Institute for Wine Biotechnology
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- ItemCarnitine metabolism and biosynthesis in the yeast Saccharomyces cerevisiae(Stellenbosch : University of Stellenbosch, 2009-12) Franken, Jaco; Bauer, Florian; Strauss, Erick; University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: Carnitine plays an essential role in eukaryotic metabolism by mediating the shuttling of activated acyl residues between intracellular compartments. This function of carnitine, referred to as the carnitine shuttle, is supported by the activities of carnitine acyltransferases and carnitine/acylcarnitine transporters, and is reasonably well studied and understood. While this function remains the only metabolically well established role of carnitine, several studies have been reporting beneficial effects associated with dietary carnitine supplementation, and some of those beneficial impacts appear not to be directly linked to shuttle activity. This study makes use of the yeast Saccharomyces cerevisiae as a cellular model system in order to study the impact of carnitine and of the carnitine shuttle on cellular physiology, and also investigates the eukaryotic carnitine biosynthesis pathway. The carnitine shuttle of S. cerevisiae relies on the activity of three carnitine acetyltransferases (CATs), namely Cat2p (located in the peroxisome and mitochondria), Yat1p (on the outer mitochondrial membrane) and Yat2p (in the cytosol), which catalyze the reversible transfer of activated acetyl units between CoA and carnitine. The acetylcarnitine moieties can be transferred across the intracellular membranes of the peroxisomes and mitochondria by the activity of the carnitine/acetylcarnitine translocases. The activated acetyl groups can be transferred back to free CoA-SH and further metabolised. In addition to the carnitine shuttle, yeast can also utilize the glyoxylate cycle for further metabolisation of in particular peroxisomally generated acetyl-CoA. This cycle results in the net production of succinate from two molecules of acetyl-CoA. This dicarboxylic acid can then enter the mitochondria for further metabolism. Partial disruption of the glyoxylate cycle, by deletion of the citrate synthase 2 (CIT2) gene, generates a yeast strain that is completely dependent on the activity of the carnitine shuttle and, as a consequence, on carnitine supplementation for growth on fatty acids and other non-fermentable carbon sources. In this study, we show that all three CATs are required for the function of the carnitine shuttle. Furthermore, overexpression of any of the three enzymes is unable to crosscomplement deletion of any one of the remaining two, suggesting a highly specific role for each CAT in the function of the shuttle. In addition, a role for carnitine that is independent of the carnitine shuttle is described. The data show that carnitine can influence the cellular response to oxidative stresses. Interestingly, carnitine supplementation has a protective effect against certain ROS generating oxidants, but detrimentally impacts cellular survival when combined with thiol modifying agents. Although carnitine is shown to behave like an antioxidant within a cellular context, the molecule is unable to scavenge free radicals. The protective and detrimental impacts are dependent on the general regulators of the cells protection against oxidative stress such as Yap1p and Skn7p. Furthermore, from the results of a microarray based screen, a role for the cytochrome c heme lyase (Cyc3p) in both the protective and detrimental effects of carnitine is described. The requirement of cytochrome c is suggestive of an involvement in apoptotic processes, a hypothesis that is supported by the analysis of the impact of carnitine on genome wide transcription levels. A separate aim of this project involved the cloning and expression in S. cerevisiae of the four genes encoding the enzymes from the eukaryotic carnitine biosynthesis pathway. The cloned genes, expressed from the constitutive PGK1 promoter, were sequentially integrated into the yeast genome, thereby reconstituting the pathway. The results of a plate based screen for carnitine production indicate that the engineered laboratory strains of S. cerevisiae are able to convert trimethyllysine to L-carnitine. This work forms the basis for a larger study that aims to generate carnitine producing industrial yeast strains, which could be used in commercial applications.
- ItemCarotenoid cleavage dioxygenases (CCDs) of grape(Stellenbosch : Stellenbosch University, 2012-12) Dockrall, Samantha; Young, Philip R.; Vivier, Melane A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: Plant carotenoid cleavage dioxygenases (CCD) are a family of enzymes that catalyse the oxidative cleavage of carotenoids and/or apocarotenoids. Carotenoids are synthesised in plastids (primarily chloroplasts and chromoplasts), where they are involved in light-harvesting and protecting the photosynthetic apparatus from photo-oxidation. The carotenoid-derived apocarotenoids fulfil a number of roles in plants such as phytohormones, pollinator attractants and flavour and aroma compounds. Due to the floral and fruity characteristics that apocarotenoids contribute to wine, these C13 compounds have received interest in grapevine (Vitis vinifera L.). The CCD gene family in Arabidopsis consists of nine members, all encoding for enzymes that catalyse the cleavage of carotenoids. The enzymes in this family include 9-cis-epoxydioxygenases (NCEDs) and four classes of CCD. NCEDs and CCD7 and CCD8 are involved with plant hormone synthesis, e.g. abscisic acid (ABA) through cleavage by NCED and strigolactone (SL) through the sequential cleavage of carotenoids by CCD7 and CCD8, respectively. SLs are a fairly new class of plant hormone which are involved in several aspects of plant growth and development. The most extensively characterised role of SLs is their involvement in the inhibition of shoot-branching. CCD1 and CCD4 cleave a variety of carotenoids to form pigments and aroma compounds. For example, CCD1 forms β-ionone and β-damascenone, which are important varietal flavours of wine, and CCD4 is involved in synthesis of the pigment and aroma compounds of saffron and annatto. CCD1 enzymes symmetrically cleave the 9,10 (9’,10’) double bonds of multiple carotenoids to produce a C14 dialdehyde and two C13 products. Additional CCD1 cleavage activity at 5,6 (5’,6’) double bonds of lycopene has been reported. Previous studies have shown that CCD1 isolated from V. vinifera (VvCCD1) was able to cleave multiple carotenoid substrates in vitro, namely zeaxanthin, lutein and β-carotene at 9,10 (9’,10’) double bonds and both the 5,6 (5’,6’) and 9,10 (9’,10’) double bonds of lycopene. None of the other VvCCDs, except VvCCD4a have been isolated (but no functionality was illustrated) and characterised yet. CCD4 enzymes also cleave carotenoids at the 9,10 (9’,10’) double bond positions. The presence of plastid-target peptides implies that the CCD4 enzymes have continuous access to carotenoids. Therefore it is suggested that CCD4s are responsible for carotenoid maintenance, where CCD1s contribute towards volatile production. To test this hypothesis VvCCD1, VvCCD4a and VvCCD4b were isolated from V. vinifera (cv Pinotage) cDNA and cloned into a pTWIN1 protein expression vector. Substrate specificity of each VvCCD was tested by co-transforming a carotenoid accumulating E. coli strain with a CCD expression vector. Carotenoids synthesized by the bacteria were identified and quantified by UPLC-analysis, while the concentration of the apocarotenoids, were measured in the headspace of the bacterial cultures using HS-SPME-GC-MS. Several optimisations were done to minimize the natural degradation of the carotenoids; to ensure that the apocarotenoid formation is predominantly due to the enzymatic cleavage by the VvCCDs and not due to oxidation or other non-enzymatic degradation. The HS-SPME-GC-MS analysis indicated that all isoforms cleaved phytoene, lycopene and ε-carotene. Additionally VvCCD1 cleaved a carotenoid involved in photosynthesis, namely β-carotene, while VvCCD4a cleaves neurosporene and VvCCD4b cleaves neurosporene and ζ-carotene, carotenoids not involved in photosynthesis. This study has illustrated that VvCCD1 cleave carotenoids necessary for photosynthesis and VvCCD4s cleave carotenoids which were not present in berry tissue, suggesting their role in carotenoid maintenance. Therefore in planta substrates for CCD1 could possibly be C27 apocarotenoids generated from enzymatic cleavage through CCD4 (role in carotenoid maintenance), CCD7 and/or photo-oxidation, which are then transported from the plastid to the cytosol or possibly C40 carotenoids that are released during senescence or when the plastid membrane is damaged, thus releasing important aroma compounds. Thus the identification of the in vivo substrates has contributed to the understanding the in planta functions of these enzymes
