively homogenous topography, climate, geological substrate and soil. A total of 1389 NTUs were identified in the Stellenbosch Wine of Origin District (84 537 ha). The identified NTUs were homogenous with respect to terrain morphological unit, altitude, aspect and soil type. Each of the identified units was further described with respect to the extent of the expected sea breeze effect and, for certain of the soil types, the associated parent material. As a second phase of data acquisition, a network of plots of Sauvignon blanc and Cabernet Sauvignon were delimited in commercial vineyards in proximity to weather stations and their viticultural and oenological response monitored for a period of seven years. Regression tree analyses were performed on the complete data set and the relative importance of the environmental and management related variables determined for each dependent variable. Excepting for scion clone, which had a high relative importance for bunch mass of Sauvignon blanc and yield to pruning mass index of Cabernet Sauvignon, no other non-environmental variable included in the analyses appeared to have a strong effect on grapevine performance and wine character. The performance of Cabernet Sauvignon was affected by the potassium content of the subsoil and the climate of the season. The performance of Sauvignon blanc appeared to be related to soil texture, wind exposure and temperature, both during the green berry growth stage and the month prior to ripening. From the results presented, it appears that environmental parameters have an overriding effect on the performance of both Cabernet Sauvignon and Sauvignon blanc, but that these two cultivars react differently to environmental stimuli. A knowledge-driven model used the rules generated in the regression tree analyses to directly classify natural terroir units with respect to expected response of Cabernet Sauvignon and Sauvignon blanc in the Stellenbosch Wine of Origin District. The natural terroir units were thus grouped into terroir units that were homogenous with respect to predicted viticultural and oenological response for each cultivar. The use of representative sites to determine the response of the grapevine to its environment is time consuming and costly and limits terroir studies to research related investigations. Vineyard managers were therefore surveyed with respect to the functioning of established Sauvignon blanc vineyards in the Stellenbosch Wine of Origin District in an attempt to obtain the necessary data. Comparison of the data generated with these questionnaires to measured data in commercial vineyards suggested that the vineyard managers were able to characterise the performance of vineyards with respect to vigour, signs of drought stress and yield. Each vineyard was mapped and the responses were linked to modelled environmental variables. Classification and regression trees were used to construct decision trees, which could be applied to environmental data in a geographic information system to determine viticultural terroirs for production of Sauvignon blanc. These terroirs, although fewer, were comparable to those generated using field data. Data gathered during terroir studies, and the identified viticultural terroirs for Cabernet Sauvignon and Sauvignon blanc, were used to revisit the boundaries of the Stellenbosch Wine of Origin District and the Simonsberg-Stellenbosch ward. Modifications were proposed based on expected wine characteristics. Boundaries for two new wards in the Helderberg basin were proposed. It was also possible to identify vineyards within a ward for the production of terroir specific wines. / AFRIKAANSE OPSOMMING: Toenemende verbruikersaanvraag vereis produkte waarvan die etikette nie net die oorsprong aandui nie, maar ook kan dien as ‘n waarborg vir die produk se kwaliteit en kenmerkendheid. Hierdie tendens verklaar die toenemende wereldwye fokus op afgebakende areas van oorsprong. Die integriteit van die afgebakende areas van oorsprong sowel as hul verdedigbaarheid kan gewaarborg word deur terroirs as basis vir afbakening te gebruik. Die doelstellings van die studie was om die oorheersende omgewingseienskappe wat wingerdprestasie en wynkarakter van twee belangrike cultivars, naamlik Cabernet Sauvignon en Sauvignon blanc, in die Stellenbosch Wyn van Oorsprongsdistrik bemvloed, te bepaal; tweedens om ‘n toepaslike metodologie te gebruik om terroirs in die die distrik te bepaal wat gebaseer is op die geidentifiseerde omgewingseienskappe met die gebruik van ‘n geografiese inligtingstelsel; en, ten slotte, om hierdie terroirs vir wingerbou te gebruik