Doctoral Degrees (Viticulture and Oenology)

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Browsing Doctoral Degrees (Viticulture and Oenology) by browse.metadata.advisor "Du Toit, M."

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    Characterization and evaluation of glucose oxidase activity in recombinant Saccharomyces cerevisiae strains
    (Stellenbosch : Stellenbosch University, 2010-03) Malherbe, Daniel Francois; Van Rensburg, P.; Pretorius, I. S.; Du Toit, M.; Divol, Benoit; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology, 2010.
    ENGLISH ABSTRACT: Popular wine styles prepared from fully-ripened, more mature grapes are characterized by intense fruitiness and varietal flavors. However, lengthy maturation of grapes in the vineyard does not only translate into higher flavor intensity but also into higher sugar levels, which, in turn, leads to wines with higher concentrations of alcohol. Excessive alcohol levels can compromise wine flavor and render wine unbalanced. This, along with health issues and anti-social behavior linked to high-risk alcohol consumption patterns, stricter legislation and increased tax rates associated with high-alcohol wines, have increased demand for wines with reduced alcohol concentrations, without loss of the intense fruity aromas. Although low-alcohol wines can be made using physical post-fermentation processes, such approaches are often expensive and can impact adversely on wine flavor. As an alternative strategy, yeast strains are being developed by several research groups to convert some of the grape sugars into metabolites other than ethanol. Based on promising results from previous preliminary work, this study focused on the development of an industrial Saccharomyces cerevisiae wine strain producing glucose oxidase (GOX; b-D-glucose:oxygen oxidoreductase, EC 1.1.3.4). GOX oxidizes b-D-glucose to D-glucono-d-lactone and gluconic acid (GA) extracellularly, thus preventing its entry into glycolysis, thereby diverting a portion of the sugar carbon away from ethanol. The GOX-encoding gene from the foodgrade fungus, Aspergillus niger was used to construct three cassettes (GOX1, GOX2 and GOX2LOX). In these gene cassettes, the A. niger GOX gene was placed under the regulation of the S. cerevisiae phosphoglycerate-kinase-1 gene promoter (PGK1P) and terminator (PGK1T ). To facilitate secretion, in GOX1 the yeast mating pheromone-factor a secretion signal (MFa1S) was fused to the GOX gene, and in GOX2 the native A. niger secretion signal of GOX was used. These gene cassettes were each integrated into the genome of two laboratory yeast strains (BY4742 and S1278b) and one industrial wine yeast strain (VIN13). An additional integration cassette, designated GOX2LOX, was constructed to knock out the IME1 gene in S. cerevisiae. In GOX2LOX, GOX2 was fused to a loxP cassette. VIN13-D1 was obtained by integrating a single copy of GOX2LOX into the IME1 locus. To generate an asporogenic, GOX-producing wine yeast, VIN13-D2 was created by sporulation, micromanipulation and re-diploidisation of VIN13-D1. Comparative analysis indicated that (i) GOX2 resulted in higher levels of extracellular glucose oxidase activity than GOX1; and that (ii) the levels of secreted glucose oxidase activity in the wine yeast transformants were sufficiently high to conduct follow-up small-scale wine fermentation trials. The wine yeast transformant, VIN13-D1 was evaluated under red and white experimental winemaking conditions. Results from this work indicated that glucose oxidase was produced and secreted by VIN13-D1 that dominated the fermentation to the end, but also that the enzyme was not highly active under the evaluated winemaking conditions. Consequently, no significant decrease in ethanol concentrations was observed in the wine made from VIN13-D1 when compared to that from VIN13. Wine samples were analyzed by Fourier transform-middle infrared spectrometry (FT-MIR) to determine the chemical composition and Gas chromatography with a flame ionization detector (GC-FID) to evaluate the concentrations of aroma compounds. The levels of gluconic acid were determined by enzymatic assays. Multivariate data analysis (PCA and PLS1-discrim) was applied to highlight significant differences between the wines made by VIN13 (wild-type) and VIN13- D1. Chemometric projections of the score plots for all results allowed insight into all significant variation up to three principal components (PCA) or PLS components, which showed very clearly that GA is a key factor in evaluating the effect of GOX in VIN13-D1 fermentation with regard to VIN13 fermentations. The VIN13- D1 effect manifestations were best shown on PLS1-discrim score plots that revealed that, of the restricted variable subsets the FT-MIR-compounds and GC-compounds yielded better results, with the GC-compounds displaying greater discriminability between cultivars and VIN13 / VIN13-D1. It can be concluded from these results that the greatest influence of VIN13-D1 produced wines could be observed in the aroma components, but, because there were also discriminability effects discernable in the FT-MIR-compounds, thus the flavor components were also affected. The activity of GOX in grape juice was further investigated in controlled small scale fermentations performed in a bio-reactor. It was confirmed that GOX is active under aerobic conditions, inactive under anaerobic conditions, and can be activated instantly when an anaerobic culture is switched to aerobic conditions (simulated micro-oxygenation). These fermentations showed that glucose oxidase is active in grape juice, and that oxygen play a key-role in the enzyme’s activation. Finally, it was shown with the help of a simplified model, that under ideal conditions, GOX secreted from VIN13-D1, can be employed to reduce the ethanol by a predefined concentration for the production of low alcohol wines. This work gives more insight into how to employ a GOX-producing wine yeast during winemaking and strongly suggests the use of micro-oxygenation to activate the enzyme in order to reduce available glucose, thereby diverting a portion of the sugar carbon away from ethanol production.