- ItemCell differentiation in response to nutrient availability : the repressor of meiosis, RME1, positively regulates invasive growth in Saccharomyces cerevisiae(Stellenbosch : Stellenbosch University, 2003-03) Hansson, Guy Robert, 1974-; Bauer, Florian; Pretorius, I. S.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: Yeasts, like most organisms, have to survive in highly variable and hostile environments. Survival therefore requires adaptation to the changing external conditions. On the molecular level, specific adaptation to specific environmental conditions requires the yeast to be able: (i) to sense all relevant environmental parameters; (ii) to relay the perceived signals to the interior of the cell via signal transduction networks; and (iii) to implement a specific molecular response by modifying enzyme activities and by regulating transcription of the appropriate genes. The availability of nutrients is one of the major trophic factors for all unicellular organisms, including yeast. Saccharomyces cerevisiae senses the nutritional composition of the media and implements a specific developmental choice in response to the level of essential nutrients. In conditions in which ample nutrients are available, S. cerevisiae will divide mitotically and populate the growth environment. If the nutrients are exhausted, diploid S. cerevisiae cells can undergo meiosis, which produces four ascospores encased in an ascus. These ascospores are robust and provide the yeast with a means to survive adverse environmental conditions. The ascospores can lie dormant for extended periods of time until the onset of favourable growth conditions, upon which the spores will germinate, mate and give rise to a new yeast population. However, S. cerevisiae has a third developmental option, referred to as pseudohyphal and invasive growth. In growth conditions in which nutrients are limited, but not exhausted, the yeast can undergo a morphological switch, altering its budding pattern and forming chains of elongated cells that can penetrate the growth substrate to forage for nutrients. The focus of this study was on elements of the signal transduction networks regulating invasive growth in S. cerevisiae. Some components of the signal transduction pathways are well characterised, while several transcription factors that are regulated via these pathways remain poorly studied. In this study, the RMEt gene was identified for its ability to enhance starch degradation and invasive growth when present on a multiple copy plasmid. Rme1 p had previously been identified as a repressor of meiosis and, for this reason, the literature review focuses on the regulation of the meiotic process. In particular, the review focuses on the factors governing entry into meiosis in response to nutrient starvation and ploidy. Also, the transcriptional regulation of the master initiator of meiosis, IMEt, and the action of Ime1 p are included in the review. The experimental part of the study entailed a genetic analysis of the role of Rme1 p in invasive growth and starch metabolism. Epistasis analysis was conducted of Rme1 p and elements of the MAP Kinase module, as well as of the transcription factors, Mss11p, Msn1p/Mss10p, Tec1p, Phd1p and F108p. Rme1p is known to bind to the promoter of CLN2, a G1-cyclin, and enhances its expression. Therefore, the cell cyclins CLN1 and CLN2 were included in the study. The study revealed that Rme1 p functions independently or downstream of the MAP Kinase cascade and does not require Cln1 p or Cln2p to induce invasive growth. FL011/MUC1 encodes a cell wall protein that is required for invasive growth. Like the above-mentioned factors, Rme1 p requires FL011 to induce invasive growth. We identified an Rme1 p binding site in the promoter of FL011. Overexpression of Rme1p was able to induce FL01t expression, despite deletions of mss11, msn1, ttos, tee1 and phd1. In the inverse experiment, these factors were able to induce FL011 expression in an rme1 deleted strain. This would indicate that Rme1 p does not function in a hierarchical signalling system with these factors, but could function in a more general role to modify transcription.
- ItemCharacterisation of grapevine berry samples with infrared spectroscopy methods and multivariate data analyses tools(Stellenbosch : Stellenbosch University, 2015-04) Musingarabwi, Davirai M.; Vivier, Melane A.; Nieuwoudt, Helene; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: Grape quality is linked to the organoleptic properties of grapes, raisins and wine. Many advances have been made in understanding the grape components that are important in the quality of wines and other grape products. A better understanding of the compositional content of grapes entails knowing when and how the various components accumulate in the berry. Therefore, an appreciation of grape berry development is vitally important towards the understanding of how vineyard practices can be used to improve the quality of grapes and eventually, wines. The more established methods for grape berry quality assessment are based on gravimetric methods such as colorimetry, fluorescence and chromatography. These conventional methods are accurate at targeting particular components, but are typically multi-step, destructive, expensive, polluting procedures that might be technically challenging. Very often grape berries are evaluated for quality (only) at harvest. This remains a necessary exercise as it helps viticulturists and oenologists to estimate some targeted metabolite profiles that are known to greatly influence chemical and sensory profiles of wines. However, a more objective measurement of predicting grape berry quality would involve evaluation of the grapes throughout the entire development and maturation cycle right from the early fruit to the ripe fruit. To achieve this objective, the modern grape and wine industry needs rapid, reliable, simpler and cost effective methods to profile berry development. By the turn of the last millennium, developments in infrared instrumentation such as Fourier-transform infrared (FT NIR) and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR FT-IR) in combination with chemometrics resulted in the development of rapid methods for evaluating the internal and external characteristics of fresh fruit, including grapes. The advancement and application of these rapid techniques to fingerprint grape compositional traits would be useful in monitoring grape berry quality. In this project an evaluation of grape berry development was investigated in a South African vineyard setting. To achieve this goal, Sauvignon blanc grape berry samples were collected and characterised at five defined stages of development: green, pre-véraison, véraison, post-véraison and ripe. Metabolically inactivated (frozen in liquid nitrogen and stored at -80oC) and fresh berries were analysed with FT-IR spectroscopy in the near infrared (NIR) and mid-infrared (MIR) ranges to provide spectral data. The spectral data were used to provide qualitative (developmental stage) and quantitative (metabolite concentration of key primary metabolites) information of the berries. High performance liquid chromatography (HPLC) was used to separate and quantify glucose, fructose, tartaric acid, malic acid and succinic acid which provided the reference data needed for quantitative analysis of the spectra. Unsupervised and supervised multivariate analyses were sequentially performed on various data blocks obtained by spectroscopy to construct qualitative and quantitative models that were used to characterise the berries. Successful treatment of data by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) gave statistically significant chemometric models that discriminated the berries according to their stages of development. The loadings from MIR models highlighted the important discriminant variables responsible for the observed developmental stage classification. The best calibration models to predict metabolite concentrations were obtained from MIR spectra for glucose, fructose, tartaric acid and malic acid. The results showed that both NIR and MIR spectra in combination with multivariate analysis could be reliably used to evaluate Sauvignon blanc grape berry quality throughout the fruit’s development cycle. Moreover, the methods used were fast and required minimal sample processing and no metabolite extractions with organic solvent. In addition, the individual major sugar and organic acids were accurately predicted at the five stages under investigation. This study provides further proof that IR technologies are robust and suitable to explore high-throughput and in-field application of grape compound profiling.