om afgebakende areas van oorsprong in dieselfde omgewing te identifiseer. ‘n Terroir kan gedefinieer word as ‘n samestelling van homogene omgewingseenhede, of natuurlike terroir-eenhede (NTE), wat gebaseer word op die kenmerkende eienskappe van die produkte wat daaruit verkry word. Identifisering en karakterisering van terroirs sal afhang van kennis van die omgewingsparameters, die funksionering van die wingerdstok en die eienskappe van die finale produk. Veldstudies waaruit puntdata verkry word, is noodsaaklik om die funksionering van die wingerdstok te ondersoek. Dit is egter noodsaaklik om eers hierdie inligting in ruimtelike konteks te plaas alvorens die inligting vir soneringstudies gebruik kan word. As 'n eerste fase van datagenerering, was die Stellenbosch Wyn van Oorsprongsdistrik gekarakteriseer en NTE’s geidentifiseer deur gebruik te maak van bestaande digitale data en ‘n geografiese inligtingstelsel. ‘n NTE kan gedefinieer word as ‘n landseenheid wat gekarakteriseer word deur ‘n relatiewe homogene topografie, klimaat, geologiese substraat en grondtipe. ‘n Totaal van 1389 NTE’s is geidentifiseer in die Stellenbosch Wyn van Oorsprongsdistrik (84 537 ha). Die geidentifiseerde NTE’s was homogeen met betrekking tot die terrein morfologiese eenheid, hoogte bo seespieSI, hellingsaspek en grondtipe. Elk van die geidentifiseerde eenhede was verder beskryf volgens die omvang van die seewindinvloed en, vir toepaslike grond tipes, die geassosieerde moedermateriaal. As ‘n tweede fase van datagenerering is ‘n netwerk van persele van Cabernet Sauvignon en Sauvignon blanc afgebaken binne bestaande kommersiele wingerde in die nabyheid van weerstasies. Hul wingerd- en wynkundige respons is vir ‘n periode van sewe jaar gemonitor. Regressieboomanalises is gebruik om die volledige stel data te analiseer en om die relatiewe belang van omgewings- en bestuurspraktykverbonde veranderlikes te bepaal. Die bostokkloon (wat 'n hoe relatiewe belang vir die trosmassa van Sauvignon blanc en die oes- tot snoeimassa verhouding van Cabernet Sauvignon het) is die enigste van die nie-omgewingsparameter wat ‘n sterk invloed op wingerdprestasie of wynkarakter blyk te he. Die prestasie van Cabernet Sauvignon is beTnvloed deur die kaliuminhoud van die ondergrond sowel as die seisoensklimaat. By Sauvignon blanc het dit voorgekom of die prestasie verband hou met grondtekstuur, windblootstelling en temperatuur tydens die groen fase van korrelgroei sowel as die maand voor rypwording. Alhoewel dit blyk uit die resultate dat omgewingsparameters 'n oorheersende invloed op die prestasie van beide Cabernet Sauvignon en Sauvignon blanc uitoefen, reageer die twee cultivars verskillend op omgewingsprikkels. ‘n Kennisgedrewe model waarvan die riglyne uit resultate van die regressieboomanalise saamgestel is, word gebruik om NTE direk te klassifiseer ten opsigte van die verwagte respons van Cabernet Sauvignon en Sauvignon blanc in die Stellenbosch Wyn van Oorsprongsdistrik. Die NTE is dus gegroepeer om terroir eenhede te vorm wat homogeen was ten opsigte van die verwagte wingerd- en wynkundige respons vir elke cultivar. Die gebruik van verwysingspersele om die respons van die wingerdstok teenoor sy onmiddelike omgewing te bepaal, is tydrowend en duur en beperk sodoende terroir studies tot navorsing. Gevolglik is ‘n opname onder wingerdbestuurders gemaak om inligting oor die prestasie van Sauvignon blanc in die Stellenbosch Wyn van Oorsprongsdistrik in te win en sodoende die nodige data te verkry. Na vergelyking van die ingewinde data, wat uit die opname verkry is, met gemete data vanaf kommersiele wingerde, kon afgelei word dat wingerdbestuurders by magte is om die prestasie van wingerde ten opsigte van groeikrag, tekens van droogtestres en opbrengs te karakteriseer. Elke wingerd is gekarteer en die respons is gekoppel aan die gemodeleerde omgewingsparamters. Klassifikasie en regressiebome is gebruik om besluitnemingsmodelle saam te stel wat toegepas kon word op omgewingsdata in ‘n geografiese inligtingstelsel om terroirs vir die produksie van Sauvignon blanc te bepaal. Hierdie terroirs, alhoewel minder, was vergelykbaar met die wat gegenereer was met behulp van veldstudies. Data verkry met terroirstudies, sowel as gei'dentifiseerde terroir-eenhede, was gebruik om die grense van die Stellenbosch Wyn van Oorsrongsdistrik en die Simonsberg-Stellenbosch wyk te herbepaal. Voorgestelde modifiserings was gebaseer op verwagte wyneienskappe. Grense vir twee nuwe wyke in die Helderbergkom is voorgestel. Dit was ook moontlik om wingerde binne ‘n wyk te identifiseer vir die produksie van terroir-spesifieke wyne.