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    Directed evolution of wine-related lactic acid bacteria and characterisation of evolved strains
    (Stellenbosch : Stellenbosch University, 2020-03) Tenyane, Seipati Precious; Bauer, Florian; Du Toit, M.; Rossouw, D.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: Microorganisms form part of complex ecological networks, governed by either metabolic, physical or molecular processes that have positive, neutral or negative effects on microbial interactions. Understanding microbial interactions provides the opportunity to control and manipulate microbes for different biotechnological and industrial applications. For example, the production of beverages such as wine shows how microbial interactions can be controlled and manipulated to achieve desired outcomes. One example is the deliberate inoculation of lactic acid bacteria (LAB) such as Oenococcus oeni or Lactobacillus plantarum to inhibit the growth of spoilage bacteria by depleting available carbon sources such as L-malic acid in a process known as malolactic fermentation (MLF). Indeed, wine provides a good model to study microbial interactions because grape must is inhabited by multiple species of filamentous fungi, yeast, acetic acid bacteria (AAB) and LAB in an anthropogenic and relatively controlled environment. In this study, I investigated the impact of the interaction between the wine yeast Saccharomyces cerevisiae and the LAB L. plantarum. Briefly, the impact of the yeast on the evolution of the bacteria was evaluated after 50 and 100 generations first phenotypically, followed by a genome-wide analysis to identify genetic targets of evolution. A serial transfer method was used for the directed evolution (DE) experiments, introducing bottlenecks and fluctuation between nutrient rich and poor environments after each transfer. This strategy results in a ‘feast-and-famine’ regime, which results in conflicting selective pressures, resembling what normally occurs in dynamic natural environments, which was important here to generate robust and resilient bacteria. Additionally, two yeast strains were used to investigate whether microbial interactions result in yeast-specific adaptations or generic adaptations. Therefore, the yeast strains were kept constant by discarding the yeast at the end of each DE cycle and re-inoculating the mother culture at the start of each DE cycle. The data show yeast strain-specific phenotypes for isolates evolved for 50 generations. Genome-wide analysis showed that broadly targeted pathways are peptidoglycan biosynthesis and degradation, nucleic acid processing, and carbohydrate transport and metabolism in isolates evolved for 50 and 100 generations. These data show that yeast-driven DE results in yeast-specific phenotypic variations and high genetic diversity, but also in convergent evolution over time. The results obtained in this study suggest that yeast drive the evolution of bacteria by dominating the metabolic landscape, showing that strong competitive interactions promote positive selection in mixed species communities, and weak competitive interactions results in no adaptation. This work enriches our understanding of yeast-bacteria interactions over time. Moreover, an isolate that is superior to the parent strain in terms of growth and MLF was obtained, showing potential as a starter culture for winemaking.
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    The effect of oxygen on the composition and microbiology of red wine
    (Stellenbosch : University of Stellenbosch, 2006-03) Du Toit, Wessel Johannes; Du Toit, M.; Marais, Jeannine; Pretorius, I. S.; Lonvaud-Funel, A.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology.
