Masters Degrees (Institute for Wine Biotechnology)

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Carotenoid 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
Cell 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.
Characterisation 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.
Characterization 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.
Coevolution of Saccharomyces cerevisiae and Lactobacillus plantarum : engineering interspecies cooperation
(Stellenbosch : Stellenbosch University, 2018-03) Du Toit, Sandra Christine; Bauer, Florian; Rossouw, Debra; Du Toit, Maret; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Microbial interactions are ubiquitous in nature and play a vital role in economically important industrial processes like winemaking. Saccharomyces cerevisiae and Lactobacillus plantarum are important species responsible for the completion of alcoholic and malolactic fermentation (AF and MLF) respectively. Understanding how these species interact with each other and their environment is important to better manage successful completion of AF and MLF. However, the complexity of the wine matrix makes it nearly impossible to study these interactions in a natural environment and synthetic ecological systems can therefore be used to overcome these difficulties. This study was designed to establish a co-dependent, mutualistic relationship between S. cerevisiae and Lb. plantarum in order to gain insights into the cooperation between species, how pH, temperature, and inoculation dosages influences the interaction, and how the interaction evolves over time. The interaction, centered on the reciprocal exchange of amino acids, was established between the lysine auxotrophic strain S. cerevisiae THI4 and the isoleucine, alanine, valine, and methionine auxotrophic strain Lb. plantarum B038. Different combinations of amino acids were omitted from the chemically defined synthetic grape juice-like media in order to find an amino acid treatment which promoted the best growth for both microorganisms. B038 showed excellent growth when cocultured with THI4 for all the amino acid treatments, but THI4 struggled to grow under these conditions. The two treatments selected for further experiments were the Lys-Ile (lysine and isoleucine omitted) and Lys-Val (lysine and valine omitted) treatments since THI4 showed the best growth under these conditions. Lower temperature and pH conditions had a negative effect on the growth and malic acid consumption of B038, but when co-cultured with THI4 the yeast appeared to stimulate the growth of the bacteria under both selective and control conditions. THI4 continued to show poor growth performance and sugar consumption under these conditions. However, when THI4 and B038 were inoculated at cell densities with similar biomass, the growth of THI4 improved significantly. It was expected that THI4 and B038 would show poor growth when grown in the absence of their respective auxotrophic amino acids and support of their respective partner. This proved true for all the amino acid treatments except when B038 was grown in the absence of lysine and valine. B038’s ability to grow under these conditions was hypothesized to be linked to the uptake of glutamine and the production of γ-Aminobutyric acid (GABA), but further research is still required to investigate this. Over continuous rounds of fermentation, THI4 adapted to the imposed selective conditions by increasing its consumption of glucose while cell density remained the same. Whether this is linked to increased ethanol production still needs to be determined. No significant changes were observed in B038 after coevolving the strains. This study provides relevant insights into the industrially important interaction between S. cerevisiae and Lb. plantarum and also provides a basis for future work to create optimised yeast-bacteria pairings for both industrial applications in winemaking and to investigate the genetic changes involved in the establishment of cooperative interactions between species.
Construction 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.
Consumer 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.
The 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.
Effect 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.
The 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.
The 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.
Engineered 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.
Engineering 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.
Evaluating 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.
