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Recent exponential increases in attendance at wine tasting rooms resulted in growing research in this subject area as producers seek to learn more about wine tasting room customers and identify ways to capitalize on additional revenue-generating opportunities. Direct wine sales are big business with $3.4 billion in sales in the United States in 2010. Research has shown that small and medium-sized wineries have become financially dependent on direct sales linked to wine tasting rooms with an average of 70 percent of winery sales coming from the tasting room. With limited sources outlining best practices within wine marketing, there is a clear need to identify and classify the literature on this topic. This research applies a marketing theoretical approach using the 4Ps (product, place, price, and promotion) of the marketing mix in conjunction with a comprehensive citation-based global literature review, with the goal of assessing those factors, if any, which may impact wine tasting room profitability. Our findings highlight key differences in individual wine tasting room marketing mix strategies which emphasize the need to understand consumer tastes and preferences for the wine tasting experience. Research shows that investing in the product and promotion of the wine tasting room has a positive impact on profitability. Key profit drivers include investing in branding, tasting room staff, and aggressively pursuing word-of-mouth recommendations to enhance wine tasting room profitability. / Master of Science

Master of Science

It was concluded that there is a free potential for future growth in the quality sector. Price trend analysis for different qualities is consistent with the expected growth. Envisaged changes in production patterns will bring increased revenues for the industry. However, if government intervention goals are to increase substantially industry profitability and farmers income, a balance between government incentives to expand the area of quality wine and government and private investment in market promotion are required. The non-adoption of marketing strategies to increase demand or a failure to promote quality wine demand will have negative effects on prices and on the industry profitability. It was concluded that there is a free potential for future growth in the quality sector. Price trend analysis for different qualities is consistent with the expected growth. Envisaged changes in production patterns will bring increased revenues for the industry. However, if government intervention goals are to increase substantially industry profitability and farmers income, a balance between government incentives to expand the area of quality wine and government and private investment in market promotion are required. The non-adoption of marketing strategies to increase demand or a failure to promote quality wine demand will have negative effects on prices and on the industry profitability.

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The production of glycerol by Saccharomyces cerevisiae under anaerobic conditions is essential for maintaining the intracellular redox balance thereby allowing continuous energy generation through conversion of sugars into ethanol. In addition, glycerol can act as an osmolyte and is synthesized to maintain turgor pressure under hyperosmotic conditions. The production of ethanol from sugars can be a redox-neutral process, where the NAD+ (nicotinamide adenine dinucleotide) that is consumed during the glycolytic conversion of glyceraldehyde-3-phosphate to pyruvate is later regenerated by the reduction of acetaldehyde to ethanol. However, in particular the redirection of metabolic flux of pyruvate to biomass formation leads to excess NADH formation. The intracellular redox balance in these conditions is then primarily maintained through formation of glycerol which is control by two main enzymes, namely Gpd1p and Gpd2p. Deletion of the genes coding for these two proteins leads to accumulation of NADH and renders the cells incapable of maintaining their fermentative ability and growth under anaerobic conditions. The goal of this study was to investigate the growth, fermentative ability and metabolite synthesis of various gpd1Δgpd2Δ double mutant (DM) strains in which the redox balancing potential was partially restored through expression of native or heterologous genes. Strains were constructed by introducing alternative NADH oxidizing pathways or manipulating existing pathways to favour the oxidation of excess NADH. More specifically, the modifications included (i) sorbitol formation; (ii) establishing a pathway for propane-1,2-diol formation; and (iii) increasing ethanol formation. Apart from genetically manipulating the gpd1Δgpd2Δ double mutant, the addition of pyruvate during growth was also investigated. The experiments were carried out under oxygen limited conditions in a high sugar medium and the fermented product was analyzed for total sugar consumed, biomass and primary and secondary metabolites formed by the different strains. The relationships between sugar consumption, growth and metabolite production by different strains were investigated