Masters Degrees (Institute for Wine Biotechnology)

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Browsing Masters Degrees (Institute for Wine Biotechnology) by browse.metadata.advisor "Du Toit, Maret"

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    Coevolution of Saccharomyces cerevisiae and Lactobacillus plantarum : engineering interspecies cooperation
    (Stellenbosch : Stellenbosch University, 2018-03) Du Toit, Sandra Christine; Bauer, Florian; Rossouw, Debra; Du Toit, Maret; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Microbial interactions are ubiquitous in nature and play a vital role in economically important industrial processes like winemaking. Saccharomyces cerevisiae and Lactobacillus plantarum are important species responsible for the completion of alcoholic and malolactic fermentation (AF and MLF) respectively. Understanding how these species interact with each other and their environment is important to better manage successful completion of AF and MLF. However, the complexity of the wine matrix makes it nearly impossible to study these interactions in a natural environment and synthetic ecological systems can therefore be used to overcome these difficulties. This study was designed to establish a co-dependent, mutualistic relationship between S. cerevisiae and Lb. plantarum in order to gain insights into the cooperation between species, how pH, temperature, and inoculation dosages influences the interaction, and how the interaction evolves over time. The interaction, centered on the reciprocal exchange of amino acids, was established between the lysine auxotrophic strain S. cerevisiae THI4 and the isoleucine, alanine, valine, and methionine auxotrophic strain Lb. plantarum B038. Different combinations of amino acids were omitted from the chemically defined synthetic grape juice-like media in order to find an amino acid treatment which promoted the best growth for both microorganisms. B038 showed excellent growth when cocultured with THI4 for all the amino acid treatments, but THI4 struggled to grow under these conditions. The two treatments selected for further experiments were the Lys-Ile (lysine and isoleucine omitted) and Lys-Val (lysine and valine omitted) treatments since THI4 showed the best growth under these conditions. Lower temperature and pH conditions had a negative effect on the growth and malic acid consumption of B038, but when co-cultured with THI4 the yeast appeared to stimulate the growth of the bacteria under both selective and control conditions. THI4 continued to show poor growth performance and sugar consumption under these conditions. However, when THI4 and B038 were inoculated at cell densities with similar biomass, the growth of THI4 improved significantly. It was expected that THI4 and B038 would show poor growth when grown in the absence of their respective auxotrophic amino acids and support of their respective partner. This proved true for all the amino acid treatments except when B038 was grown in the absence of lysine and valine. B038’s ability to grow under these conditions was hypothesized to be linked to the uptake of glutamine and the production of γ-Aminobutyric acid (GABA), but further research is still required to investigate this. Over continuous rounds of fermentation, THI4 adapted to the imposed selective conditions by increasing its consumption of glucose while cell density remained the same. Whether this is linked to increased ethanol production still needs to be determined. No significant changes were observed in B038 after coevolving the strains. This study provides relevant insights into the industrially important interaction between S. cerevisiae and Lb. plantarum and also provides a basis for future work to create optimised yeast-bacteria pairings for both industrial applications in winemaking and to investigate the genetic changes involved in the establishment of cooperative interactions between species.
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    Influence of winemaking practices on the chemical characteristics of winery wastewater and the water usages of wineries.
