Masters Degrees (Institute for Wine Biotechnology)

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    South African de-alcoholised sparkling wines: a study focused on sensory and chemical profiles
    (Stellenbosch : Stellenbosch University, 2022-12) Maesela, Lethabo Mologadi; Nieuwoudt, Helene; Brand, Jeanne; Pentz, Chris; Muller, Magdalena; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: De-alcoholised sparkling wines contain a maximum of 0.5% v/v ethanol and a minimum carbon dioxide (CO2) pressure of 300 kPa. The wines are produced through yeast-mediated alcoholic fermentation to full ethanol-strength of table wines (circa 12 to 14% v/v), followed by the physical removal of the volatile aroma fraction and reduction of the ethanol concentration. De-alcoholised base wines are reconstituted by re-introducing the volatile fraction, adding permitted chemical compounds, and sparging with external CO2. Although South Africa (SA) only recently joined the global trend of producing de-alcoholised sparkling wines in response to consumer preference for the products, no published information was available on their sensory quality and chemical composition when this study was undertaken. This study used a quantitative and qualitative approach to investigate the sensory quality of nine commercially available South African de-alcoholised white and rosé sparkling wines. A panel of 51 South African wine industry professionals tasted the wines and evaluated their quality using a 20- point scoring system with maximum points for each sensory modality: appearance 3, aroma 7, and palate 10. Qualitative wine sensory profiles were generated using the free listing method, whereby the professionals described their perceptions of the different modalities. Text data mining included standardisation of raw text, lemmatisation to form sensory groups, and categorising of the groups as positive, neutral, or negative wine attributes. Correspondence analysis identified the sensory categories that best described the wine profiles. The wines’ average total quality scores ranged between 11 and 14 points out of 20, with palate quality scoring, on average, from 4.9 to 6.0 out of 10 points. The free listing method produced an information-rich dataset with 2414, 2110, and 3321 word counts for appearance, aroma, and palate, respectively. The lemmatised text data resulted in 10 appearance, 29 aroma and 61 palate sensory categories. Examples of neutral sensory categories included ‘muscat’, ‘citrus’, and ‘fruit’ aroma, whie positive sensory categories included ‘wine-like’ aroma and ‘nose-palate follow-through’ of wine flavours. Negative palate categories (22 in total) included ‘watery’, ‘short finish’, ‘unbalanced’ and ‘acidic’. The volatile aroma profile and basic wine oenological parameters were generated and compared to published data on full-ethanol strength sparkling wines, in the absence of published data on de-alcoholised sparkling wines. Glycerol concentrations were markedly higher in the de- alcoholised wines, ranging from 14.30 to 20.20 g/L. Volatile compounds’ concentration showed lower ranges than in full-strength sparkling wines. For example, ethyl acetate and isoamyl alcohol ranged from 0.427 to 4.677 mg/L and 0.373 to 5.636 mg/L in this study, respectively, versus 8.000 to 45.200 mg/L and 16.317 to 167.080 mg/L in full-strength wines. The results showed that de-alcoholised sparkling wines are unique products with distinct sensory and chemical profiles. Future research topics to be pursued on best de-alcoholisation and re-constitution practices were pointed out. As a first exploratory study into this technically challenging product category, the results generated meaningful and informative feedback for the South African wine industry.