- ItemCharacterisation, evaluation and use of non-Saccharomyces yeast strains isolated from vineyards and must(Stellenbosch : Stellenbosch University, 2004-03) Jolly, N. P. (Neil Paul); Pretorius, I. S.; Augustyn, O. P. H.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: Wine is the product of a complex biological and biochemical interaction between grapes and different microorganisms (fungi, yeasts, lactic acid bacteria and acetic acid bacteria, as well as the mycoviruses and bacteriophages affecting them) in which yeasts play the most important role regarding the alcoholic (primary) fermentation. These wine-associated yeasts can be divided into Saccharomyces and non-Saccharomyces yeasts. During fermentation, there is a sequence of dominance by the various non-Saccharomyces yeasts, followed by Saccharomyces cerevisiae, which then completes the fermentation. This is especially evident in spontaneously fermenting must, which has a low initial S. cerevisiae concentration. Some non- Saccharomyces yeasts can also be found throughout the fermentation. The non- Saccharomyces presence in the fermentation can affect wine quality, either positively or negatively. A positive contribution could be especially useful to improve wines produced from grape varieties with a neutral flavour profile due to non-optimal climatic conditions and/or soil types. As part of a comprehensive South African research programme, the specific objectives of this study were: the isolation of indigenous non-Saccharomyces yeasts from vineyards and musts; the identification of these isolates; the characterisation and evaluation of predominant species under winemaking conditions; and the development of a protocol for their use in enhancing wine quality. Initially, 720 isolates representing 24 different species, were isolated from grape (vineyard) and must samples taken over three vintages from four distinctly different wine producing regions. The isolates were characterised and grouped utilising biochemical profiles and DNA karyotyping, whereupon representative isolates were identified. The yeast species that had the highest incidence of predominance in the vineyard was Kloeckera apiculafa. However, some vineyard samples were characterised by low numbers or absence of this yeast, which is not according to generally accepted norms. Other species that also predominated in a few of the vineyard samples were Candida pulcherrima, Kluyveromyces thermofolerans, Rhodotorula sp. and Zygosaccharomyces bailii. Generally, there was a greater diversity of yeasts in the processed must than from the vineyard samples. Furthermore, while each sample showed a different yeast population, no pattern linking species to climatic zone was observed. Four species i.e. Candida collieulosa, Candida pulcherrima, Candida stel/ata and Kloeckera apiculata, were found to predominate in grape must samples. Representative strains consequently received further attention during laboratory and small-scale winemaking trials. A protocol was developed whereby individual species could be used in co-inoculated fermentations with S. cerevisiae in the small-scale production of wine. An improvement in wine quality was achieved and it was found that there was a link between specific species and grape cultivar. The ability of C. pulcherrima to improve Chenin blanc wine quality was investigated further. Results over three vintages showed that the wine produced by the co-inoculated fermentation was superior to that of a reference wine (produced by S. cerevisiae only). The improvement in wine quality was not linked to increased ester content nor were the standard chemical analyses adversely affected. The effects of pH and wine production parameters i.e. 802, fermentation temperature and use of di-ammonium phosphate (DAP), on this yeast followed the same pattern as that known for S. cerevisiae. This study was successfully completed and the developed protocol can be used for the improvement of Chenin blanc wine where additional aroma and quality is needed.
- ItemCharacterization of Chenin Blanc wines produced by natural fermentation and skin contact : focus on application of rapid sensory profiling methods(Stellenbosch : Stellenbosch University, 2014-12) Weightman, Jayne; Nieuwoudt, Helene; Setati, Mathabatha Evodia ; Stellenbosch University. Faculty of AgriSciences. Dept. of Institute for Wine Biotechnology.ENGLISH ABSTRACT: Producers of South African (SA) dry and semi-dry Chenin blanc table wines are currently experimenting with winemaking techniques to modulate the flavours of the predominantly fruity styles of this genre. An important stage during wine style development is sensory profiling paired with consumer acceptance testing, before wine is produced on industrial scale. With those aforementioned goals in mind, this study was conducted in partnership with two commercial SA wine cellars. The main focus of the study was an investigation into the treatment effects of two winemaking techniques, respectively grape skin contact and natural fermentation, on the sensory profiles of experimentally produced Chenin blanc wines. Results obtained with descriptive sensory analysis (DA) of the wines were compared to those obtained by two rapid sensory profiling methods, namely projective mapping (PM) and frequency of attribute citation (FC). A consumer preference study was also done on the wines. In order to understand the treatment effects better, the dominant non-Saccharomyces yeasts that were present during the natural fermentations were identified using polymerase chain reaction (PCR), while the major volatile chemical compounds were identified with chromatography and mass spectrometry. The sensory and chemical profiles of the naturally fermented wines were significantly different from those of the inoculated wines. PCR analysis identified some of the yeasts present during alcoholic fermentation. In comparison to the inoculated fermented wines, the naturally fermented wines were generally perceived to have more intense and riper tropical fruity aromas, with enhanced sweetness and reduced intensities of sourness, bitterness and astringency. The wines fermented on the skins (FoS) had lower ester and volatile thiol concentrations than the control wines (with no skin contact) and limited skin contact wines. Sensory attributes linked to the FoS wines included aromas of dried fruit, dried grass and a vegetative character, with an increased sour and bitter taste and astringent mouthfeel. In contrast, the wines that were produced with limited skin contact (12 hours) retained their tropical fruity aromas better than the FoS wines. Limited skin contact seemed to have had a less harsh effect on the taste and mouthfeel than the FoS wines. A consumer study was done to establish a Generation Y consumer group’s (18-35 years) preference for the different treated wines. Overall, the naturally fermented wines, which were described as having a strong tropical fruit character, were preferred. The FoS wines were generally disliked by the consumers. Results obtained from the three sensory analysis methods, respectively DA, PM and FC, were similar, thereby confirming the suitability of the rapid methods PM and FC, to extract qualitative information from the sensory profiling of white wine. The results of this study made a significant contribution towards validation of rapid sensory methods for wine evaluation, which are particularly valuable in the context of sustainability and technology transfer to research and industry alike. The knowledge gained on the chemical profiles of SA Chenin blanc is novel and this is one of the first reports on the volatile thiol content of SA Chenin blanc wine.
- ItemCoevolution 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.