by comparing the data generated from the different strains by using multivariate data analysis tools. Analysis of the pathways involved in the production of primary (acids, ethanol and other metabolites) and secondary metabolites (aroma compounds) were also carried out in order to establish flux modification in comparison to the wild type (WT) strain. The results revealed that these manipulations improved the fermentative capacity of the gpd1Δgpd2Δ double mutant, suggesting a partial recovery of NAD+ regeneration ability, albeit not to the extent of the WT strain. As expected a significant correlation was found between sugar consumption and ethanol and biomass formation. Ethanol yields but not final concentrations were increased by the genetic manipulations. Sorbitol by DM(srlD) and DM(SOR1) strains and propane-1,2-diol by DM(gldA, GRE3, mgsA) strain were formed in significant amounts although at lower molar yields than glycerol. Furthermore, by genetic manipulation the yield of secondary metabolites (isobutanol, isoamyl alcohol, 2-phenyl ethanol and isobutyric acid) was increased whereas the ethyl acetate concentration and yield decreased. The results indicate that aroma compound properties of wine yeasts could be favourably changed by manipulating the glycerol synthesizing pathway. The addition of pyruvate during the growth of gpd1Δgpd2Δ double mutant contributes to excess NADH re-oxidation through additional ethanol formation. / AFRIKAANSE OPSOMMING: Die produksie van gliserol deur Saccharomyces cerevisiae onder anaërobiese toestande is noodsaaklik vir die onderhouding van die intrasellulêre redoksbalans en maak dus ononderbroke energie-ontwikkeling tydens die omsetting van suikers in etanol moontlik. Daarbenewens kan gliserol as ‘n osmoliet optree en word dit gesintetiseer om turgordruk onder hiperosmotiese toestande te onderhou. Die produksie van etanol uit suikers kan ‘n redoksneutrale proses wees, waar die NAD+ (nikotinamiedadenien-dinukleotied) wat tydens die glikolitiese omskakeling van gliseraldehied-3-fosfaat na piruvaat verbruik word, later deur die reduksie van asetaldehied na etanol regenereer word. Die nasending van die metaboliese vloeiing van piruvaat na biomassavorming lei egter na ‘n oormaat NADH-vorming. Onder hierdie toestande word die intrasellulêre redoksbalans dan hoofsaaklik deur die vorming van gliserol onderhou. Laasgenoemde word veral deur twee ensieme beheer, naamlik Gpd1p en Gpd2p. Die delesie van die gene wat vir hierdie twee proteïene enkodeer, lei tot ‘n akkumulasie van NADH en veroorsaak dat die selle nie hulle gistingsvermoë en groei onder anaërobiese toestande kan onderhou nie. Die doelwit van hierdie studie was om die groei, gistingsvermoë en metabolietsintese van verskeie gpd1Δgpd2Δ dubbelmutant (DM) rasse te ondersoek waarin die redoksbalanseringspotensiaal gedeeltelik herstel is deur die uitdrukking van inheemse of heteroloë gene. Rasse is gekonstrueer deur alternatiewe NADH-oksiderende weë in te voer of deur bestaande weë te manipuleer om die oksidasie van oormaat NADH te bevoordeel. Meer spesifiek het die modifikasies die volgende ingesluit: (i) sorbitolvorming; (ii) die vestiging van ‘n weg vir propaan-1,2-diol-vorming; en (iii) die verhoging van etanolvorming. Buiten die genetiese manipulering van die gpd1Δgpd2Δ dubbelmutant, is die byvoeging van piruvaat tydens groei ook ondersoek. Die eksperimente is onder suurstofbeperkte toestande in ‘n hoë-suiker medium uitgevoer en die gegiste produk is ondersoek vir totale suikerverbruik, biomassa en primêre en sekondêre metaboliete wat deur die verskillende rasse gevorm is. Die verhoudings tussen suikerverbruik, groei en metabolietproduksie deur die verskillende rasse is ondersoek deur die data wat deur die verskillende rasse gegeneer is deur middel van meerveranderlike data-analise te vergelyk. Analise van die weë wat in die produksie van primêre (sure, etanol en ander metaboliete) en sekondêre metaboliete (aromaverbindings) betrokke is, is ook uitgevoer om die verandering in vloei te bepaal in vergelyking met die wildetipe (WT) ras. Die resultate het gewys dat hierdie manipulasies die gistingsvermoë van die gpd1Δgpd2Δ-dubbelmutant verbeter het, wat ‘n gedeeltelike herstel van NAD+- regenerasievermoë voorstel, hoewel nie tot dieselfde mate as in die WT-ras nie. Soos verwag, is ‘n beduidende korrelasie tussen suikerverbruik en etanol- en biomassavorming gevind. Etanolopbrengs is deur genetiese manipulasies verhoog, maar nie die finale konsentrasies van etanol nie. Sorbitol is in beduidende hoeveelhede deur die DM(srlD) en DM(SOR1)-rasse gevorm en propaan-1,2-diol deur die DM(gldA, GRE3, mgsA) -rasse, hoewel teen laer molare opbrengste as gliserol. Verder is die opbrengs van sekondêre metaboliete (isobutanol, iso-amielalkohol, 2-fenieletanol en isobottersuur) deur genetiese manipulasie verhoog, terwyl die etielasetaatkonsentrasie en -opbreng verlaag is. Die resultate dui aan dat die aromaverbindingseienskappe van wyngiste voordelig verander kan word deur die gliserolsintetiseringsweg te manipuleer. Die byvoeging van piruvaat tydens die groei van die gpd1Δgpd2Δ-dubbelmutant dra by tot uitermate NADH-reoksidasie tydens die bykomende vorming van etanol.