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    In-Line Monitoring of Red Wine Fermentation
    (Stellenbosch : Stellenbosch University, 2021-03) Lambrecht, Kiera Nareece; Aleixandre-Tudo, Jose Luis; Du Toit, Wessel J.; Nieuwoudt, Helene; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Phenolic compounds may only account for a small percentage of the final composition of a finished red wine but are vital to its sensory attributes. During red wine making, extraction of these phenolic compounds takes place, whereby there is mass transfer from the solids of the grape into the liquid phase. The rate and the extent to which this extraction occurs is dependent on many factors. There are many different methods employed in the wine industry which can influence the composition of the wine. These techniques are varied and can involve manipulating process conditions such as temperature or the addition of certain oenological products. As the final composition of the wine is a major contributing factor to the quality of the wine, it is vital to be able to monitor and control this process. It has been demonstrated through a variety of studies that the use of infrared spectroscopy along with chemometrics provides an avenue for implementation of monitoring and control systems in wineries. However, the limiting factors in these studies are the extensive sample pre-treatment to remove solids before scanning as well as their discrete and off-sight sampling. In the contents of the first research chapter (Chapter 3), a system was designed for the purpose of automatic sampling directly from vessels containing fermenting wines. This was an extensive design process which required separate sampling pumps and sampling lines which delivered samples to a single instrument. Another requirement was automation of different components and synchronisation of these in an individual system. The resulting design was put through a series of stress tests to ensure functionality and reliability. The results showed that the automated system was capable of full-time operation without experiencing component failures and, therefore, it was applied to actual fermentations. For this, 24 hours of real time monitoring was achieved. The turbidity remained a challenge as a perfectly clarified sample was not achievable. This led to the development of partial least squares (PLS) calibrations for three different spectroscopy techniques where the samples used incorporated differing degrees of sample pre-treatment to reduce turbidity. The results of this endeavour compiled in Chapters 4 and 5 showed favourable results for samples with different levels of turbidity as well as for contactless methods of conducting analysis. With further optimisation of the models using spectral pre-processing and wavenumber selection, it was possible to develop models suitable for application in an industrial setting. Finally, in Chapter 6, these models were deployed for use with a series of fermentations, where the ability to monitor phenolic extraction of fermentations receiving different treatments was explored. The results show that the system can be used to monitor trends in phenolic extraction in an industrial set-up. In addition to this, the system has the capacity for updated models and different methods of process control, thereby allowing it to be tailored to each unique scenario.
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    Protoplast-based biotechnology methods for Vitis vinifera
    (Stellenbosch : Stellenbosch University, 2020-12) Derman, Shannon Skye; Vivier, Melane A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: As a major fruit crop worldwide, grapevine production yields the raw materials for the table grape, wine grape, raisin and grapeseed oil industries alike and mostly rely on commercial varieties of the European grape, Vitis vinifera L. As with most widely planted crops, the potential impacts of rapidly changing climatic conditions and associated biotic and abiotic stressors demand a renewed focus on plant improvement strategies. One of the techniques that has been prominent in the recent wave of novel crop improvement methods is that of protoplast biotechnology. Grapevines exhibit recalcitrance towards several biotechnological procedures, including protoplast methodologies. Although some successes have been reported, the potential benefits of protoplast-based methods are far from routine in grapevine science. This study aims to contribute to an existing body of grapevine protoplast research, by evaluating the standard methods of protoplast isolation from both Vitis embryogenic calli and other grapevine explants as productive sources of viable protoplasts and test their usefulness towards a number of applications. Somatic embryogenic cultures from three Vitis vinifera cultivars, namely Chardonnay, Pinotage and Muscat were established from immature influorescence-derived explants (anthers, ovaries and whole flower). Genotype-specific variability was obvious in the ability to form callus and specifically the extent of embryogenic callus recovery. Productive somatic embryogenic cultures were recovered from all cultivars, as well as regenerated plantlets, confirming the regeneration ability of the cultures. Chardonnay is known to respond well to culturing, which was confirmed in this study, and therefore was used for protoplast isolations, optimisations and application analysis. Two established enzymatic methods of protoplast isolation were first compared to identify the more superior of methods. The method using the higher concentration of enzymes (Cellulase (2%), Macerozyme (1%), Pectolyase (0.05%)) was higher yielding with a good viability of protoplasts recorded, and this method was then used to further evaluate and