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: During winemaking, a number of biochemical changes occur as a result of the metabolic activity of wine lactic acid bacteria (LAB) associated with malolactic fermentation (MLF). The latter process, which occurs mostly after alcoholic fermentation by wine yeasts, involves the conversion of L-malate to L-lactate and CO2, thus resulting to wine acidity reduction, microbiological stabilization and alterations of wine organoleptic quality. Although Oenococcus oeni is predominantly the most preferred species suitable for carrying out MLF in wine owing to its desirable oenological properties, Lactobacillus plantarum has also been considered as a potential candidate for MLF induction. Other species in the genera of Lactobacillus and Pediococcus are often associated with wine spoilage. These microorganisms induce wine spoilage by producing off-flavours derived from their metabolic activity. It is therefore of paramount importance to understand the mechanism by which wine microbiota cause spoilage. The purpose of this study was to investigate the presence of genes encoding enzymes of oenological relevance in wine-associated LAB strains. In order to achieve this, different sets of specific primers were designed and employed for a wide-scale genetic screening of wine LAB isolates for the presence of genes encoding enzymes involved in various metabolic pathways, such as citrate metabolism, amino acid metabolism, hydrolysis of glycosides, degradation of phenolic acids as well as proteolysis and peptidolysis. PCR detection results showed that the majority of the tested strains possessed most of the genes tested for. It was also noted that, among the O. oeni strains tested for the presence of the pad gene encoding a phenolic acid decarboxylase, only two strains possessed this gene. None of the O. oeni strains has previously been shown to possess the pad gene, and this study was the first to report on the presence of this gene in O. oeni strains. In an attempt to genetically characterize this putative gene, DNA fragments from the two positive O. oeni strains were sequenced. The newly determined sequences were compared to other closely related species. Surprisingly, no match was found when these sequences were compared to the published genomes of three O. oeni strains (PSU-1, ATCC BAA-1163 and AWRI B429). This reinforced a speculation that the pad gene in these two strains might have been acquired via the horizontal gene transfer. In addition, it remains to be further determined if the presence of this gene translates to volatile phenol production in wine. In this study, a novel strain isolated from South African grape and wine samples was also identified and characterized. The identification of this strain was performed through the 16S rDNA sequence analysis, which indicated that this strain belongs to Lactobacillus florum (99.9% sequence identity). A novel PCR assay using a species-specific primer for the rapid detection and identification of Lb. florum strains was also established. For further characterization, this strain was also investigated for the presence of genes encoding enzymes of oenological relevance. PCR detection results indicated that the Lb. florum strain also possess some of the genes tested for. In addition to genetic screening of wine LAB isolates for the presence of different genes, this study was also aimed at evaluating the regulation of the mleA gene encoding malate decarboxylase in three oenological strains of O. oeni. The regulation of this gene was tested in a synthetic wine medium under various conditions of pH and ethanol. From the expression analysis, it was observed that the mleA gene expression was negatively affected by high ethanol content in the medium. On the other hand, low pH of the medium seemed to favour the expression of this gene as the mleA gene expression was more pronounced at pH 3.2 than at pH 3.8. The findings from this study have shed more light on the distribution of a wide array of enzyme-encoding genes in LAB strains associated with winemaking. However, it remains unknown if the enzymes encoded by these genes are functional under oenological conditions, given that wine is such a hostile environment encompassing a multitude of unfavourable conditions for the enzymes to work on. Evaluating the expression of these genes will also help give more insights