Thesis (MBA)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: The wine industry in South Africa has the potential to produce wine of world-class quality, but simultaneously large volumes of inferior quality wine are produced annually and sold in the market in cheap, low-quality packaging, making up the bottom end of the alcoholic beverage market. The study investigated the South African wine industry to determine where the industry is headed. The document focussed on inexpensive wine that is sold in cheap packaging to determine who drinks it and what the effect is on the wine industry. The methodology followed was to perform a literature study of the current situation of the South African wine industry. Personal interviews were conducted with members of wineries and companies to determine if the sales of foil bags influenced the sales on their products. The study found that the packaging, in which the inexpensive wine is sold is not the problem, but rather the quality of the wine that goes into the cheap packaging. The banning of wine sold in foil bags from the market in July 2005 shows a commitment from the industry to improve the image of the industry. It will, however, not solve the social problem of alcohol abuse in South Africa, as the profile of the inexpensive wine drinker indicated that if cheap wine is not available, they would change to any other form of cheap alcohol that is available. It is recommended that the quality of the products sold in cheap packaging must be improved. To improve the quality of the product, quality standards must be put into place in the industry that suppliers must adhere to. Policing in the industry needs to be improved to ensure that everybody adheres to the regulations. The industry must help with policing by mobilising industry resources. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse wynindustrie het die potensiaal om wyn van wêreldklas gehalte te produseer. Tesame hiermee word wyn van lae kwaliteit jaarliks geproduseer wat in goedkoop, lae kwaliteit verpakkings in die mark verkoop word. Dit maak die laagste deel van die alkoholiese mark uit. Die navorsing het gekyk na waar die Suid-Afrikaanse wyn industrie heen op pad is. Die dokument het gefokus op die goedkoop wyn wat verkoop word in goedkoop verpakkings, om te bepaal wie dit drink en wat die effek daarvan op die mark is. Die metodologie wat gevolg is, was om 'n literatuur studie te doen van die huidige situasie in die Suid-Afrikaanse wynindustrie. Persoonlike onderhoude met lede van kelders en maatskappye is gedoen om te bepaal of die verkope van goedkoop wyn in goedkoop verpakkings enige invloed het op hulle markte. Die studie het gevind dat die verpakkings waarin die goedkoop wyn verkoop word nie soseer die probleem is nie, maar eerder die kwaliteit van die wyn wat in die goedkoop verpakking gebruik word. Die verbod op die verkope van wyn in papsakke vanaf Julie 2005 wys die verbintenis van die industrie om die beeld van die industrie te verbeter. Dit sal egter nie die sosiale probleem in Suid-Afrika oplos nie, aangesien die profiel van die goedkoop wyndrinker aandui dat wanneer goedkoop wyn nie beskikbaar is nie, hulle enige ander vorm van goedkoop alkohol sal drink wat wel beskikbaar is. Dit word aanbeveel dat die kwaliteit van die produk wat in goedkoop verpakkings verkoop word, verbeter word. Om die kwaliteit van die produk te verbeter moet daar kwaliteitstandaarde in plek wees waaraan die verskaffers moet voldoen. Polisiëring in die industrie moet verbeter word om te verseker almal aan die regulasies voldoen. Die industrie moet help met polisiëring deur die mobilisering van industrie hulpbronne.

