P. van Rensburg

Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts

Aims: The objective of this study was to investigate what types of enzymes are being produced by non‐Saccharomyces yeasts isolated from grapes in South Africa vineyards and clarified grape juice. These enzyme profiles could pave the way for attributing specific effects in wine to some of these enzymes produced by so‐called wild yeasts associated with grape must.

Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production

There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; β-d-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria and lactic acid bacteria, during fermentation. The A. niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone α-factor secretion signal (MFα1S) and the phosphoglycerate-kinase-1 gene promoter (PGK1P) and terminator (PGK1T). The PGK1P-MFα1S-gox-PGK1T cassette (designated GOX1) was introduced into a laboratory strain (Σ1278) of S. cerevisiae. Yeast transformants were analysed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid bacteria and acetic acid bacteria. This might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide, a known antimicrobial agent. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8–2.0% less alcohol. This was probably due to the production of d-glucono-δ-lactone and gluconic acid from glucose by GOX. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.

Genetic screening of wine-related enzymes in Lactobacillus species isolated from South African wines.

Aims:  The objective of this study was to investigate the presence of genes coding for enzymes of oenological relevance in wine Lactobacillus strains isolated from South African grape and wine samples during the 2001 and 2002 harvest seasons.

Fermentation-derived Aroma Compounds in Varietal Young Wines from South Africa

The volatile composition of 925 single cultivar young Sauvignon blanc, Chardonnay, Pinotage, Merlot, Shiraz and Cabernet Sauvignon wines of vintages 2005 to 2007, was determined using gas chromatography – flame ionisation detection. Compositional data were compared to published data on young wines from South Africa and other countries. South African young wines analysed in this study had a largely similar volatile composition to that reported in the literature. Significant between-vintage and between-cultivar differences were observed in the volatile composition of the wines investigated in this study. The concentration ranges of four compounds in red wines, hexanol, propanol, diethyl succinate and ethyl lactate, and four compounds in white wines, 2-phenylethanol, hexanoic acid, isoamyl acetate and propanol, were not influenced by vintage effects. This finding was interpreted as the first indication that typical concentration ranges for some aroma compounds can be established for South African young cultivar wines. A trend was observed in the white wines that the alcohols and their respective acetate esters, as well as fatty acids and their ethyl esters, were responsible for the vintage-related effects. Differences in volatile composition between Chardonnay and Sauvignon blanc wines could also largely be explained on the same basis. Classification models were established to discriminate between individual red wine cultivars and between the two white wine cultivars and correct classification rates of respectively, 79 % and 85 % were achieved.

Different genetic backgrounds influence the secretory expression of the LKA1-encoded Lipomyces kononenkoae α-amylase in industrial strains of Saccharomyces cerevisiae

A haploid laboratory strain and four industrial (baking, brewing, wine, ATCC) strains of Saccharomyces cerevisiae were transformed with the Lipomyces kononenkoae α-amylase-encoding gene (LKA1). These transformants displayed significant differences in terms of the level of secretory expression of LKA1 under control of the PGK1 promoter and terminator, as well as their ability to produce and secrete the LKA1-encoded rawstarch-degrading α-amylase and to ferment starch. These results demonstrate the importance of the selection of appropriate host strains for yeast development pursuant to starch conversion into commercially important commodities via consolidated bioprocessing.

The heterologous expression of polysaccharidase-encoding genes with oenological relevance in Saccharomyces cerevisiae

Aims:  The main objective of this study was to develop polysaccharide‐degrading wine strains of Saccharomyces cerevisiae, which are able to improve aspects of wine processing and clarification, as well as colour extraction and stabilization during winemaking.

The production of reduced-alcohol wines using Gluzyme Mono® 10.000 BG-treated grape juice.

High alcohol wines have become a major challenge in the international wine trade. Several physical processes are used to produce wines with reduced-alcohol content, all of which involve the selective extraction of ethanol based on volatility or diffusion. In this study, the possibility of Gluzyme Mono® 10.000 BG (Gluzyme) (Novozymes, South Africa) to reduce the glucose content of synthetic grape juice before fermentation was investigated in order to produce wine with reduced-alcohol content. Gluzyme is a glucose oxidase preparation from Aspergillus oryzae, currently used in the baking industry. Glucose oxidase catalyses the oxidation of glucose to gluconic acid and hydrogen peroxide (H2O2) in the presence of molecular oxygen. Gluzyme was initially used in synthetic grape juice, where different enzyme concentrations and factors influencing its efficiency were investigated under winemaking conditions. The results showed up to 0.5% v/v less alcohol at an enzyme concentration of 20 kU compared to the control samples. This reduction in alcohol was increased to 1 and 1.3% v/v alcohol at pH 3.5 and pH 5.5 respectively in aerated (8 mg/L O2) synthetic grape juice using 30 kU enzyme. Secondly, Gluzyme was used to treat Pinotage grape must before fermentation. Gluzyme-treated wines at 30 kU enzyme concentration after fermentation contained 0.68% v/v less alcohol than the control wines. A decrease in acetic acid concentration of the treated compared to control wines was also observed.