    (Stellenbosch : Stellenbosch University, 2015-04) Conradie, Adel; Sigge, G. O.; Cloete, Thomas Eugene; Du Toit, Maret; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: The production of wine globally has increased over the past years, increasing the volume of water used and wastewater generated for every litre of wine produced. In the past, the small volumes of winery wastewater that were produced by wineries had little effect on the immediate environment. However, with the increasing wine production all around the world, winery wastewater is a rising concern for the contamination of soil and subsurface flow. In order to fully understand the impacts of winery wastewater, it is important to establish the volumes and chemical characteristics of the wastewater, before considering possible treatments. The first aim of this study was to determine the influence of certain winemaking practices on the water usage. Two wineries in the Stellenbosch Winelands District were monitored during two harvests and one post-harvest season. It was evident through this study that water plays a vital role during the production of wine and that water is needed at virtually all the winemaking steps. However, the volume of clean water needed differs immensely during the course of the production process. It was noticed that throughout the harvest period at both wineries the clean water demand was highest and decreased during the course of the post-harvest period and steadily increased again towards the end of the year. The harvest period contributes between 30 and 40% of the yearly water usage at the respective wineries. It was also noticed that certain winemaking practices including filtering with a bulk filter, washing of barrels and bottling contributes heavily to the water usage throughout the year. Activities that increase water usage during harvest include the washing of the press and processing a combination of red and white grapes on the same day. Furthermore, it was identified that one of the wineries used a smaller volume of water on a daily basis and per tonnage during harvest than the other, indicating that the cleaner production strategy established 10 years earlier has a positive impact on their water usage. The second aim of this study was to monitor the raw and treated winery wastewater from the two wineries during a period of 15 months, including two harvests and one post-harvest season. This was done to investigate the characteristics of the raw and treated wastewater. Firstly, to determine the impact of the different winemaking practices on the chemical composition of the wastewater and secondly, to determine the efficiency of the existing constructed wetlands on the wastewater and the characteristics of the treated wastewater. From this study it was possible to make two main observations concerning the chemical oxygen demand (COD) concentrations of the two wineries. Primarily, it was observed there were variations in the raw wastewater characteristics of the two wineries and above all, that both wineries showed a decrease in the COD of the raw wastewater produced. Not only did the decrease in the raw wastewater COD over this period show promising results when a cleaner production plan is established and managed it also seems to show a decrease in the volumes of water used by the respective wineries and increase in quality.
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    Investigating the impact of sulphur dioxide on Brettanomyces bruxellensis at a molecular and cellular level
    (Stellenbosch : Stellenbosch University, 2012-03) Duckitt, Edward; Divol, Benoit; Du Toit, Maret; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    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.
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    Investigating the influence of a wine yeast consortium on population dynamics, alcoholic and malolactic fermentation
    (Stellenbosch : Stellenbosch University, 2018-12) Janse van Rensburg, Philippe Jacques; Setati, Mathabatha Evodia; Du Toit, Maret; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture & Oenology & Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Non-Saccharomyces (NS) starter cultures of species such as Metschnikowia pulcherrima, Torulaspora delbrueckii, Pichia kluyveri and Lachancea thermotolerans, have received attention for their desirable properties and contributions in winemaking. These include the production of volatile compounds or enzymes to modify organoleptic attributes. Most studies have focussed on the use of single NS at a time with Saccharomyces cerevisiae to study the above contributions and properties; including effects on malolactic fermentation (MLF). However, there is growing interest in using complex multi-starter cultures to enhance wine aroma. Indeed, commercial products such as Anchor Alchemy II (Anchor Yeast), which comprises of different strains of S. cerevisiae, and Melody™ (CHR Hansen), comprising of S. cerevisiae, L. thermotolerans and T. delbrueckii in different ratios, are available. To develop such products, the understanding of genetic and phenotypic characteristics of strains and interactions amongst the different organisms is important. Consequently, some studies have investigated yeast-yeast interactions and their mechanisms, such as antagonistic interactions mediated by direct cell contact or through growth inhibitory metabolites. This, results in a decline of certain non-Saccharomyces like Hanseniaspora and therefore effect the final aroma composition. Conversely, synergistic effects can be observed in which species may be promoted to persist longer and therefore they contribute more to aroma. However, knowledge on population dynamics in multi-species starter cultures and their influence on alcoholic fermentation (AF) and MLF remains limited. The current study investigated a multi-species yeast consortium during AF and its effects on Oenococcus oeni viability during MLF. The consortium comprised of M. pulcherrima, L. thermotolerans, T. delbrueckii and S. cerevisiae. Fermentations were conducted in Chenin blanc and Pinotage at 15°C and 25°C, respectively. In all trials M. pulcherrima declined rapidly, while L. thermotolerans persisted until mid-fermentation. The best growth was observed for T. delbrueckii and it was able to persist until late fermentation stages. Fermentations that contained L. thermotolerans produced L-lactic acid in the Pinotage, but not in the Chenin blanc. There were no negative impacts observed on O. oeni populations during MLF for Pinotage and Chenin blanc wines. MLF kinetics were similar in all the Pinotage wines. In the Chenin blanc, the fastest L- malic acid consumption was displayed in wines that were fermented by L. thermotolerans and T. delbrueckii co-inoculations with S. cerevisiae. Different chemical profiles were detected using attenuated total reflection infrared (ATR-IR) spectroscopy. Mostly Chenin blanc wines were found to be significantly different from S. cerevisiae controls. Using gas chromatography, fold changes were observed for many volatile compounds. In conclusion, it is possible to predict a consortium population dynamic based on individual yeast performances in mixed fermentations. The volatile profiles are not additive between treatments and will be unique for each inoculation scheme. MLF seems to not be detrimentally affected by a consortium so long as each strain is regarded as compatible with lactic acid bacteria. Future work should include the evaluation of more yeast species and at alternate inoculation levels. Additionally, the inclusion of Lactobacillus plantarum for MLF needs to be investigated and quantification of detected volatiles should be performed.