solve a number of technical issues during the isolation procedure. Adaptations were introduced to reduce the number of undigested cells remaining in the isolate after digestion, and to resolve aggregation of protoplasts to each other and to cell debris. The inclusion of a pre-isolation step of coating all plastics in Bovine Serum Albumin reduced protoplast aggregation but did not solve this problem. The optimised method, with a 12-hour digestion period, yielded an average of 9.4x10 cells per 100 mg of somatic embryo calli. The protoplasts were characterised using fluorescent microscopy to evaluate their integrity and viability, to assess the presence of cellulose in remaining cell walls after isolation, as well as to confirm that the sub-cellular structures of the protoplasts could be visualised using organelle-specific markers. Two other grapevine explants were also tested for potential use as efficient and viable protoplast sources, namely zygotic embryos, obtained from using embryo rescue techniques, and meristematic bulks, formed from shoot growth tips manipulated to form meristematic bulks in culture. An extension of the digestion period resulted in an increased yield of protoplasts from zygotic embryos, whilst a pre-plasmolysis treatment of the meristematic bulks increased the yield, but at a cost to the viability of the protoplasts. Our results showed that 1.36 times more protoplasts could be isolated from meristematic bulks compared to zygotic embryos. Despite explant-specific optimisations, 5 times and 3.7 times more protoplasts could be isolated from SEC than from zygotic embryos and meristematic bulks respectively. One of the advantages of meristematic bulks is the fact that it can be established relatively easily on demand, unlike embryogenic cultures (somatic and zygotic) whose explants are highly seasonal. The culturing of the protoplasts would need further time and experimentation and was beyond the scope of this study. Preliminary analyses of protoplasts from somatic embryogenic cultures and meristematic bulks, subjected to culturing confirmed that cell divisions occurred and the appearance of microcalli was evident, but no embryos formed yet. In line with the drive towards protoplast-based genome editing techniques in grapevine, somatic embryogenic protoplasts were subjected to transfection with the YFP reporter gene. Results showed positive transfection in protoplasts deriving from both Sultana and Garganega cultivars, at a transfection efficiency of <18% in both. Stable transformation of Chardonnay embryogenic calli using the GFP reporter gene was also conducted to be used as a resource for subsequent protoplast isolation experiments and as a control system for future transgene expressing protoplast systems. Multiple points of GFP expression were detected within the calli, but these calli tend to rapidly necrotise under selection and grow very slowly. Further transformations would be needed to secure the transgenic callus lines for future experiments. This study also attempted to use flow cytometric techniques to characterise and sort protoplast populations. The method was successful in characterising the protoplasts in solution and differentiating a sub-population with “ideal” characteristics from potentially less optimal subpopulations. However, when the sorted protoplasts were “harvested”, the recovery of viable protoplasts was not possible, and this aspect therefore needs further optimisations. This study was intended towards method validation, optimisations, as well as establishing resources and workflows to make protoplasting successful in our environment. Towards those aims, the study was successful and also expanded the current body of work on grapevine protoplasting by introducing results on two additional explants towards protoplast generation and potentially regeneration, as well as providing promising evidence that cell sorting of protoplasts could be a valuable addition in protoplasting workflows to characterise the populations, but hopefully also ultimately recover only the desired fractions. It is clear that the biggest challenge remains to make regeneration of protoplasts a routine technique to realise the full potential of protoplasts in grapevine biology and biotechnology.
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    Evolution of mutualistic behaviour between chlorella sorokiniana and saccharomyces cerevisiae within a synthetic environment.
    (Stellenbosch : Stellenbosch University, 2020-12) Oosthuizen, Jennifer Rae; Bauer, Florian; Naidoo-Blassoples, Rene Kathleen; Rossouw, Debra; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Microbial symbioses are abundant in the natural environment. Mutualisms are a subset of these symbioses that still lack fundamental understanding regarding the manner in which these complex interactions form and alter microbial species over time. Phototrophic-heterotrophic microbial systems are becoming more commonplace in research due to the many benefits they can provide when different organisms are combined. Heterotrophic fungal systems are largely utilized in the production of high-value metabolites, while phototrophic microalgal systems are found primarily in the green sector such as carbon dioxide sequestration or biofuel production. Synthetic ecology implemented into thoughtfully designed artificial ecosystems provides an ideal method for both the fundamental study of mutualistic symbioses and the production of improved microbial strains for industry. Both the long- and short-term effects of microbial co-evolution on strain performance are largely unknown. Mutualistic interactions are a way to study these effects as the nature of the interaction, reliance on the survival of a partner species, prevents a single