    The winemaking process involves different complex chemical and biochemical reactions, which include those of oxygen (O2). Oxygen can come into contact with the wine through various winemaking procedures and can be used by the winemaker to enhance the quality of red wine. In wine, the main substrates for oxidation are phenolic molecules, which form quinones. These can influence the sensory characteristics of the wine. O2 can be used in fresh must to remove oxidisable phenolic molecules through a process called hyper-oxidation and can also be added to fermenting must to enhance the fermentation performance of yeast. Controlled O2 additions during ageing can lead to the wine’s colour being increased and the astringency of the wine decreased. This is due to the formation of acetaldehyde from the oxidation of ethanol, which induces the polymerisation of tannin and anthocyanin molecules. The addition of too much O2 to wine can, however, lead to unwanted over-oxidation, with certain off-odours being formed. It can also enhance the growth of unwanted spoilage microorganisms, such as Brettanomyces and acetic acid bacteria. Although research on O2 in wine was started many years ago, many questions still remain. These include the general effect of O2 on the sensory and phenolic profile of red wine especially and the microbiology of wine during ageing. An effective way of measuring oxidation, especially in red wine must also be developed. In the first part of this study, the effects of O2 and sulfur dioxide (SO2) additions on a strain of Brettanomyces bruxellensis (also known as Dekkera bruxellensis) and Acetobacter pasteurianus were investigated. Epifluorescence microscopy and plating revealed that the A. pasteurianus strain went into a viable but non-culturable state in the wine after prolonged storage under relative anaerobic conditions. This state, however, could be negated with successive increases in culturability by the addition of O2, as would happen during the transfer of wine when air is introduced. The A. pasteurianus strain was also relatively resistant to SO2, but the B. bruxellensis strain was more sensitive to SO2. A short exposure time to molecular SO2 drastically decreased the culturability of the B. bruxellensis strain, but bound SO2 had no effect on the culturability or viability of either of the two types of microorganisms. Oxygen addition to the B. bruxellensis strain also led to a drastic increase in viability and culturability. It is thus clear that SO2 and O2 management in the cellar is of critical importance for the winemaker to produce wines that have not been spoiled by Brettanomyces or acetic acid bacteria. This study should contribute to the understanding of the factors responsible for the growth and survival of Brettanomyces and acetic acid bacteria in wine, but it should be kept in mind that only one strain of each microorganism was used. This should be expanded in future to include more strains that occur in wine. The second part of this study investigated the effect of micro-oxygenation on four different South African red wines. It was found that the micro-oxygenation led to an increase in the colour density and SO2 resistant pigments of the two wines in which micro-oxygenation was started just after the completion of malolactic fermentation. In one of these wines, a tasting panel preferred the micro-oxygenation treated wines to the control. In the other two red wines, in which the micro-oxygenation was started seven months after the completion of malolactic fermentation, very little colour increase was observed. One of these two wines was also matured in an oak barrel, where the change in phenolic composition was on par with the treated wines. A prolonged period of micro-oxygenation, however, led to this wine obtaining an oxidised, over-aged character. Micro-oxygenation and maturation in an oak barrel also enhanced the survival of acetic acid bacteria and Brettanomyces in this wine. Micro-oxygenation can hence be used by the wine producer on young red wines to enhance the quality of the wine, but should be applied with care in older red wines. Future research into micro-oxygenation should focus on whether it can simulate an oak barrel. More research into the effect of micro-oxygenation on the sensory profile of the wine is needed. As mentioned, the addition of O2 can lead to oxidative degradation of wine. The brown colour in wine is often used as an indication of oxidation, but oxidative aromas can be perceived before a drastic increase in the brown colour has been observed in red wine. The third part of this study was to assess the possible use of Fourier Transform Infrared Spectroscopy (FTIR) to measure the progression of oxidation in Pinotage red wines. Three wines were used in this study and clear separation between the control and aerated wines was observed by using Principle Component Analysis (PCA). Sensory analysis of these wines confirmed this observation, with a reduction especially in berry fruit and coffee characters and an increase first in potato skin and then acetaldehyde aroma characters as the oxidation progressed. PCA analysis also revealed that in certain wines the visible spectrum of light did not indicate the progression of oxidation as sensitively as with the use of FTIR. This also correlated with the inability of the panel to observe a drastic colour change. FTIR should be further investigated as a possible means of monitoring oxidation in wine and this study should be expanded to wines made from other cultivars as well.