- ItemConstruction of recombinant Saccharomyces cerevisiae strains for starch utilisation(Stellenbosch : Stellenbosch University, 2002-12) Eksteen, Jeremy Michael; Pretorius, I. S.; Van Rensburg, P.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology .ENGLISH ABSTRACT: Starch-containing agricultural crops are widely available as feedstocks for the production of fuel ethanol, potable spirits or beer, single-cell protein (animal feed) and high-fructose corn syrups (sweeteners). Starch-rich crops, such as maize, rye, barley and wheat, are usually used for the production of whisky. One of the first steps in the production of whisky is to boil the raw starch at temperatures exceeding 100°C. This gelatinisation step is performed to disrupt and solubilise the starch granules to make them more accessible for enzymatic hydrolysis. After this cooking process, the starch is liquefied by a-amylase and then saccharified by glucoamylase and a debranching enzyme. Lipomyces kononenkoae and Saccharomycopsis fibuligera secrete highly effective a-amylases and glucoamylases, making them two of the most efficient raw-starchdegrading yeasts known. However, L. kononenkoae and S. fibuligera cannot be used in existing industrial fermentations because of their low ethanol tolerance, slow growth rate, catabolite repression, poorly characterised genetics and lack of GRAS (Generally Regarded As Safe) status. This study is divided into two sections. The aim of the first section was to clone a gene (LKA2) encoding a novel starch-degrading enzyme, a second a-amylase (Lka2p) from L. kononenkoae. LKA2 was cloned into a multicopy plasmid, the yeast episomal plasmid, YEp352, under the control of the phosphoglycerate kinase promoter (PGK1 p) and terminator (PGKh) expression cassette. This recombinant plasmid was designated pJUL3 and transformed into a laboratory strain of S. cerevisiae, I1278b. Plate and liquid assays revealed that the recombinant yeast secreted active a-amylase into the medium. The optimum pH for Lka2p was pH 3.5 and the optimum temperature 60°C. The aim of the second part of the study was to construct recombinant strains of S. cerevisiae secreting a-amylase and/or glucoamylase. The individual genes were cloned into a yeast-integrating plasmid, Ylp5, under the control of the PGK1p-PGK1.,-expression cassette. Two indigenous yeasts were selected on the basis of their ability to utilise raw starch, L. kononenkoae and S. fibuligera, as gene donors. Eight constructs containing the L. kononenkoae a-amylase genes, LKA 1 and LKA2, and the S. fibuligera a-amylase (SFA 1) and glucoamylase (SFG1) genes were prepared: four single-cassette plasmids expressing the individual coding sequences under the control of the PGK1 p-PGK1.,- expression cassette, resulting in plPLKA 1, pIPLKA2, plPSFA 1 and pIPSFG1, respectively; two double-cassette plasm ids (expressing both LKA 1 and LKA2 under the control of the PGK1p-PGK1 .,-expression cassette, and SFA 1 and SFG1 under their respective native promoters and terminators), resulting in pIPLKA1/2 and pIPSFAG, respectively, and two single-cassette plasmids expressing SFA 1 and SFG1 with their native promoters and terminators, resulting in pSFA 1 and pSFG1, respectively. The respective constructs were transformed into a laboratory strain of S. cere visiae , L1278b. By homologous recombination, each plasmid was integrated into the yeast genome at the ura3 locus. S. cerevisiae L:1278b that had been transformed with plPLKA 1/2, LKA 1 and LKA2 under the control of the PGK1 rrPGK1,expression cassette resulted in the highest levels of a-amylase activity when assayed for amylolytic activity in a liquid medium. This recombinant strain resulted in the most efficient starch utilisation in batch fermentations, consuming 80% of starch and producing 6 gIL of ethanol after 156 hours of fermentation. The strain expressing SFG1 under the control of the PGK1rrPGK1,expression cassette gave the highest levels of glucoamylase activity.' These results confirmed that co-expression of a-amylase and/or glucoamylase synergistically enhance starch degradation. This study paves the way for the development of efficient starch-degrading strains of S. cerevisiae for the production of whisky, beer and biofuel ethanol.
- ItemConsumer sensory perception of South African Chenin blanc wine(Stellenbosch : Stellenbosch University, 2018-03) Mapheleba, Andiswa; Nieuwoudt, Helene; Muller, Nina; Pentz, Chris; Oertle, Ivan; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: In the food and beverage industries, the understanding of consumer behaviour and the sensory characteristics of products are important to producers as they aid in the implementation of communication and marketing strategies. South African Chenin blanc is a white wine variety that is characterised by diverse sensory profiles, an attribute that is considered as a strength of the variety from a technical wine production perspective. Accordingly, the wine industry has become highly interested in gaining knowledge about the perceptions of consumers regarding Chenin blanc styles in particular. The main aim of this study was to investigate the sensory and overall perceptions of Chenin blanc wine among South African consumers. Sensory analysis was conducted on three different Chenin style wines, namely fresh, complex unwooded and complex wooded wines using descriptive analysis (DA) with a trained panel. Among consumers, free listing and CATA were carried out to explore their sensory perceptions through encouraging them to freely describe their tasting experience and the selection of the perceived of aroma attributes obtained from DA. Thereafter, an exploratory study with the aid of a survey questionnaire was conducted to obtain insights of the consumers’ consumption, purchase behaviour and general perceptions about Chenin blanc wine.. Industry professionals from different disciplines of the wine industry, participated in the study by providing their viewpoints on aspects regarding the position of Chenin blanc in the SA wine industry through a self-completed questionnaire. The sensory attributes that were mostly provided by the consumers in free listing were fruit, citrus, tropical, honey, wood, earthy, nutty and fresh. A significantly lower number of attributes was perceived by consumers with CATA. The important non-sensory attributes listed by the consumers included their emotions, context of usage (occasion) and pleasure. They generally displayed an inclination towards buying wines with simple sensory label descriptors. According to the industry professionals, wine labelling and initiatives by the SA wine industry should be simple and relatable. Furthermore, Chenin blanc would highly benefit from cultivar-specific shows, due to its wide wine style spectrum. This study contributed towards a better understanding of the perceptions of SA wine consumers about Chenin blanc wine of the two respondent groups to aid in the understanding of the market and the wine industry, as well as their prospective roles in the future of Chenin blanc in SA.
- ItemDefining the chemical features of wine perception(Stellenbosch : Stellenbosch University, 2018-03) Fairbairn, Samantha; Bauer, Florian; Da Silva Ferreira, A. C.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: All wines evoke a product recognition, regardless of quality and cultivar, but what is the origin of this feature? The prevalence of this wine concept suggests that its formation occurs independent of the varietal, and ageing-related aromas, and is therefore potentially a function of yeast metabolism. Yeast utilise the nutrients present in grape must to produce biomass, and metabolites which ultimately signify the conversion of grape juice to wine. Consequently, the nutrient composition is highly influential on the aromatic outcomes of alcoholic fermentation. Synthetic grape must is widely used to evaluate all facets of the fermentation process but there remains much to learn. In this study, the impact of two nutrients, namely, amino acids and anaerobic factors, were evaluated with regard to their impact on yeast growth and aroma production under fermentative conditions. This work also examines the extent to which yeast de novo metabolism, both primary and secondary metabolism, contributes to the formation of the wine-like feature. In a single amino acid context, a linear relationship was apparent between the amino acid concentration and the production of their associated volatile products. This relationship was evaluated in more complex amino acid mixtures and as expected, this linear relationship was lost. Nonetheless, a significant degree of responsiveness between the amino acid and its catabolites remained. The impact of sterol (plant or yeast derived) or unsaturated fatty acid treatments, individually, as well as in combinations, were compared for their contributions to biomass formation and aroma production. Sterols had a greater impact on biomass development, as the fermentations treated with only unsaturated fatty acids displayed a poorer response. Moreover, they differently impacted aroma production. The unsaturated fatty acid lowered the production of acetate esters, medium chain fatty acids and their esters, whereas sterol supplementation generally bolstered the production of all compounds measured. This work highlights the importance of anaerobic factor management during winemaking. Although these nutrients certainly impact wine aroma, this study also sought to examine the degree to which these nutrients contribute to wine (product) recognition. Using a novel fermentation-based approach, Saccharomyces cerevisiae converted a synthetic grape must into a wine-like product. These synthetic products underwent sensory evaluations to rate the product’s resemblance to wine as well as to describe the aroma. This sensory data was used as a decision-making tool to decide upon treatments to be studied in subsequent fermentations. Ultimately, a wine-like character was created by altering the anaerobic factor composition of a synthetic grape must. The use of this synthetic grape must would allow for the more meaningful sensory characterisation of these synthetic products, in addition to providing a wine-like matrix used to evaluate the sensory implications of wine odorants.