Thesis (MScAgric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The production of quality wine is influenced by numerous factors of which grape quality is one of the most important factors. The production of quality wine, however, is not possible without good winemaking techniques and effective quality control. Critical control points (CCP) during the winemaking process must be identified to ensure optimum wine quality. Grape must is a complex medium that contains different micro-organisms which can be either beneficial or negative to wine quality, depending on the physical and chemical conditions that prevail in the must. Yeasts are responsible for alcoholic fermentation, lactic acid bacteria (LAB) for malolactic fermentation (MLF) and acetic acid bacteria (AAB) for the production acetic acid from ethanol. Yeasts and certain LAB can also produce acetic acid and thereby increasing the volatile acidity (VA) of wine. These micro-organisms can influence each other in complex fashions by competing for growth nutrients and by producing inhibitory substances. Most winemakers nowadays use commercial yeast strains to inoculate wine fermentations. This, however, does not assure a problem-free fermentation and cases of stuck and sluggish fermentations are annually reported worldwide. In these or most cases fermentation takes longer than 21 days to complete and the wine contains a residual sugar concentration of more than 4 g/L, which can be utilised by wine spoilage micro-organisms such as certain bacteria and other wild yeasts. Stuck and sluggish fermentations also increase the chances of oxidation due to the absence of the protective CO2 layer on the surface of the wine, which is formed during alcoholic fermentation. Another effect of stuck and sluggish fermentations is that valuable tank space is wasted due to the unexpected time consumption of these fermentation problems. Many factors during the winemaking process can be responsible for stuck and sluggish fermentations. In this thesis the different factors is discussed with the emphasis on the effect of the yeast strain. The way that certain yeast strains influence AAB and LAB numbers during fermentation and MLF through the production of inhibiting by-products such as medium chain fatty acids has not been investigated in detail in the past. Certain fungicides and pesticides that are used in vineyards to control pests (e.g. mildew) contain copper which can be inhibiting to yeast growth and alcoholic fermentation. Legal limits and withholding periods on these sprays are not always strictly obeyed and can lead to stuck and sluggish fermentations. This motivated us to evaluate the growth and fermentation activities of a selection of commercial wine yeasts in the presence of copper levels in the range of maximum legal limits. The effect of these commercial strains on the LAB and AAB numbers during alcoholic fermentation and MLF were also investigated. Our results showed that there was no significant difference on numbers of the AAB obtained from fermentations inoculated with different commercial wine yeast strains. However, with regards to the LAB numbers, one of the strains produced significantly more sulphur dioxide (SO2), which led to the inhibition of MLF in that wine. Our results further indicated which commercial yeast strains were capable of effectively fermenting high sugar musts and which strains were less effective. From the strains tested VIN13, N96 & L2056 were able to utilize fructose more effectively than NT50, RJ11 & D80. We could further distinguish between yeast strains that produced the lowest (VIN13 & RJ11) and the highest (WE372, NT50 & L2056) VA concentrations in must containing high sugar levels. Strains that were more tolerant against high copper levels were also identified. We tested six yeast strains in must with added copper (0.25 mM cu2+) in the form of CuSO4 .H2O. Three Cu2+-tolerant (D80, Collection Cepage Cabernet & NT50) yeast strains were distinguished from three less Cu2+-tolerant yeast strains (VIN13, NT112 & RJ11). This study made a valuable contribution in knowledge gained about commercially available wine yeast strains that can ferment effectively under certain stress conditions. Research such as this, where wine yeasts are evaluated to ferment more effectively during strenuous winemaking conditions, will be very beneficial to winemakers. / AFRIKAANSE OPSOMMING: Die produksie van gehalte wyn word deur verskillende faktore beïnvloed waarvan druifkwaliteit seker die belangrikste is. Die produksie van gehalte wyn is egter nie moontlik sonder goeie wynmaaktegnieke en effektiewe kwaliteitsbeheer nie. Kritieke kontrole punte (KKP) tydens die wynmaakproses moet dus geïdentifiseer word om sodoende ‘n verlaging in wynkwaliteit te vermy. Druiwemos het ‘n komplekse mikrobiologiese samestelling en bestaan uit verskillende mikroörganismes wat vooren nadelig vir wynkwaliteit kan wees, afhangende van die fisiese en chemiese toestande wat in die mos bestaan. Giste