Doctoral Degrees (Viticulture and Oenology)

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

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    Evolutionary engineering of interspecies cooperation: Investigating Saccharomyces cerevisiae and Lactobacillus plantarum interactions in a synthetic ecological environment
    (Stellenbosch : Stellenbosch University, 2021-12) Du Toit, Sandra Christine; Bauer, Florian; Rossouw, Debra; Du Toit, Maret; Stellenbosch University. Faculty of AgriSciences. Faculty of Viticulture and Oenology.
    ENGLISH ABSTRACT: Saccharomyces cerevisiae and lactic acid bacteria (LAB), like Lactiplantibacillus plantarum, form part of the wine microbiome, where they each play a part in the biochemical conversion of grape must to wine. Saccharomyces cerevisiae converts grape sugars to ethanol and carbon dioxide during alcoholic fermentation (AF) and Lb. plantarum converts malic acid to lactic acid during malolactic fermentation (MLF). Physical and metabolic interactions between S. cerevisiae and LAB are often inhibitory to the growth of the bacteria, which hinders the successful completion of MLF. Despite extensive research, the interactions between these species are still poorly understood and the natural complexity of grape must hinders the study of these interactions within the natural ecological environment. This dissertation evaluated the applicability of a combined synthetic ecology and evolutionary engineering approach to better understand and improve the interactions between these species. Nutrient co-dependency between S. cerevisiae BY4742Δthi4 (lysine auxotroph) and Lb. plantarum IWBT B038 (isoleucine and valine auxotroph) was used to ensure both species co-exist during the evolutionary period and to select for improved species cooperation. Overall, the data show that this system can be used to investigate the phenotypic and genetic changes involved in the coevolution of trans-kingdom ecosystems. However, the applicability of the system for the generation of yeast- bacteria pairings with improved oenological characteristics still needs to be further investigated. Under strong selective conditions, when lysine and isoleucine are omitted from the synthetic grape juice media, the bidirectional support in the mutualistic system required optimisation. This was achieved by inoculating BY4742Δthi4 and IWBT B038 at equal biomass concentrations. It was hypothesised that the release of small peptides by BY4742Δthi4 shortly after inoculation may be important for initiating IWBT B038 growth, while the release of nutrients by IWBT B038 due to membrane damage during the later stages of fermentation may be important for BY4742Δthi4 growth. The strains were evolved under coculture and monoculture conditions using different amino acid treatments. Overall, the data show that coevolution under selective conditions selected for isolates with improved cooperative interactions, relative to isolates coevolved under non-selective conditions (no amino acids omitted) and isolates evolved in monoculture. Three evolved yeast isolates showing improved growth and sugar consumption characteristics were subjected to whole-genome sequencing. Although various genetic mutations could be identified in these isolates, the mutations could not be linked to the observed phenotypes. Regardless, the FLO9 and ECM21 genes showed interesting mutations and should be investigated further to determine what role they play in the adaptation of BY4742Δthi4 to the imposed selective conditions.
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    Molecular screening of lactic acid bacteria enzymes and their regulation under oenological conditions
    (Stellenbosch : University of Stellenbosch, 2011-03) Mtshali, Phillip Senzo; Du Toit, Maret; Divol, Benoit; University of Stellenbosch. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    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.