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    Lactobacillus plantarum : amino acid utilization
    (Stellenbosch : Stellenbosch University, 2018-03) Botma, Izak Johannes; Du Toit, Maret; Bauer, Florian; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Amino acid metabolism serves as a source of sulphur, carbon and nitrogen for bacteria growing in wine or grape juice. The metabolism of amino acids controls the rate of growth and malic acid degradation and also results in the formation of various aromatic compounds which may positively or negatively influence the aroma profile of wine. L. plantarum, a lactic acid bacterium (LAB), may be used as co-inoculant in high pH (≥ 3.5) grape juice for fast malic acid degradation and high aroma production. Since the research on L. plantarum nitrogen metabolism is scarce, the overall goal of this study was to better understand it. The first aim was to determine the amino acid requirements in L. plantarum for growth and malic acid degradation, through single amino acid omissions. This entailed inoculation of nitrogen starved L. plantarum strains into chemically defined media (in this case synthetic grape juice) in which one amino acid is removed at a time. The data suggests that amino acid trophic requirements in L. plantarum are highly strain dependent, although Leu, Ile, Val, Glu and Met were shown under our conditions to be essential amino acids and Gln, Gly, His, Lys and Trp were non-essential amino acids. In a subsequent experiment, 5 single amino acid omissions (Ala, Arg, Gln, Trp and Val) were selected to evaluate their effect on growth and malic acid uptake in synthetic grape juice. During malolactic fermentation (MLF) the removal of Ala and Val had completely repressed MLF induced by L. plantarum while the removal of Trp and Arg had somewhat repressed MLF. Only the removal Gln did not hinder MLF for at least one strain. The second aim was to determine the order of amino acid uptake by L. plantarum in synthetic grape juice using HPLC. Asp, Thr, Ser and Ala tends to be assimilated at a high rate within the first 72 h while the branched chain amino acids, aromatic amino acids (AAA) and Met are assimilated after 72 h. The third aim determined the amino acid uptake in Chardonnay grape juice. The assimilation pattern differed considerably between synthetic grape juice and Chardonnay grape juice. In contrast to synthetic grape juice Arg, Leu, Phe and Ala were preferred amino acid sources. It is thought that the differences could be attributed to mainly two factors: initial nitrogen concentration (40 mg N/L in SGJ vs 240 mg N/L in grape juice) and the pre-culture conditions. This study confirmed that higher nitrogen concentrations resulted in higher growth and quicker malic acid degradation. The high nitrogen requirement of certain amino acids combined with the harsh wine parameters experienced in sequential MLF might explain why L. plantarum struggles with MLF in this scenario. Further research should be directed towards identifying the preferred amino acids in dried and fresh L. plantarum starter cultures to assess if there is a difference. If nitrogen requirements continues to be investigated in L. plantarum successful tailored supplements can be created to aid the growth of L. plantarum in wine or grape juice.