is verantwoordelik vir alkoholiese fermentasie, melksuurbakterieë (MSB) vir appelmelksuurgisting (AMG) en asynsuurbakterieë (ASB) vir die produksie van asynsuur vanaf etanol. Asynsuur word egter ook deur giste en MSB geproduseer en dra so by tot die vlugtige suurheid (VS) van ‘n wyn. Hierdie mikroörganismes kan mekaar op komplekse wyses beïnvloed deur o.a. te kompeteer vir voedingstowwe asook deur die produksie van inhiberende verbindings. Die meeste wynmakers maak gebruik van kommersiële gisrasse om alkoholiese fermentasies mee uit te voer. Gevalle van sogenaamde slepende en gestaakte alkoholiese fermentasies, waar suiker nie volledig na etanol en CO2 omgeskakel word nie, kom egter nog gereeld in die wynbedryf voor. In sulke gevalle neem die fermentasie gewoonlik langer as 21 dae om te voltooi met ‘n suiker konsentrasie van meer as 4 g/L wat in die wyn oorbly. Dit is nadelig vir wynkwaliteit aangesien dit nie net die kanse vir bederf deur bakterieë en giste verhoog nie, maar ook die kanse vir oksidasie verhoog a.g.v. die verlies van die beskermende CO2 lagie bo-oor die wyn. Hoe sekere gisrasse, ASB en MSB getalle gedurende fermentasie en AMG beïnvloed deur die produksie van inhiberende verbindings soos medium ketting vetsure en SO2, is ook nie baie in die verlede ondersoek nie. Sommige spuitstowwe wat gebruik word in die bekamping van swamsiektes bevat koper wat inhiberend kan wees vir gisgroei en alkoholiese fermentasie. Wetlike maksimum limiete en onthoudingsperiodes op spuitstofresidue word egter nie altyd gehoorsaam nie en kan lei tot slepende en gestaakte fermentasies. Dit het ons gemotiveer om ‘n seleksie van kommersiële gisrasse te evalueer in terme van gisgroei en fermentasie in die teenwoordigheid van kopervlakke naby die maksimum limiet. Ons resultate het gewys dat daar nie noemenswaardige verskille in AAB getalle tydens alkoholiese fermentasie tussen behandelings met verskillende kommersiële gisrasse was nie. Een van die gisrasse het wel noemenswaardig meer SO2 geproduseer wat gelei het tot inhibering van AMG in hierdie wyn. Ons het verder uitgewys watter kommersiële gisrasse instaat is om meer effektief in hoër suiker mos te fermenteer en watter van die rasse minder suksesvol was. Ons het ook rasse geïdentifiseer wat meer weerstandbiedend is teen hoë kopervlakke in mos en sodoende groter kans op ‘n suksesvolle fermentasie sal hê in mos wat koperresidue bevat wat afkomstig is van sekere spuitstowwe. Die effek van die ASB en MSB getalle gedurende fermentasie en AMG is ook ondersoek. Ons resultate het verder gewys watter kommersiële gisrasse instaat was om mos met hoë suikervlakke meer effektief te fermenteer. Vam die gisrasse wat getoets was het VIN13, N96 & L2056 fruktose meer effektief benut as NT50, RJ11 & D80. Ons kon verder onderskei tussen gisrasse wat die laagste (VIN13 & RJ11) en die hoogste (WE372, NT50 & L2056) vlakke van VS produseer in mos met hoë inisiële suikervlakke. Gisrasse wat meer tolerant was teen koperresidue in mos is ook geidentifiseer. Ons het ses gisrasse getoets in mos met bygevoegde koper (0.25 mM Cu2+) in die vorm van CuSO4 .5H2O. Daar is onderskei tussen drie Cu2+-tolerante (D80, Collection Cepage Cabernet & NT50) en drie minder Cu2+-tolerante gisrasse (VIN13, NT112 & RJ11). Hierdie studie lewer ‘n waardevolle bydrae in die invordering van kennis oor kommersieel beskikbare wyngisrasse wat meer effektief sal fermenteer onder sekere streskondisies wat in mos voorkom. Inligting soos hierdie is belangrik om die wynmaker se keuse uit die reeks bestaande kommersiële gisrasse te vergemaklik.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2005. / The quality of wine is influenced by a variety of factors, most noticeably the quality of the grapes, winemaking practices and the yeast strains used for alcoholic fermentation. Although several yeast strains are present in the must at the beginning of fermentation, strains of S. cerevisiae quickly dominate and survive alcoholic fermentations. This dominance of S. cerevisiae prompted research that led to the development of a multitude of industrial yeast starter cultures. Starter cultures are usually capable of quick and complete fermentations, with minimal production of deleterious substances such as volatile acidity, H2S, SO2 and ethyl carbamate. Yeast strains should be able to survive the stressful environment created during alcoholic fermentation, whilst possibly offering novel oenological benefits such as pectinolytic activity, killer activity and malic acid degradation. The increased production of volatile esters and higher alcohols may also be desirable, as this will allow the production of wines that are more aromatic. In this study, VIN13 was crossed with S. paradoxus strain RO88 and WE14 by using a micomanipulator. VIN13 was chosen for its fast and complete fermentation ability and moderate aroma production potential. Other factors such as the presence of killer activity and low production of volatile sulphur compounds also favoured the selection