Evaluating the vitamin requirements of wine-related yeasts and the resultant impact on population dynamics and fermentation kinetics
(Stellenbosch : Stellenbosch University, 2019-04) Julies, Jerobiam Marvin; Bauer, Florian; Divol, Benoit; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: Within the vineyard environment, grape berries serve as a habitat to various microorganisms including bacteria, filamentous fungi and yeasts, of which some play distinct roles in winemaking. Studies on yeast species other than Saccharomyces cerevisiae, commonly referred to as non-Saccharomyces (NS) yeasts in oenology, have evaluated the ability of these yeast to modulate the sensory profile of wine. In the early stages of spontaneous fermentation when the ethanol concentrations are low, the NS yeast population increases, but is progressively replaced by S. cerevisiae, which is better adapted to the environmental conditions associated with fermenting grape juice. The overall sensory profile of wine is in part a result of the metabolite production of yeasts, and the extent of the contribution of each species will depend on the total metabolic activity of each species. Metabolic activity is directly related to the availability of nutrients such as carbon, nitrogen, vitamins and trace elements. These nutrients are indeed converted to biomass and other metabolites, many of which are aroma and flavour active by-products. Only limited information regarding the nutrient requirements of wine-related yeasts other than S. cerevisiae has been published. Several studies have explored the carbon and nitrogen requirements of some NS species, but the vitamin requirements of many biotechnologically relevant species remains to be determined. Vitamins are organic compounds, mostly of a complex chemical nature, and serve as cofactors in metabolic reactions. Vitamins occur in small quantities in grapes and grape juice, but some data suggest that they may in some cases be limiting for yeast growth in this environment, affecting metabolism and ultimately impact the final wine. This knowledge gap motivates the current study, which focuses on the growth and fermentation kinetics of different NS yeasts when presented with varying concentrations of the relevant vitamins: biotin, pantothenate, inositol, thiamine and pyridoxine. In a first section, a high-throughput microtiter plate assay was optimised to allow for the rapid screening of the vitamin requirements of NS yeasts. The results of this assay showed differences in the vitamin requirements amongst the different yeasts. The statistically most significant vitamin-dependent yeast phenotypes from the screen were selected for further investigation. These included the dependence of Viniflora® P. kluyveri Frootzen ™ on biotin and thiamine and of Viniflora ® L. thermotolerans Concerto ™ on inositol. The data obtained from this study provide a better understanding of the vitamin requirements of NS yeasts and how these requirements can potentially enhance the growth performance of NS yeasts. The data suggest that targeted nutrient additions may lead to a better modulation of the overall sensory profile of wine.
The evaluation of bacteriocins and enzymes for biopreservation of wine
(Stellenbosch : Stellenbosch University, 2002-03) Du Toit, Corina; Du Toit, M.; Pretorius, I. S.; Van Rensburg, P.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: The winemaking process involves a number of microorganisms, each with its own role. Yeasts are responsible for the alcoholic fermentation, the lactic acid bacteria (LAB) are Gram-positive bacteria associated with must and wine and perform the malolactic fermentation (MLF), while the acetic acid bacteria (AAB) are Gram-negative bacteria converting ethanol to acetic acid. These microorganisms are present in the cellar and fermentation tanks and can be seen either as beneficial or as wine spoilage microorganisms because, under certain circumstances, they affect the wine quality if they should grow in the wine or must. Strict measures need to be implemented in the cellar during the winemaking process to ensure microbiological stability. This can be achieved through good microbiological practices and, additionally, chemical preservatives. Sulphur dioxide (S02) is widely used as the primary preservative in winemaking. However, consumer resistance has been building up against the use of chemical preservatives, due to the possible health risks and a decrease in nutritional value and sensorial quality of certain foods and beverages. Biopreservation as an alternative to the traditionally-used chemical preservation is a new approach and has been attracting much attention. This implies the use of the natural microflora and/or their antibacterial products, such as bacteriocins and bacteriolytic enzymes (e.g. lysozyme). Bacteriocins from LAB are proteins or protein complexes, produced by Gram-positive bacteria, with antibacterial activity against closely-related Gram-positive species. Lysozyme occurs in substances such as hen egg white and has lytic activity against Gram-positive bacteria. ' The bacteriocins nisin, of the class I lantibiotics, and pediocin PA-1 and leucocin BTA 11a, of the class lIa Listeria-active bacteriocins, have been investigated for the biopreservation of wine. Nisin, however, is the only bacteriocin that has been approved for use as a preservative, while pediocin is likely to follow in approval. Lysozyme has been approved for use in winemaking by the Office International de la Vigne et du Vin (OIV). The main objectives of this study