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    The influence of base wine composition and wood maturation on the quality of South African brandy
    (Stellenbosch : Stellenbosch University, 2004-03) Snyman, Caroline Lucie Charlotte; Lambrechts, M. G.; Du Toit, M.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Brandy production is a multi-step process that involves grape harvesting, base wine fermentation, distillation, wood maturation and blending. Within each of these production process steps there are a number of factors that can influence the composition and resultant quality of the base wine, unaged and wood matured distillates. These factors include geographic and climatic features of the origin of grapes used, viticultural practices, grape maturity, grape variety, vintage variation, vinification techniques, storage of the base wine prior to distillation, distillation technique, age and origin of oak wood used for maturation and barrel toasting levels. The composition of flavour is extremely complex in wine and distilled beverages such as brandy. A multitude of compounds can take part in the formation of flavour, and it is rare that a particular compound, that is solely responsible for nuances of a specific flavour, is identified. Thus, taking brandy production factors and the nature of aroma and flavour into account, it is clear that thorough understanding of the complexities affecting the perception of quality in brandy is difficult. In commercial brandy production, standard operating procedures do not allow for the separate distillation of brandy base wines from different producers and mixing of base wines from different regions and producers thus occurs. This makes it difficult to determine whether the quality determination of the base wine is in fact an indication of the quality of the resultant distillate after the required period of wood maturation. Therefore, the aim of this study is to determine whether there is any merit in storing and distilling brandy base wines from different producers and regions separately. This was done by firstly determining the demographic and production factors that influence brandy base wine composition. Secondly, the influence of brandy base wine composition on the quality of the respective unaged potstill distillates as well as the ultimate style and quality of the threeyear old wood matured potstill distillates was determined. For this purpose, four potstills with a capacity of 2000 L each were isolated and used for commercial scale distillations of 33 and 25 brandy base wines in 1999 and 2000, respectively, at the DisteIl distillery in Worcester. The experimental outlay used in the study closely emulated Distell's standard operating procedures for commercial brandy production. Chenin blanc and Colombar are the two most popular grape varietals used in the making of brandy base wine in South Africa. Due to the complexity of the brandy production process, the first part of the study only focussed on South African young Chenin blanc wines, with a predominantly fermentation derived aroma. The concentration of iso-amyl acetate, hexyl acetate, ethyl caprylate, ethyl caprate, 2-phenethyl acetate and octanoic acid was significantly higher in wines awarded gold and silver medals and decreased significantly with subsequent decreases in quality categories. Ethyl lactate exhibited the opposite pattern. A quality predictor model based on the behaviour of 21 volatile compounds quantified in these wines had a resonable prediction accuracy when having to predict the quality of wines made in the same vintages as it had been trained on. It was significantly poorer when tested on a completely different vintage of wines to the ones it had been trained on. The number of vintages, number of wines per vintage and the number of compounds quantified for the model influence its prediction accuracy. Twenty-seven volatile compounds were quantified in 33 and 25 brandy base wines, their unaged and three year old distillates from 1999 and 2000, respectively. ANOVA and CART analysis showed that vintage, region, harvest time, choice of cultivar and yeast strain can have a significant influence on the volatile compound composition of brandy base wines, their unaged and three year old distillates. These factors as well as the volatile compound composition were also found to influence the sensory quality of these products. Base wines, unaged and three year old distillates originating from the De Dooms region, which predominantly cultivates table grapes, were of significantly lower quality than those from the remaining regions. Products made from grapes harvested early in the season were of significantly higher quality. The volatile aroma compound composition was found to differ significantly between the 1999 and 2000 base wines and distillates, irrespective of the exclusion of those samples that had undergone partial or complete malolactic fermentation. Consequently, quality indicating compounds may vary from vintage to vintage. The relationship between the quality of brandy base wines and the concentration of n-butanol, iso-amyl acetate, ethyl lactate, ethyl caprylate, octanoic- and decanoic acid was the same as that reported in young Chen in blanc wines in this study. In unaged distillates, increased levels of ethyl lactate also exert a negative influence on distillate quality. Iso-amyl acetate, hexyl acetate, ethyl caproate, ethyl caprylate, n-butanol, octanoic acid, ethyl caprate and decanoic acid showed some positive correlation, whilst iso-butanol, ethyl lactate, acetic acid, acetaldehyde and ethyl acetate showed a significant negative correlation to three-year old distillate quality. Sensory descriptive analysis on selected good, average and poor quality distillates using the South African brandy aroma wheel showed that there are small differences in profile between the good and average distillates, there were however significant differences between the good and poor quality distillate profiles throughout maturation. After three years of wood maturation, the aroma profile of poor quality distillates can be characterised by prominent herbaceous and woody aromas, which are more intense than the fruity aromas. Good quality distillates contained characteristically intense fruity aromas. Volatile compound concentration differences were noted during the course of and after three years of wood maturation and in barrels of varying ages. Distillates matured in new block barrels exhibited significant differences in volatile and wood compound composition after three years when compared to remaining barrels used. The style classification of the three-year old potstill distillate was influenced by demographic and production factors and volatile compound composition, but not by the sensory quality of the distillates. In summary, vintage, region, cultivar, harvest time and choice of yeast strain have a significant influence on the volatile composition of brandy base wines, their unaged and three year old potstill distillates, which in turn affects the sensory quality of these products. These effects cannot be viewed in isolation as they jointly exert an influence on the composition and quality of these products. From a commercial perspective, this study has provided a valuable indication as to which production and demographic factors can influence the quality and style of potstill brandy. Thus, future brandy base wine intake should, as far as possible, take place in such a manner to allow base wines originating from the same cultivar or region or harvest time or combination thereof (and to a lesser extent yeast strain) to be received simultaneously at the distillery for distillation.