species from outcompeting the other. The clear benefits of mutualistic interactions for industrial applications, such as increased growth of both species and/or the production of novel metabolites, also provide clear incentives to investigate these interactions. This study employed synthetic ecology principles and designed an artificial ecosystem to investigate the effects of co-evolution on a mutualistic yeast-microalgal pairing. The yeast, Saccharomyces cerevisiae, and microalga, Chlorella sorokiniana, were co-evolved in an environment that imposed an obligate mutualism between the two microbial partners for approximately 100 generations. The obligate mutualistic interaction was based upon the reciprocal exchange of carbon (CO2 from S. cerevisiae) and nitrogen (ammonia from C. sorokiniana). Strains were isolated from the 50th and 100th generation for further phenotypic, metabolic and transcriptional analysis compared to the parental strains. Phenotypic screening of isolates took place in both mono- and co-culture (multiple pairwise combinations of evolved yeast and microalgae) with various carbon and nitrogen sources to test the limits and effects of co-evolution. This study clearly demonstrated how even short periods of co-evolution can cause changes to the phenotypic growth and metabolite usage of co-evolved isolates. All co-evolved yeast and microalgal strains showed changes to growth rate and a wide variety of growth patterns when compared to the parental strains. Importantly, changes in the expression of key carbon and nitrogen genes were also observed in the evolved isolates of both species. These observed changes assist in highlighting potential underlying mechanisms that occur during co-evolution. These results, when taken together show that even short periods of co-evolution, can produce strains with different characteristics to the parental strains. Harnessing techniques such as co-evolution in combination with synthetic ecology and artificial ecosystems will allow for the creation of functional ecosystems with applications in a wide variety of sustainable industries such as the bioremediation, carbon capture and biofuel industries.
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    Impact of exogenous thiamine on Kluyveromyces marxianus under oenological conditions
    (Stellenbosch : Stellenbosch University, 2020-12) Labuschagne, Pieter Willem Jacobus; Divol, Benoit; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Managed inoculation of non-Saccharomyces yeast species is regarded as a practical way to introduce new characteristics to wine. However, these yeasts struggle to survive until fermentation is complete. The non- Saccharomyces strain, Kluyveromyces marxianus IWBT Y885, was recently isolated from South African grape juice and was partially characterised for winemaking purposes. This yeast strain displays relatively good fermentation ability as well as commercially relevant characteristics such as the production of strong pectinase activity and high phenylethanol and phenylethyl acetate production levels. In order to consider this strain for industrial winemaking, it is important to ensure its prolonged survival and good fermentation performance. A key factor for survival, growth and sustained metabolic activity of all yeasts is their nutrient requirements. Thus, identifying nutrients that are essential for maximising fermentation performance, and subsequently ensuring adequate levels of nutrients, is a means to ensure significant contribution of yeast to wine properties. This study aimed to identify essential nutrients, other than previously studied sugars and nitrogen, for maximum impact of K. marxianus Y885, as well as to characterise the outcomes of their utilisation. A multifactorial experimental design was employed to investigate the impact of nutrient concentrations on fermentation performance with K. marxianus Y885 in synthetic must. Exogenous thiamine concentration was determined to be the most significant factor impacting fermentation performance of K. marxianus Y885. The response to exogenous thiamine concentration for K. marxianus Y885 and S. cerevisiae EC1118 was compared in terms of population viability, fermentation rate, total sugars utilised, thiamine assimilation kinetics, and final wine composition. A saturation effect for initial thiamine concentration of K. marxianus Y885 fermentations was characterised, with a maximum fermentation rate and over 90% of available sugars utilisation obtained at 0.25 mg/L. A delayed uptake of thiamine at higher concentrations for K. marxianus Y885 suggested differential regulation of thiamine uptake compared to S. cerevisiae EC1118. In addition, different trends in metabolites produced between species suggest that thiamine concentration impacts the carbon metabolic flux differently in these two yeasts. To assess how this knowledge would translate to industrial winemaking conditions, the effect of maximum legal thiamine concentration to K. marxianus Y885/S. cerevisiae EC1118 sequential fermentations at semi-industrial scale in real grape must was investigated. No effect of thiamine supplementation on fermentation performance was observed, most likely as a result of the high native must thiamine content and lack of indigenous organisms that may compete for thiamine. However, thiamine concentration clearly differentially affected major volatile production of K. marxianus Y885 and S. cerevisiae EC1118. Notably, an increased fusel alcohol production of K. marxianus Y885 resulted from thiamine addition, which positively affects wine aroma. This study provides relevant information that may be used to ensure optimal utilisation of K. marxianus Y885 in oenology and aids in our understanding of how thiamine alters yeast metabolism.