of VIN13. S. paradoxus strain RO88 was selected for its ability to degrade malic acid and the favourable impact on aroma production during fermentation. Hybrids between these yeasts may have the potential to produce more aromatic wines, with the added bonus of pectinolytic activity and a strong fermentation capacity. The first crossing yielded 5 hybrids between VIN13 and S. paradoxus strain RO88. Two of these hybrids stood out in the sense that they were able to degrade more malic acid than VIN13 and they also possessed killer and pectinolytic activity. Cinsaut wine was made and the 2 hybrids were shown to have higher aroma compound capacity than the parental yeasts. This was also confirmed during sensory evaluation. The second crossing between VIN13 and WE14 yielded 10 hybrids with low H2S production potential and killer activity. WE14 was selected for its ability to produce very aromatic wines and also the slower fermentation capacity. Hybrids between these yeast may have the potential to produce wines with an increased aromatic content and the fermentation rate might be slower, thereby improving the aroma profile of the wine. After microvinification, 5 hybrids were selected on the basis of fermentation rate differing from that of the parental yeasts and favourable oenological traits, such as fast and complete fermentation, high production of glycerol and low production of volatile acidity. Pinotage wine was made and it was shown that some of the hybrids produced more esters and higher alcohols than the parental yeasts. Sensory evaluation also showed the aroma production potential of the hybrids, as some of the hybrids were shown to score higher for banana, cherry and tobacco characteristics.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2007. / The discrimination between wine yeast strains as well as between their fermented wines has been investigated in this pilot study. The study was divided in two parts, the first to investigate the discrimination between wines fermented with five different Saccharomyces cerevisiae yeast strains, analysed by gas chromatography (GC) and Fourier transform infrared spectroscopy (FTIR) and the second part to investigate discrimination between wine yeast strains in different liquid media and in dried form using FTIR in transmission and attenuated total reflectance (ATR) modes. Wines from three cultivars (Clairette Blanche, Pinotage and Cabernet Sauvignon) that were fermented by five Saccharomyces cerevisiae yeast strains (VIN13, WE372, VIN13-EXS, VIN13-PPK and ML01) were analysed by GC and FTIR. This analysis was done on individual sample sets that consisted of the wines of each of the mentioned cultivars and also on samples drawn throughout the ageing process of these wines. The data obtained were analysed by PLS-Discrimination (PLS-discrim), a chemometric method. Using the data from both the analytical methods, discrimination was observed between wines fermented with different yeast strains in each of the two vintages (2005 and 2006) for all the cultivars. When combining the data from the two vintages no discrimination could be observed between the fermented wines. The discrimination of the fermented wines was found to be similar when using data from GC and FTIR, respectively. Since analysis with FTIR is considerably faster than analysis by GC, it would be recommended that FTIR is used for future studies of similar nature. Combining the samples into one set consisting of wines fermented with commercial wine yeast strains and wines fermented from closely related wine yeast strains (the parental strain and two genetically modified versions thereof (VIN13, VIN13-EXS and VIN13- PPK), those fermented with closely related stains did not show good discrimination from each other. Discrimination was found between wines fermented with genetically modified (GM) wine yeast strains and those fermented with non-GM wine yeast strains. This was done on a limited number of yeast strains and a larger study is needed to confirm these results. As this is the first study of this nature and differences seen could be as result of the different phenotypes. It was shown that it is possible to use both FTIR-transmission and FTIR-ATR (attenuated total reflectance) to discriminate between different wine yeast strain phenotypes. It was shown that when using FTIR-transmission there is discrimination between yeast samples suspended in yeast-peptone-dextrose (YPD) and in water. Dried yeast samples could be discriminated when the yeast samples were in a granular, powder form or in a pellet form, using FTIR-ATR. It was possible to discriminate between the closely related yeast strain phenotypes using FTIR-ATR. In this pilot study it was shown that there can be discriminated between different wine yeast strains and also between the wines fermented with different wine yeast strains. It is recommended that further studies be conducted to refine and expand the study.

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.