- ItemThe deletion and overexpression of two esterase genes, IAH1 and TIP1, in Saccharomyces cerevisiae to determine their effects on the aroma and flavour of wine and brandy(Stellenbosch : Stellenbosch University, 2002-12) Hignett, Jason Satch; Du Toit, M.; Pretorius, I. S.; Lambrechts, M. G.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology .ENGLISH ABSTRACT: No single chemical constituent can be accredited with giving wine and brandy their overall aroma and flavour. The aroma and flavour of wine and brandy are rather attributed to a number of chemical constituents reacting together and it is these reactions that give the beverage its character. Certain chemicals within wine and brandy do, however, make larger contributions to the flavour. These include the esters, terpenes and volatile acids, although others also exist. Esters are a large group of volatile compounds with variable aroma and flavour characteristics, including banana-like (isoamyl acetate), apple-like (ethyl caproate) and chemical/solvent-like (ethyl acetate). Esters are produced as secondary metabolites during the conversion of sugar to ethanol and are formed when an alcohol binds with a fatty acid. Chemically, ester metabolism is well documented and understood; however, much work still needs to be done on a genetic level. The yeast strain used during fermentation is one of the most important factors contributing to the type and quantity of esters produced. This is due to differences in genetic makeup. The metabolism of esters is controlled largely on a genetic level, with numerous genes being involved. The alcohol acetyltransferase genes are involved in ester anabolism, whilst esterase genes are involved in ester catabolism. Esterases have a negative effect on the overall level of esters within an alcoholic beverage, as they are capable of reducing the number of esters and are thus capable of altering the beverage's aroma and flavour profile. The IAH1 and the TIP1 gene products are believed to encode for two such esterases. The objective of this study was to investigate the contribution of the IAH1 and TIP1 genes to the level of esters in both wine and brandy. This was accomplished by using two approaches. Firstly, the above genes were disrupted using a polymerise chain reaction (PCR)-generated disruption cassette homologous to either the IAH1 or the TIP1 gene. These cassettes were integrated into the industrial wine yeast, Saccharomyces cerevisiae strain VIN13. The integrations were verified by Southern blot analysis to produce yeasts VIN13-~IAH1 and VIN13-~TIP1; however, only a single copy of each was disrupted. Secondly, the IAH1 and the TIP1 genes were cloned from S. cerevisiae using PCR into plasmid pj between the phosphoglycerate kinase gene (PGK1) promoter and terminator, producing plasmids pJ-IOE1 and pJ-TOE1. The PGK1 promoter has previously been shown to constitutively express genes at high levels. These new constructs were then used as template for PCR to produce two overexpression cassettes, one for IAH1 and the other for TlP1. These cassettes were integrated into S. cerevisiae VIN13 and verified by Southern blot analysis to produce strains VIN13-IOE1 and VIN13-TOE1. The above yeast strains including VIN13 were used for the production of wines and base wines from Colombard must. Reverse-transcriptase (RT-PCR) confirmed that the VIN13-IOE1 and VIN13-TOE1 strains overexpressed the appropriate gene at a higher level than the control VIN13 strain. The VIN13-AIAH1 disrupted strain showed no difference in expression level to that of the control strain, whilst VIN13-ATIP1 showed lower levels of expression than that of the control strain. VIN13-IOE1 behaved as expected, with a decrease of between 30% and 60% in the total ester level in the wine and base wine respectively, a 30% decrease in the total acid level and no change in the higher alcohol level. The VIN13-AIAH1 strain showed no difference to the control wine, most likely as this strain still expressed the IAH1 gene at levels consistent with the control strain. VIN13-TOE1 behaved in an unexpected manner - instead of hydrolysing esters, it appeared to produce them. This increase in the total ester level was most noticeable during distillation, when a 20% increase took place. Another unexpected occurrence was a large decline in the total acid level, with acetic acid being the most significant contributor, decreasing by up to 78%. This is a very favourable finding, as acetic acid is a known spoilage molecule and is a cause of sluggish/stuck fermentations. VIN13-ATIP1 behaved in an opposite manner to VIN13-TOE1, with higher total acid levels and slightly decreased total ester levels, especially during distillation. Neither affected the total higher alcohol levels. Sensorially, the only significant difference in the wine samples was for the fruity flavour. A panel of judges distinguished that VIN13-TOE1 was fruitier than the other wines, with VIN13-ATIP1 being the least fruity. This study again proves the significant impact that a single gene can have on the chemical makeup of wine and brandy. The relatively simple genetic alteration of an organism can drastically change and improve not only the organoleptic properties of the organism, but its viability as well. These alterations can produce more favourable organisms with more desirable characteristics for the fermenting beverage industry to produce products of higher quality and better suitability.
- ItemEffect of foliar Nitrogen and Sulphur spraying on white wine composition ( Vitis vinifera L.cv. Chenin Blanc and Sauvignon Blanc)(Stellenbosch : Stellenbosch University, 2018-12) Bruwer, Freda Aléta; Buica, Astrid; Stellenbosch University. Faculty of AgriSciences. Dept. Viticulture & Oenology & Institute for Wine Biotechnology.ENGLISH ABSTRACT: Chenin Blanc and Sauvignon Blanc, as the most planted wine cultivars in South Africa, are of great interest to researchers worldwide, due to its increased high wine quality. Wine quality is interlinked with wine aroma. Vine nitrogen fertilization influence the vine physiology and composition of the grapes, and enhanced aroma expression. By addressing Yeast Assimilable Nitrogen (YAN) deficiency with foliar fertilization, during the ripening season, to low nitrogen containing vines, the aroma potential of the wines can be potentially influenced. The main aim of this research study was to assess the influence of different foliar fertilization treatments on Chenin Blanc and Sauvignon Blanc vines planted in various locations in South Africa. For each season, two vineyards with a history of producing low nitrogen content grapes were used, one vineyard per cultivar. The vineyards received sulphur and nitrogen foliar treatments twice before véraison. During winemaking, the juices and wines underwent analysis for non-volatile and volatile content. The wines underwent maturation for three and nine months, and then sensorially and chemically analysed. In Chapter 2 the various wine compounds and classes of compounds present in Chenin Blanc and Sauvignon Blanc wines were analysed. The specific characteristics, aroma composition and its implications on the sensory perception of the cultivars were reviewed. The influence and contribution of different fertilization practices on the chemical compounds and resulting wine’s aromatic expression were investigated. The first part of the research study investigated the effect of foliar fertilization on the non-volatile content in the juices and wines. In Chapter 3, the nitrogen containing foliar fertilization applications increased the YAN levels. This increase is relevant not only for yeast metabolism, but also for the aromatic potential of a wine, as certain amino acids being precursors of aroma compounds. Glutathione were also influenced by the treatments for both years and both cultivars, but the trends were not as evident as with YAN. The second part of the study assessed the effect of various fertilization treatments on the volatile content of the juices and aged wines. Sensory analysis and chemical analysis were used to assess the wines after three and nine months of bottle maturation. Chapter 4 highlighted that sulphur containing foliar treatments influenced the volatile content of major volatiles and volatile thiols. The overall volatile content of the wines was very similar but identified a clear vintage and age effect during maturation. Sensory analysis classified the Chenin Blanc wine with ‘tropical’ and ‘fruity’ aromas, while Sauvignon Blanc wines had prominent ‘tropical’, ‘passion fruit’, and ‘grapefruit’ aromas. During bottle maturation, some notes and aroma characters were maintained but their frequency of citations changed. The results of this research study contributed to the knowledgebase on South African Chenin Blanc and Sauvignon Blanc wines, but also concluded that foliar fertilization can influence the non-volatile and volatile content of wines. South African winemakers and the industry can use this information to make decisions at the viticulture and winemaking level to produce wines with more desirable sensory attributes.