on the regulation of the genes under winemaking conditions. / AFRIKAANSE OPSOMMING: Gedurende wynmaak, sal 'n aantal biochemiese veranderinge plaasvind as gevolg van die metaboliese aktiwiteit van wyn melksuurbakterieë (MSB) wat betrokke is by appelmelksuurgisting (AMG). Die laasgenoemde proses, wat meestal na alkoholiese fermentasie deur wyngiste plaasvind, behels die omskepping van L-malaat na L-laktaat en CO2, om sodoende die wyn se suur te verminder, mikrobiologiese stabiliteit en verandering van wyn organoleptiese kwaliteit. Alhoewel Oenococcus oeni hoofsaaklik die mees gewenste spesies is wat geskik is vir die uitvoering van AMG in wyn weens sy geskikte wynkundige eienskappe, Lactobacillus plantarum word ook beskou as 'n potensiële kandidaat vir AMG induksie. Ander spesies in die genera Lactobacillus en Pediococcus word dikwels geassosieer met wynbederf. Hierdie mikro-organismes veroorsaak wynbederf deur die produksie van wangeure as gevolg van hul metaboliese aktiwiteite. Dit is dus van kardinale belang dat die meganisme van die wynbederf verstaan word. Die doel van hierdie studie was om die teenwoordigheid van koderend ensieme gene van wynkundige belang in wynverwante MSB stamme te ondersoek. Ten einde dit te bereik, was verskillende stelle van spesifieke peilers ontwerp en toegepas vir 'n groot skaal se genetiese toetsing van wyn MSB isolate vir die teenwoordigheid van ensiemkoderende gene betrokke by verskeie metaboliese paaie, soos sitraat metabolisme, aminosuur metabolisme, hidrolise van glikosiede, agteruitgang van fenoliese sure sowel as proteolise en peptidolise. PKR opsporings resultate het getoon dat die meerderheid van die stamme getoets, die meeste van die gene getoets voor besit. Dit is ook opgemerk dat, onder die O. oeni stamme getoets vir die teenwoordigheid van die pad geen, slegs twee stamme hierdie geen besit. Geen O. oeni stamme het voorheen gewys dat hul die pad geen besit, en hierdie studie was die eerste bewys oor die teenwoordigheid van hierdie geen in O. oeni stamme. In 'n poging om die geen geneties te karakteriseer, is DNA-fragmente van die twee positiewe O. oeni stamme se sekwens volgorde bepaal. Die DNA volgorde is vergelyk met ander nouverwante spesies. Verrassend, was geen passende DNA volgorde gevind met die gepubliseerde genome van drie O. oeni stamme (PSU-1, ATCC BAA-1163 en AWRI B429) nie. Dit versterk die spekulasie dat die pad geen in hierdie twee stamme via die horisontale geen-oordrag verkry is. Verder moet dit nog bepaal word of die teenwoordigheid van hierdie geen lei na vlugtige fenol produksie in wyn. In hierdie studie, is ongeïdentifiseerde stam geïsoleerd van Suid-Afrikaanse druiwe en wyn monsters ook geïdentifiseer en karakteriseer. Die identifisering van hierdie stam is uitgevoer deur middel van die 16S rDNA volgorde analise, wat aangedui het dat hierdie stam behoort aan Lactobacillus florum (99.9% volgorde identiteit). PKR toetse met behulp van die spesie-spesifieke peiler vir die vinnige opsporing en identifikasie van Lb. florum stamme is ook ontwikkel. Vir verdere karakterisering, was hierdie stam ook ondersoek vir die teenwoordigheid van koderende ensiem gene van wynkundige belang. PKR opsporings resultate het aangedui dat die Lb. florum stam ook oor 'n paar van die gene getoets voor besit. Bykomend tot genetiese toetsing van wyn MSB isolate vir die teenwoordigheid van verskillende gene, het die studie ook die evaluering van die regulering van die mleA geen, kodering malaatdekarboksilase in drie wyn stamme van O. oeni. Die regulering van hierdie geen was getoets in die sintetiese wynmedium onder verskillende pH en etanol kondisies. Van die uitdrukkingsresultate, is daar waargeneem dat die mleA geenuitdrukking is negatief geraak deur hoë etanol-inhoud in die medium. Aan die ander kant, in die lae pH medium was die uitdrukking van hierdie geen bevoordeel by pH 3.2 as by pH 3.8. Die bevindinge van hierdie studie het meer lig gewerp op die verspreiding van die wye verskeidenheid van ensiem-koderende gene in MSB stamme wat verband hou met wynmaak. Dit bly egter steeds onbekend of die ensieme gekodeer deur hierdie gene funksioneel is onder wynkondisies, gegewe dat wyn so 'n vyandige omgewing is menigte ongunstige toestande vir die werking van ensieme. Evaluering van die uitdrukking van hierdie gene sal ook help om meer insigte gee oor die regulering van die gene onder wynmaak toestande.