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    Investigation of the impact of commercial malolactic fermentation starter cultures on red wine aroma compounds, sensory properties and consumer preference
    (Stellenbosch : University of Stellenbosch, 2011-03) Malherbe, Sulette; Nieuwoudt, Helene; Du Toit, M.; Tredoux, A. J. G.; Naes, T.; University of Stellenbosch. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Wine is the result of a variety of biochemical reactions and microbial interactions which contribute to the organoleptic properties of wine. Wine aroma and flavour encapsulate the sensory experience of wine and could ultimately determine wine quality and consequently influence consumer acceptance and preference of a product. A thorough understanding of potential factors influencing wine aroma and flavour is therefore needed in order to exploit such factors. The aim of this study was to evaluate the influence of commercial malolactic fermentation (MLF) starter cultures on wine composition, aroma and flavour and the potential impact on consumer preference of experimentally produced red wines. An analytical platform was established to capture the compositional changes induced by different MLF bacteria in experimentally produced red wines. A fast gas chromatography flame ionisation detection (GC-FID) method was developed to determine 39 wine volatile compounds in less than 15 minutes per sample. A 3-fold reduction in analysis time was achieved in comparison to a conventional GC-FID method (40 minutes). Analytes quantified comprise a large boiling point and polarity range which illustrates the robustness of the method. A method was also developed for the direct quantification of carbonyl compounds including diacetyl, acetoin, 2,3-pentanedione and certain aldehydes using headspace solid phase microextraction coupled to gas chromatography mass spectrometry (HS-SPME GC-MS). Both analytical methods showed satisfactory linearity, repeatability and limits of quantification. The contribution of four commercial Oenococcus oeni malolactic fermentation (MLF) starter cultures to the volatile composition, organic acid content and infrared spectral properties of experimentally produced South African red wines, showed significant strain-specific variations in the organic acid profiles, especially for the production of citric acid and lactic acid during MLF. Subsequently, concentrations of compounds related to citric acid metabolism, namely ethyl lactate, acetic acid, diacetyl and acetoin, were influenced accordingly. Bacterial metabolic activity increased the concentration of higher alcohols, fatty acids and esters, with a larger increase observed in ethyl esters compared to acetate esters. A strain-specific tendency to reduce total aldehyde concentrations was found at the completion of MLF, however, further investigation is needed to clarify this observation. Infrared spectral fingerprints were used to characterise the different bacteria and in addition, the prediction of MLF related compounds, diacetyl, acetoin and 2,3-pentanedione, from mid-infrared spectra was explored by partial least squares (PLS) models. Quantitative descriptive analysis (QDA) results depicted significant differences between wines fermented with different starter cultures, in terms of sensory attributes including buttery, fruity, nutty and yoghurt/buttermilk aroma as well as smoothness and mouth-feel attributes. Consumer preference testing results indicate that sensory differences imparted by different MLF bacteria could influence consumer-liking. Preference mapping revealed interesting relationships between sensory attributes and consumer-liking, that can be used for preliminary interpretative purposes. In conclusion, this study illustrated the potential impact of bacterial strains on wine aroma and flavour, resulting sensory properties and consumer preference through an integrative approach combining compositional, spectral, sensory and consumer data. The results presented in this study are of significance to the wine industry since they illustrate and reiterate the potential of different MLF starter cultures as an additional tool to contribute to wine aroma and flavour, and potentially influencing consumer preference and product liking.