- ItemThe effect of grape temperature on the phenolic extraction and sensory perception of Méthode Cap Classique wines(Stellenbosch : Stellenbosch University, 2017-03) Mafata, Mpho; Van Jaarsveld, Francois; Du Toit, Wessel J.; Buica, Astrid; Stellenbosch University. Faculty of AgriScience. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: The first sparkling wine in South Africa was released in 1971. The South African Cap Classique Producers Association (CCPA), formed for the appreciation of Méthode Cap Classique (MCC) traditional style sparkling wines (TSW), was established in 1992 and has since contributed to the growth of these wines on a competitive footing with the international market. Generally, studies on TSW have focused primarily on the foam capability, volatile composition and autolytic character of the wines and very little on phenolic content of the wines. Phenolic compounds are important quality indicators of wine. Their composition in wine is determined by various factors including grape variety, terroir, viticultural practice, and oenological practices. In this project, MCC wines were made by the traditional method using Chardonnay and Pinot noir grapes harvested from two regions (Robertson and Darling) and stored at 0, 10, 25 and 30ºC, over two vintages (2014 and 2015). The phenolic concentration of the wine samples throughout the winemaking process was analysed by spectrophotometer and the aroma and taste of the final 9 month old sparkling wines performed. The study was aimed at investigating the effect of the grape storage temperature on the phenolic content and the sensory properties of MCCs through a quantitative phenolic analysis. The study found that MCCs made from grapes stored at lower temperatures (0 and 10ºC) had lower total phenolic content, colour intensity and total hydroxycinnamates than wines made from grapes stored at higher temperatures (25 and 30ºC) showing that there was greater phenolic extraction from grapes stored at 25 and 30ºC. The total phenolics, as measured by spectrophotometer, was below the range cited in literature for Champagne made from the same cultivars. The sensory evaluation of the MCCs comprised a sorting analysis similar to that used for beers. Separating the aroma and taste sorting of the MCCs, the study showed a grouping of the MCCs according to temperature treatments for both vintages. There were, however, clear vintage differences in terms of the attributes cited and the frequency of citations. Based on frequency of citation, 2014 MCCs made from grapes stored at 0 and 10°C were described by judges as being fruity, fresh and crisp whilst those made from grapes stored at 25 and 30°C were described as having oxidised fruit, volatile acidity and solvent-like aromas. The judges perceived less oxidation and VA (in terms of the frequency of citation) in the aroma of 2015 MCCs, although higher temperature treatments were still associated with less desirable attributes compared to lower temperature treatments. Judges were better able to separate the Darling wines according to treatments compared to the Robertson wines. This study has shown that the grape storage temperature has an effect on the phenolic extraction and the sensory perception of MCCs aged 9-months with no changes in the phenolic content observed throughout winemaking.
- ItemEffect of non-Saccharomyces yeasts and lactic acid bacteria interactions on wine flavour.(Stellenbosch : Stellenbosch University, 2018-03) Du Plessis, Heinrich Wilbur; Jolly, Neil; Du Toit, Maret; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: Wine aroma and flavour are important indicators of quality and are primarily determined by the secondary metabolites of the grape, by the yeast that conducts the primary fermentation and also the lactic acid bacteria (LAB) that performs malolactic fermentation (MLF). This is a complex environment and each microorganism affects the other during the wine production process. Therefore, the overall aim of this study was to investigate the interactions between Saccharomyces, non-Saccharomyces yeasts and LAB, and the effect these interactions had on MLF and wine flavour. Contour-clamped homogeneous electric field gel electrophoreses (CHEF) and matrix-assisted laser desorption ionization using time-of flight mass spectrometry (MALDI-TOF MS) were useful tools for identifying and typing of Hanseniaspora uvarum, Lachancea thermotolerans, Candida zemplinina (synonym: Starmerella bacillaris) and Torulaspora delbrueckii strains. Hanseniaspora uvarum strains had β-glucosidase activity and Metschnikowia pulcherrima strains had β-glucosidase and protease activity. Only Schizosaccharomyces pombe and C. zemplinina strains showed mentionable malic acid degradation. Candida stellata, C. zemplinina, H. uvarum, M. pulcherrima and Sc. pombe strains were slow to medium fermenters, whereas L. thermotolerans and T. delbrueckii strains were found to be medium to strong fermenters, comparable to S. cerevisiae. The effect of non-Saccharomyces yeast species on MLF varied and inhibition was found to be strain dependent. In a Shiraz winemaking trial where seven non-Saccharomyces strains were evaluated in combination with S. cerevisiae and three MLF strategies, the C. zemplinina and the one L. thermotolerans isolate slightly inhibited LAB growth in wines where yeast and LAB were inoculated simultaneously. However, the same effect was not observed during sequential inoculation of LAB. Mixed culture fermentations using non-Saccharomyces yeasts contained lower alcohol levels, and were more conducive to MLF than wines produced with S. cerevisiae only. Yeast treatment and MLF strategy resulted in wines with significantly different flavour and sensory profiles. Yeast selection and MLF strategy had a significant effect on berry aroma, but MLF strategy also had a significant effect on acid balance and astringency of wines. In a follow up trial, H. uvarum was used in combination with two S. cerevisiae strains, two LAB (Lactobacillus plantarum and Oenococcus oeni) species and three MLF strategies. One of the S. cerevisiae strains had an inhibitory effect on LAB growth, while H. uvarum in combination with this S. cerevisiae strain had a stimulatory effect on MLF. Simultaneous MLF completed faster than sequential MLF and wines differed with regard to their chemical and sensory characteristics. Isoamyl acetate, ethyl hexanoate, ethyl octanoate, ethyl-3-hydroxybutanoate, ethyl phenylacetate, 2-phenyl acetate, isobutanol, 3-methyl-1-pentanol, hexanoic acid and octanoic acid were important compounds in discriminating between the different wines. Yeast treatment had a significant effect on fresh vegetative and spicy aroma, as well as body and astringency of the wines. The LAB strain and MLF strategy had a significant effect on berry, fruity, sweet associated and spicy aroma, as well as acidity and body of the wines. Mid-infrared (MIR) spectroscopy was used to differentiate between wines produced with the selected Saccharomyces and non-Saccharomyces yeast combinations, LAB species and MLF strategies. This study provides valuable information about the interactions between non-Saccharomyces, Saccharomyces yeast, LAB and MLF strategies, and how important pairing of strains are to ensure successful AF and MLF. Furthermore, the results also showed how these interactions can be applied to diversify wine flavour.
- ItemThe effect of wind on the performance of the grapevine(Stellenbosch : Stellenbosch University, 2005-12) Pienaar, Jacobus Wilhelm; Carey, Victoria; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology .ENGLISH ABSTRACT: Wind, as a component of the environment, contributes to the viticultural concept of terroir in the South Western Cape region of South Africa. Many other components also contribute to terroir (e.g. soil, altitude, direction of slope, etc.) and it is difficult to quantify the contribution of each. A good terroir promotes slow and complete ripening of the berries. A vineyard on such a terroir produces good quality crops over time and the effects of climatic extremes on the performance of the vine are limited by this terroir. Although grapevines in the South Western Cape are exposed to strong synoptic southerly and southeasterly winds during the growing season and sea breezes add to the effect of wind in coastal areas, little is known about the effect of wind on grapevine performance. This preliminary study was undertaken to obtain more information on the effect of wind on some morphological and reproductive characteristics of Merlot nair. The aim was to obtain preliminary data which can serve as a basis for future studies on the effect of wind on grapevine performance. Important differences .in wind speed were measured spatially in a selected vineyard and exposure to wind was observed to result in essential viticultural differences. The effect of wind on vegetative parameters, canopy density, yield, berry composition and wine quality was investigated. Wind caused leaves on primary shoots of exposed vines to be smaller, but increased lateral growth in their fruiting zones. Sheltered vines had longer shoots but no significant differences were measured concerning cane diameter and pruning mass. Although it was expected that sheltered vines would have denser canopies, similar canopy densities were measured for both treatments. Vines exposed to wind responded with decreased stomatal conductance. As a result, leaf temperature was affected, showing differences between sheltered and exposed vines. Sheltered vines had more bunches per vine but fewer berries per bunch. As a result, bunches of sheltered vines were smaller than those of exposed vines. No significant difference was observed concerning the yield under the two treatments. The effect of wind on stomatal conductance had an essential impact on berry composition, thus directly influencing the quality of wine. Grapes from exposed vines showed a higher colour index. Berries from sheltered vines had significantly lower pH values and potassium concentrations and the malic acid content was lower than in berries from exposed vines. Wine from sheltered vines had more vegetative undertones in comparison with the stronger fruity character of exposed vines. A better acid balance, together with superior complexity (fullness/mouth feel), contributed to the better overall quality identified during the evaluation of wine produced from sheltered vines.