were to determine whether these substances showed any antimicrobial action against wine-associated microorganisms, namely LAB, AAB and yeasts. The stability and suitability of the bacteriocins and lysozyme as antimicrobials in wine was researched, especially when used in combination. Possible synergistic or antagonistic interactions between the bacteriocins were also investigated by means of the microtitre broth dilution method and scanning electron microscopy, as well as at what concentration and combinations the bacteriocins were most effective against increasing LAB concentrations. It was found that nisin, pediocin and leucocin were effective to varying degrees against a test panel of LAB type and reference strains, as well as wine isolates. Nisin repeatedly had the highest level of inhibition against all the LAB tested, followed by pediocin and leucocin. There was no inhibition of the wine-associated AAB and yeasts tested. Pediocin stability was evaluated in simulated wine must and proved to be stable for at least 20 days, without being affected by the sulphur or alcohol content. A low pH, however, led to a more rapid decrease in activity. The same was found for nisin and leucocin in other studies. Combinations of bacteriocins at increasing concentrations were evaluated against increasing concentrations of a LAB wine isolate. When used in pairs (namely, nisinleucocin, nisin-pediocin and pediocin-Ieucocin), the combinations were most effective against lower concentrations of bacteria, namely 102 and 104 cfu/ml. At lower concentrations, the pairs of bacteriocins were much less effective against the higher bacterial concentrations of 106 and 108 cfu/ml. Leucocin-pediocin combinations were the least effective, while nisin-Ieucocin combinations were marginally more effective than the nisin-pediocin combinations. The most pronounced effect was observed when all three the bacteriocins were used together. Combinations of bacteriocins had no inhibitory effect against AAB. Pediocin and lysozyme was used in combination against the same wine isolate, but no conclusive conclusions could be drawn in this experiment. __ Scanning electron microscopy was used to investigate any disturbances in cell morphology when bacteriocins were added to LAB. The above-mentioned LAB was subjected to bacteriocins used singularly and also in combinations of equal amounts of bacteriocins. The action of the bacteriocins led to major disturbances in cell morphology. Once again, the combination of leucocin-pediocin was the least effective, even less so than when the single bacteriocins were used. The nisin-pediocin and nisin-Ieucocin combinations seemed to be more effective in causing cell disturbances and perturbations. The microtitre broth dilution methodwas used to further characterise the nature of the interaction of the pairs of bacteriocins. This test showed clearly that the bacteriocins had definite interactions. By adding one bacteriocin to varying concentrations of another bacteriocin, the inhibitory action of the second bacteriocin was affected, either increasing or decreasing its effectiveness. The most important factor to consider seems to be the ratio at which the bacteriocins should be used together, leading either to synergism or antagonism, and this also implies a very complex interaction. This project indicated that it is indeed possible to use both bacteriocins and lysozyme in "Vine preservation, both being stable in wine environments and effective against LAB without affecting the yeast fermentation. Bacteriocins could also be used in combination, to broaden the inhibition spectrum, as well as possibly increasing the inhibitory potential of the individual antimicrobials. The underlying interactions in such combinations should be carefully researched, however, when considering using combinations of antimicrobials in food and beverage products. Further attention can also be given to finding biopreservatives against the Gram-negative AAB, as well as to research the interaction of the pairs of bacteriocins over time. Another point to consider would be the engineering of yeasts or bacteria to produce these antibacterial substances in situ as part of their metabolism.
The evaluation of Fourier transform infrared (FT-IR) spectroscopy and multivariate data analysis techniques for quality control at aniIndustrial cellar
(Stellenbosch : Stellenbosch University, 2015-04) Hoon, Ansunette; Nieuwoudt, Helene; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: The alcoholic beverage industry needs objective analysis of grape juice and liqueur quality. Fourier transform infrared (FT-IR) spectroscopy with multivariate data analysis techniques is widely used in wine laboratories across South Africa for accurate, fast and high sample throughput analyses. In this study the potential of FT-IR spectroscopy is evaluated for the quantification of ammonia in freshly pressed grape juice. FT-IR spectroscopy is evaluated, using two different spectrometers, in respectively attenuated total reflection (ATR) - and transmission scanning modes for the quantification of alcohol, pH and invert sugar in spirit-based liqueurs. The ultimate aim was to implement the PLS regression algorithms developed at an industrial cellar and replace the complex and lengthy reference methods used at the time of this study. Principle component analysis (PCA) was performed prior to the calibration step to identify groupings and patterns within the spectra. The PLS calibration models were developed from samples collected at the cellar and using partial least square (PLS) regression. The models were evaluated using the performance criteria coefficient of determination (R2) and root mean squared error of cross validation (RMSECV) at calibration stage, and root mean square error of prediction (RMSEP) and residual predictive deviation ratio (RPD) at validation stage. The average RMSEP (1.88 mg/L) of the ammonia PLS calibration model was in agreement with the standard error of laboratory (SEL = 1.54 mg/L). The R2 (92.05) and average RPD (3.3) proposed a model with excellent precision for screening purposes that was ready to be transferred for use by the laboratory. The r2 values for the alcohol, pH and invert sugar PLS calibration models obtained in ATR and transmission, indicated good to excellent precision (80