Thesis (PhD (Wine Biotechnology))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Alcoholic fermentation, and especially wine fermentation, is one of the most ancient microbiological processes utilized by man. Yeast of the species Saccharomyces cerevisiae are usually responsible for most of the fermentative activity, and many data sets clearly demonstrate the important impact of this species on the quality and character of the final product. However, many aspects of the genetic and metabolic processes that take place during alcoholic fermentation remain poorly understood, including the metabolic processes that impact on aroma and flavour of the fermentation product. To contribute to our understanding of these processes, this study took two approaches: In a first part, the initial aim had been to compare two techniques of transcriptome analysis, DNA oligo-microarrays and Serial Analysis of Gene Expression (SAGE), for their suitability to assess wine fermentation gene expression changes, and in particular to assess their potential to, in combination, provide combined quantitative and qualitative data for mRNA levels. The SAGE methodology however failed to produce conclusive data, and only the results of the microarray data are shown in this dissertation. These results provide a comprehensive overview of the transcriptomic changes during model wine fermentation, and serve as a reference database for the following experiments and for future studies using different fermentation conditions or genetically modified yeast. In a second part of the study, a screen to identify genes that impact on the formation of various important volatile aroma compounds including esters, fatty acids and higher alcohols is presented. Indeed, while the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the genetic regulation of the network and the physiological roles of individual pathways within the network. Various factors that directly or indirectly affect and regulate the network have been proposed in the past, but little conclusive evidence has been provided. To gain a better understanding of the regulations and physiological role of this network, we took a functional genomics approach by screening a subset of the EUROSCARF strain deletion library, and in particular genes encoding decarboxylases, dehydrogenases and reductases. Thus, ten genes whose deletion impacted most significantly on the aroma production network and higher alcohol formation were selected. Over-expression and single and multiple deletions of the selected genes were used to genetically assess their contribution to aroma production and to the Ehrlich pathway. The results demonstrate the sensitivity of the pathway to cellular redox homeostasis, strongly suggest direct roles for Thi3p, Aad6p and Hom2p, and highlight the important role of Bat2p in controlling the flux through the pathway. / AFRIKAANSE OPSOMMING: Alkoholiese fermentasie, en veral die maak van wyn, is een van die vroegste mikrobiologiese prosesse wat deur die mensdom ingespan is. Die gisspesie Saccharomyces cerevisiae is gewoonlik grotendeels verantwoordelik vir die fermentasie and verskeie vorige studies het gedemonstreer dat hierdie spesie ‘n baie belangrike rol speel in die uiteindelike kwaliteit en karakter van die voltooide produk. Nieteenstaande die feit is daar steeds baie aspekte van beide die genetiese en metaboliese prosesse wat plaasvind tydens alkoholiese fermentatsie wat nog swak verstaan word, insluitende metaboliese padweë wat ‘n impak het op die smaak en aroma van die fermentasie produk. Om ons kennis van die veld uit te brei het die studie twee aanslae geneem: In die eerste geval is gepoog om twee tegnieke van transkriptoom analiese, nl. DNA oligomikro- arrays en Serial Analysis of Gene Expression (SAGE) te bestudeer vir hul vermoë om geen ekspressie veranderinge tydens wynfermentasie te ondersoek en meer spesifiek om hul potensiaal om ‘n kombinasie van kwantitatiewe sowel as kwalitatiewe data met betreking to mRNA vlakke te produseer. Die SAGE metode kon egter geen betroubare resultate produseer nie en dus word slegs die resultate van die mikro-array eksperimente in die tesis bespreek. Die resultaat is ‘n geheeloorsig oor die geenekspressie veranderinge wat so ‘n wyngis tydens alkoholiese fermentasie ondergaan en dien as ‘n verwysingsraamwerk vir toekomstige studies met geneties gemodifiseerde gis of selfs verskillende fermentasieparameters. Die tweede deel van die studie het gefokus op die identifikasie van gene wat ‘n impak het op die vorming van belangrike, vlugtige aroma komponente, o. a. Esters vetsure en hoër alkohole d.m.v. ‘n siftingseksperiment. Alhoewel daar redelik baie inligting is oor die onderligende metaboliese netwerke wat lei tot die vorming van die verbindings, is daar min kennis van die genetiese regulasie van die netwerk en die fisiologiese rol van individuele padweë wat die netwerk vorm. Verskeie faktore – wat of die netwerk direk of indirek affekteer – is al voorgestel, meer met min konkrete bewyse. Dus het ons gepoog om meer lig op die onderwerp te laat m.b.v. ‘n funksionele genoom aanslag deur ‘n siftingseksperiment te doen op ‘n subgroep (spesifiek gene wat kodeer vir dekarboksilase, dehidrogenase en reduktase ensieme) van die EUROSCARF delesiebiblioteek. Dus is tien gene geïdentifiseer – die delesie waarvan ‘n merkbare effek het op die aroma produksie netwerk en spesifiek die van hoër alkohole. Ooruitdrukkings en enkel en meervoudige delesie rasse van die tien gene is gemaak om d.mv. genetiese analiese, hulle rol in aroma produksie en die Ehrlich padweh uit te pluis. Die resultate toon dat hierdie padweg sensitief is teenoor die sellulêre redoks balans en dui op direkte rolle vir Thi3p, Aad6p en Hom2p, asook dat Bat2p ‘n baie belangrike rol speel in die werking van die padweg.