- ItemEngineered yeast and microalgae mutualisms: Synthetic ecology applied to species isolated from winery wastewater(Stellenbosch : Stellenbosch University, 2018-03) Simpson, Zoe Faith; Bauer, Florian; Naidoo, Rene K.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: Large volumes of winery wastewater, classified as biodegradable industrial effluent, are generated annually. The development of a cost-effective treatment system is difficult due to the variable and batch nature of winery wastewater. Research has focused on the development of dynamic biological treatment systems using microorganisms including yeast and microalgae, as it has become apparent that these microbes have bioremediation capabilities in various wastewaters. However, no biological winery wastewater treatment system, employing multi-species ecosystems with known species of yeast and microalgae, currently exists. In this study, yeast and microalgae were previously isolated from natural winery wastewater. The first aim of this study was to characterise the bioremediation potential of the yeast, Saccharomyces cerevisiae, and microalga, Parachlorella beijerinckii, in synthetic and raw winery wastewater. P. beijerinckii was physiologically characterised and was able to tolerate salinity and ethanol levels commonly associated with winery wastewater, making it a suitable candidate for further bioremediation studies. Both S. cerevisiae and P. beijerinckii were able to decrease the chemical oxygen demand of winery wastewater and P. beijerinckii monoculture was able to increase the pH of the acidic wastewater. S. cerevisiae out-competed P. beijerinckii in co-culture growth experiments. Interestingly, yeast growth was improved in the presence of the microalgae in this system, suggesting a potential for symbiotic association. The increased yeast growth however had no impact on the bioremediation potential of the co-culture system. To overcome this drawback, a synthetic ecology approach was used to engineer stable symbiotic associations between these evolutionarily unrelated strains of yeast and microalgae. Engineered mutualisms between S. cerevisiae and P. beijerinckii were established under strongly selective conditions based on the nutrient exchange of carbon and nitrogen. These mutualistic associations were relatively easy to establish as the complementary metabolic abilities of each organism were key elements in the mutualism design. The impact of temperature and pH were assessed in these obligatory mutualistic conditions to determine whether the co-culture functions optimally in specific environmental conditions and whether such conditions are similar or different from the optimal conditions required for single species growth. Experiments were first conducted in small scale and continued in larger scale bioreactor studies. The bioreactor conditions were evaluated to generate a more constant continuous culture system. Such continuous culture system would provide an ideal tool to conduct studies on the evolutionary development of mutualistic associations, and may be the first step in developing a multi-species approach to winery wastewater treatment with enhanced bioremediation capabilities. We propose that in the long run such co-culture systems might serve to overcome the limitations associated with single culture system and might improve biotechnological processes.
- ItemEngineering yeast for the production of optimal levels of volatile phenols in wine(Stellenbosch : Stellenbosch University, 2002-12) Smit, Annel; Van Rensburg, Pierre; Cordero Otero, R. R.; Lambrechts, M. G.; Pretorius, I. S.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology .ENGLISH ABSTRACT: Phenolic acids (principally p-coumaric and ferulic acids), which are generally esterified with tartaric acid, are natural constituents of grape must and wine, and can be released as free acids during the winemaking process by certain cinnamoyl esterase activities. Free phenolic acids can be metabolised into 4-vinyl and 4-ethyl derivatives by several microorganisms present in wine. These volatile phenols contribute to the aroma of the wine. The Bretfanomyces yeasts are well known for their ability to form volatile phenols in wine. However, these species are associated with the more unpleasant and odorous formation of the ethylphenols and the formation of high concentrations of volatile phenols. Other organisms, including some bacterial species, are responsible for the formation of volatile phenols at low concentrations, especially the 4-vinylphenols, and this enhances the organoleptic properties of the wine. The enzymes responsible for the decarboxylation of phenolic acids are called phenolic acid decarboxylases; and several bacteria and fungi have been found to contain the genes encoding these enzymes. The following genes have been characterised: PAD1 from Saccharomyces cerevisiae, fdc from Bacillus pumilus, pdc from Lactobacillus plantarum and padc from Bacillus subtilis. PadA from Pediococcus pentosaceus was also identified. S. cerevisiae contains the PAD1 (phenyl acrylic acid decarboxylase) gene, which is steadily transcribed in yeast. The activity of the PAD1-encoded enzyme is low. Phenolic acid decarboxylase from B. subtilis, as well as p-coumaric acid decarboxylase from L. plantarum displays substrate inducible decarboxylating activity with phenolic acids. Both the p-coumaric acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) genes were cloned into PGK1 PT expression cassette. The PGK1 PT expression cassette consisted of the promoter (PGK1 p) and terminator (PGK1 T) sequence of the yeast phosphoglyceratekinase I gene (PGK1). Episomal and yeast integration plasmids were constructed for the PAD1 gene under the control of the PGK1 PT for overexpresion in yeast. Industrial strains with the PAD1 gene disrupted were also made. Overexpression of pcoumaric acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) in S. cerevisiae showed high enzyme activity in laboratory strains. The overexpressed PAD1 gene did not show any higher enzyme activity than the control strain. Both bacterial genes, under the control of the PGK1 PT cassette, were also cloned into a yeast-integrating plasmid, with the SMR1 gene as selective marker. The cloning and transformation of pdc and padc into industrial wine yeast strains can therefore be used to detect the effect of phenolic acid decarboxylase genes in the winemaking process for the possible improvement of wine aroma. Wine was made with all three strains (the bacterial genes overexpressed and PAD1 disrupted). The effect of these genes in wine was determined through GC analysis. The results showed that the bacterial genes could effectively produce higher levels of volatile phenols in the wine. The manipulated strains also produced enzymes capable of producing large amounts of favourable monoterpenes in the wine. This study paves the way for the development of wine yeast starter culture strains for the production of optimal levels of volatile phenols, thereby improving the sensorial quality of wine.
