Masters Degrees (Institute for Wine Biotechnology)

Permanent URI for this collection

https://scholar.sun.ac.za/handle/10019.1/5685

Browse

Browsing Masters Degrees (Institute for Wine Biotechnology) by browse.metadata.advisor "Cordero Otero, R. R."

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    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.
  • Thumbnail Image
    Item
    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.