The evaluation of malolactic fermentation starter cultures under South African winemaking conditions
(Stellenbosch : Stellenbosch University, 2008-03) Van der Merwe, Hanneli; Du Toit, M.; Du Toit, Wessel J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: With ever increasing pressure on wine producers to lower the financial costs involved in winemaking to be able to compete in the market, all while maintaining a high level of wine quality, the focus on maintaining control over all aspects of the winemaking process are greatly emphasized. Malolactic fermentation (MLF) is one of the important processes in red wine production. The advantages of this process, when performed successfully, is widely known and accepted. One way to gain control over MLF is the use of MLF starter cultures. Starter cultures usually consist of Oenococcus oeni that has been isolated from grapes or wines and is in most cases available in a freeze-dried form ready for direct inoculation into the wine when MLF is desired. Starter cultures are induced into wine and usually ensure the immediate onset as well as a fast and clean execution of the process. Starter cultures used in South Africa are in most cases isolated from cooler viticultural regions in the Northern hemisphere. The constitution of wines from cooler viticultural regions, differ from those in South Africa, which has a warm climate. The most important difference is the acid content of the wines which is lower in South African must/wines and results into a higher pH. The three most important changes that develop in wine during MLF are a decrease in acidity due to the conversion of malic acid to the less harsh lactic acid, enhanced flavour and aroma of wine and an increase in the microbiological stability of wine. The decrease in acidity is very important for wines produced for grapes grown in cool viticulture regions. In South Africa though, the climate is warm and higher pH’s are present in the musts and wines and the de-acidification due to MLF is not the main aim but rather the microbiological stabilisation. One of the compounds that could be produced by lactic acid bacteria (LAB) is biogenic amines (BA’s). These compounds can be hazardous to human health. This thesis focussed on the performance of MLF starter cultures in high pH South African red wines. The first objective of the study was to stretch MLF starter cultures in high pH red wines of South Africa. Stretching means to use less than the prescribed dosage or the re-use of starter cultures. The difference in MLF rate, the influence of the natural occurring LAB and the levels of biogenic amines formed during MLF were determined for the different stretching treatments. The results showed that different rates in malic acid degradation were experienced between the treatments, but in all cases MLF fermentation was completed. Biogenic amines were formed at various levels and the influence of the natural occurring LAB also played a role. The second objective of the study was the evaluation of the effect of a wine isolated LAB (Lactobacillus) and an acetic acid bacteria (AAB), inoculated with a MLF starter culture had on MLF at different wine pH’s. It was found that especially in the case where the Lactobacillus was inoculated in combination with the MLF starter culture a possible stimulatory effect was experienced with regards to malic acid degradation rate. Biogenic amine concentration was measured at the end of MLF and it was found that no histamine and tyramine were formed in any of the treatments, while the putrescine and cadaverine levels were found to be at approximately similar levels for the different treatments. The third objective was to evaluate the possible influence of commercial tannin additions and a pectolytic enzyme on rate of MLF and phenolic composition of high pH red wine. The commercial tannins had possible inhibitory as well as stimulatory effects on the rate of malic acid degradation especially during the initial stages of MLF, with the highest dosage having the significant effect. The BA results showed difference in the levels produced due to tannin additions as well as strain differences could exist. The phenolic content showed a decrease in colour density, total red pigments, total phenolics and anthocyanins between AF and MLF. The fourth objective was to evaluate inoculation time of MLF starter cultures. The results showed that the fastest AF/MLF time was with simultaneous inoculation of the yeast and MLF starter cultures. It was also for this treatment where no histamine or tyramine was detected at the end of MLF compared to the other inoculation strategies (before the end of AF and after AF). This study generated a large amount of novel data which made a valuable contribution with regards to MLF in high pH red wines of South Africa.