- ItemEspacement studies on unirrigated grafted Pinot noir (Vitis vinifera L.)(Stellenbosch : Stellenbosch University, 1990) Archer, Eben; Goussard, P. G.; Stellenbosch University. Faculty of AgriSciences. Department of Viticulture and Oenology and Institute for Wine Biotechnology.ENGLISH ABSTRACT: The effect of vine spacing on root distribution, plant and soil water status, some physiological aspects as well as vegetative and reproductive growth of Vitis vinifera L. cv. Pinot noir, grafted onto 99 Richter (Vitis Berandieri var. Las Sorres x Vitis rupestris var. du Lot) was investigated. Vine spacings used were 1,0 m x 0 .5 m, 1,0 m x 1,0 m, 2,0 m x 1,0 m, 2,0 m x 2,0 m, 3,0 m x 1,5 m, and 3,0 m x 3,0 m. This trial was conducted on a medium potential soil without irrigation in the Stellenbosch district. Root density was directly affected by vine spacing and it increased and decreased with closer and wider spacing respectively. The larger contact surface between roots and soil in the case of closer spaced vines, increased the utilization of soil water and nutrients. This increased depletion of soil water, induced water stress earlier in the growth season with the result of a timely arrestment of shoot growth. This phenomenon brought about advantageous characteristious in canopy density and canopy microclimate. Vine spacing also affected cordon length per vine as well as total cordon length per hectare, thus affecting the distribution and orientation of the canopy. Closely spaced vines induced shorter cordons per vine, but the total cordon length per hectare was higher than that of widely spaced vines. Although a smaller crop was produced per vine in the case of closer spacings, the total yield per hectare was higher than in the case of wider spewed vines. In addition, a better potential for bunch nutrition was ensured by a higher leaf surface : fruit mass ratio in the case of closer spaced vines. The low canopy densities of closer spaced vines, together with a better water-supply early in the season (bud break to pea size) ensured a higher physiological activity than in the case of wider spaced vines. As the season progressed, however, this situation was reversed so that the shoot elongation of closer spaced vines ceased before that of wider spaced vines. This phenomenon was advantageous for bunch nutrition, affecting both grape composition and wine quality. The subterranean and above-ground changes brought about by closer vine spacing augmented both yield and quality. A better wine quality was obtained through a higher sugar concentration, lower acid concentration and better colour of the grapes. A higher yield per hectare stemmed from an increase in total cordon length per hectare. These results were realized for a medium potential soil without irrigation. More luxurious conditions will probably induce more vegetative growth, necessitating wider in-row vine spacing for the best yield and quality. The ideal vine spacing for a given locality is dictated by soil potential, rootstock and scion cultivar and cultivation practices such as irrigation and fertilization. It is probably wrong to standardise vine spacing for all vineyards on a specific farm.
- ItemEvaluating the effect of oxygen addition on yeast physiology, population dynamics and wine chemical signature in controlled mixed starter fermentations(Stellenbosch : Stellenbosch University, 2017-12) Sekhawat, Kirti; Setati, Mathabatha Evodia ; Bauer, Florian; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH SUMMARY: The use of commercial starter cultures of non-Saccharomyces yeast, usually together with Saccharomyces cerevisiae, has become a trend in the global wine industry in the past decade. Depending on the specific species of non-Saccharomyces yeast, the procedure may aim at enhancing aroma and flavour complexity of the wine, reduce acetic acid levels, and/or lower the ethanol yield. However, the contribution of non-Saccharomyces yeast strains depends on several factors, and in particular on the strains ability to establish significant biomass and to persist for a sufficient period of time in the fermentation ecosystem. For an effective use of these yeasts, it is therefore important to understand the environmental factors that modulate the population dynamics of such environments. In this study, we evaluated the effect of oxygen addition on yeast physiology, population dynamics and wine chemical signature in controlled mixed starter fermentations. The population dynamic in co-fermentations of S. cerevisiae and three non-Saccharomyces yeast species namely, Torulaspora delbrueckii, Lachancea thermotolerans, and Metschnikowia pulcherrima, revealed that oxygen availability strongly influences the population dynamics and chemical profile of wine. However, results showed clear species-dependent differences. Further, experiments were confirmed in Chardonnay Grape juice, inoculated with L. thermotolerans and S. cerevisiae with different oxygen regimes. The results showed a trend similar to those obtained in synthetic grape juice, with a positive effect of oxygen on the relative performance of L. thermotolerans. The results in this study also indicates that continuous stirring supports the growth of L. thermotolerans. We further analysed the transcriptomic signature of L. thermotolerans and S. cerevisiae in single and mixed species fermentations in aerobic and anaerobic conditions. The data suggest the nature of the metabolic interactions between the yeast species, and suggests that specific stress factors are more prominent in mixed fermentations. Both yeasts showed higher transcript levels of genes whose expression is likely linked to the competition for certain metabolites (copper, sulfur and thiamine), and for genes involved in cell wall integrity. Moreover, the transcriptomic data also aligned with exo-metabolomic data of mixed fermentation by showing higher transcripts for genes involved in the formation of aroma compounds found in increased concentration in the final wine. Furthermore, the comparative transcriptomics analysis of the response of the yeasts to oxygen provides some insights into differences of the physiology of L. thermotolerans and S. cerevisiae. A limited proteomic data set aligned well with the transcriptomic data and in particular confirmed a higher abundance of proteins involved in central carbon metabolism and stress conditions in mixed fermentation. Overall, the results highlight the role of oxygen in regulating the succession of yeasts during wine fermentations and its impact on yeasts physiology. The transcriptomics data clearly showed metabolic interaction between both yeasts in such ecosystem and provide novel insights into the adaptive responses of L. thermotolerans and S. cerevisiae to oxygen availability and to the presence of the other species.
- ItemEvaluating the impact of yeast co-inoculation on individual yeast metabolism and wine composition(Stellenbosch : Stellenbosch University, 2014-12) Mains, Arlene Olive; Bauer, Florian; Divol, Benoit; Stellenbosch University. Faculty of AgriSciences. Dept. of Institute for Wine Biotechnology.ENGLISH ABSTRACT: The use of non-Saccharomyces yeasts together with Saccharomyces cerevisiae in mixed starter cultures has become an accepted oenological tool to enhance the organoleptic properties of wine. Recent studies have indeed demonstrated the positive contribution that non- Saccharomyces yeasts may have on the bouquet of wine. These mixed starter cultures are characterized by high inoculation levels of individual strains into the must, and each strain in turn is characterized by its own specific metabolic activity. These factors lead to a multitude of interactions occurring between the individual populations within the must. The fundamental mechanisms which drive these interactions are still largely unknown, but several studies have been conducted in order to investigate the metabolic outcome of these interactions. In this study, we endeavour to further characterize the interactions which occur between four individual non-Saccharomyces yeast strains in mixed culture fermentation with S. cerevisiae. Metschnikowia pulcherrima IWBT Y1337, Lachancea thermotolerans IWBT Y1240, Issatchenkia orientalis Y1161 and Torulaspora delbrueckii CRBO LO544 were used in mixed culture fermentations with a commercial strain of S. cerevisiae at an inoculation ratio of 10:1 (non-Saccharomyces: S. cerevisiae). The biomass evolution and fermentation kinetics of both participating species were affected by the high cell density of the other, with neither population reaching the maximal density attained by the pure culture fermentation. The final wine composition of each individual mixed fermentation showed clear differences, from the pure cultured S. cerevisiae and from each other, based on the concentrations of the major volatile compounds found in the wine. Upon further characterization of these specific mixed culture fermentations, it was found that each individual combination of non-Saccharomyces and S. cerevisiae produced similar increases and decreases of certain major volatile compounds as demonstrated by previous authors, using the same combination of non-Saccharomyces species together with S. cerevisiae. From a winemaking perspective, the use of these non- Saccharomyces yeast strains in combination with S. cerevisiae could be a useful strategy to diversify the chemical composition of wine, by increasing the concentration of certain desirable volatile compounds and by modulating the concentration of undesirable metabolites. Furthermore, this research serves as a foundation for further elucidation of the interactions which drive these metabolic outcomes in response to the high cell density of two yeast populations in mixed culture fermentations.