Gibberellic acid sprays, girdling, flower thinning and potassium applications affect fruit splitting and yield in the ‘Ellendale’ tangor

SummaryThis study quantified the effect of fruit set treatments on preharvest fruit splitting, a serious problem in the ‘Ellendale’ tangor, as well as treatments aimed at alleviating fruit splitting incidence. Treatments, during the full bloom period, aimed at increasing fruit set, viz. GA3 sprays and girdling, increased fruit splitting to between 30 to 60% of final yield. This related to a significant shift/delay in the normal fruit drop pattern. Only the girdling treatment during fullbloom significantly (P<0.05) increased final yield. The blossom GA3 sprays increased yield only slightly while the postbloom GA3 sprays, varying from 10 to 20mgl–1, did not affect final yield at all. In this study various approaches to reduce fruit splitting in ‘Ellendale’ tangor were tested: (i) specific chemical and mechanical flower thinning treatments reduced fruit splitting, without adversely affecting yield; (ii) potassium sprays, applied from ten weeks before fullbloom to six weeks after full bloom achieved limited s...

The influence of different winemaking techniques on the extraction of grape tannins and Anthocyanins

The aim of this study was to determine the effect of different maceration techniques on the extraction of grape tannins and anthocyanins. Two cultivars (Cabernet Sauvignon and Shiraz) were harvested in two different climatic regions (Durbanville and Simondium) at two different ripeness levels for the 2008 and 2009 harvest seasons. Five basic winemaking processes were applied, namely a normal alcoholic fermentation (C), enzyme treatment (E), cold soaking (CM), post-maceration (PM), and a combination of cold and post-maceration (CM+PM). At harvest the phenolic ripeness was determined with the Glories method, while the tannin concentration was determined with the methyl cellulose (MCP) method. The grapes in the warmer area had higher tannin levels than grapes harvested in the cooler area in both years. In the 2009 harvest season, the enzyme treatment extracted the highest levels of tannin. CM+PM showed the best results of tannin extraction with early ripeness (Cabernet Sauvignon), and CM with fuller ripeness in the warm area. CM showed the best results with both early and fuller ripeness levels in the cooler area. PM showed the best results with the early ripeness levels, and the E treatment with the fuller ripeness levels, in the warm area. CM+PM showed the best results with the early ripeness level in the cooler area, and varied results with the fuller ripeness levels. In both years, grapes from the cooler area contained more anthocyanin than those from the warmer area. At a fuller ripeness level (2009) the treatments had no effect.

The influences of different winemaking techniques on the mouthfeel of Shiraz grapes.

The objective of this study was to determine the effect of ripeness and of different tannin extraction methods on the sensory properties of wine, with a specific focus on mouthfeel properties. Quantitative descriptive analysis (QDA) was performed to evaluate the sensory properties of 20 young Shiraz wines in two phases. In Phase 1, wines from a cool area were evaluated and, in Phase 2, wines from a warm area were evaluated. Clear differences were found between the wines from the two regions. Wines from the cooler region were generally associated with higher levels of total non-flavonoids and total anthocyanins, and more intense numbing and puckering sensations. In contrast, the wines from the warmer region as a group were associated with a more drying and grippy mouthfeel, as well as less total anthocyanins and total non-flavonoids. In the set of wines from the cooler region, the effect of ripeness was more pronounced than in the set of wines from the warmer region. In both cases, riper grapes resulted in a coarser surface smoothness, a more numbing sensation, a bitter aftertaste and less adhesive mouthfeel. The wines from the cooler region that were harvested at a riper stage were associated with many of the anthocyanins/ anthocyanin derivatives and were negatively associated with hydroxycinnamate, procyanidin B1 and delphinidin-3-glucoside-p-coumaric acid. In the warmer area, the riper grapes were again associated with anthocyanins/anthocyanin derivatives, but this time were strongly associated with procyanidin B2, caffeic acid, p-coumaric acid, catechin, coutaric acid and total non-flavonoids. The effect of tannin extraction method on the sensory properties of the wines from the warmer region was more pronounced than in the wines from the cooler region. Unfortunately, the differentiation between treatments was not consistent from one ripeness level to the next. However, it appeared that the cold soak treatment differed the least from the control, regardless of region or ripeness, whereas the post-maceration treatment differed the most based on mouthfeel and phenolic composition. Although some mouthfeel attributes and phenolic compounds were consistently associated with region and ripeness, it is not clear if mouthfeel can be manipulated consistently by tannin extraction methods.