The evaluation of β-glucosidase activity produced by wine-isolated yeasts
(Stellenbosch : Stellenbosch University, 2004-03) Potgieter, Nydia, 1977-; Van Rensburg, P.; Pretorius, I. S.; Cordero Otero, R. R.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
ENGLISH ABSTRACT: ~-Glucosidases constitute a major group of biologically important enzymes that catalyze the hydrolysis of glycosidic linkages in ~-glucosides, as well as in glycosides that contain only carbohydrate residues, e.g. cellobiose. These enzymes occur in all living kingdoms and perform a variety of functions in organisms ranging from bacteria to highly evolved mammals. Three different types of ~-glucosidases are found in humans, each with its own function: glucocerebrosidase (a deficiency causes Gaucher disease), lactase-phlorizin hydrolase (a deficiency results in lactose intolerance) and cytosolic ~-glucosidase (responsible for the hydrolysis of ~- glucosides ingested with foods of plant and animal origin). In plants, the functions of ~-glucosidases include pigment metabolism, biomass conversion and cyanogenesis, a function it shares with insect ~-glucosidases. Microbial ~-glucosidases, as part of the cellulase enzyme system that is responsible for the hydrolysis of cellobiose and short-chain oligosaccharides into glucose, playa role in the conversion of cellulosic biomass to liquid fuel. These microbial ~-glucosidases also playa very important role in the enhancement of fruit and wine aromas through the liberation of monoterpenols. Monoterpenols play an invaluable role in the flavor and aroma of grapes and wine, and are present as free, volatile and odorous molecules, as well as flavorless, non-volatile glycosidic complexes. These complexes most often occur as 6-0-~-Dxvlopyranosyl- B-Dcqlucopyranosides, 6-0-~-D-glucopyranosyl-~-D-glucopyranosides, 6-0-a-L-arabinofuranosyl-~-D-glucopyranosides, 6-0-a-L-rhamnopyranosyl-~-Dglucopyranosides, or 6-0-~-D-apiofuranosyl-~-D-glucopyranosides of mainly linalool, geraniol, nerol, a-terpineol and hotrienol. Two mechanisms exist for the release of monoterpenes from glycosidically bound non-volatile precursors: acid hydrolysis and enzymatic hydrolysis. As high temperature acid hydrolysis causes a rearrangement of the monoterpene aglycones, the focus has shifted towards the more efficient enzymatic hydrolysis that does not result in modifications of the intrinsic aromatic character of the wine. The endogenous ~-glucosidases of grapes (Vitis vinifera), as well as of the wine yeast Saccharomyces cerevisiae, exhibit very low activity towards the glycoside precursors, and thus the focus has increasingly fallen on the addition of exogenous ~-glucosidases to enhance wine flavor. Fungal, bacterial and some yeast ~- glucosidases have been indicated as effective aroma liberators, but these enzymes are not always suitable for use under the harsh conditions that prevail during winemaking (i.e. low pH, low temperatures, and high ethanol and glucose concentrations). The limited enzyme activities of the abovementioned microorganisms have resulted in a search among non-Saccharomyces yeasts for ~- glucosidases that can withstand these conditions. The ~-glucosidase activities of 20 wine-associated non-Saccharomyces yeasts were quantified, characterized and assessed to determine the efficiency with which they could liberate monoterpenols from their terpenyl-glycosides. The Debaryomyces pseudopolymorphus l3-glucosidase from intracellular crude cell extracts exhibited the most suitable combination of properties in terms of functionality at wine pH, resistance to wine-associated inhibitory compounds (glucose, ethanol and sulfur dioxide), high substrate affinity and large aglycone-substrate recognition. This yeast strain was also used, in conjunction with S. cerevisiae VIN13, for the small-scale fermentation of Chardonnay juice. The results indicated that the l3-glucosidase of D. pseudopolymorphus had definite potential as a wine aroma-enhancing enzyme, as the concentrations of free terpenols (nerol, geraniol and citronellol) were significantly increased during fermentation. Future experimental work would include an in-depth study of the kinetic characteristics of the l3-glucosidases (both cytosolic and cell-associated) exhibiting the highest terpenol-liberating activity under winemaking conditions. The next step would then be the cloning and expression of the most efficient l3-glucosidase gene in a commercial wine yeast. Such a recombinant wine yeast would release grapederived aroma compounds from their non-volatile precursors during single culture fermentations, thereby increasing the sensorial quality of wine.
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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