Prospection of indigenous yeasts from Uruguayan Tannat vineyards for oenological applications.

Prospection of yeasts from oenological environments can provide knowledge of new native strains that are capable of fermenting must and positively influence the composition and sensory characteristics of the wine. This work addressed the biotechnological characterization of indigenous yeasts of Tannat must, an emblematic and widespread vineyard of Uruguay. Fifty-three yeast isolates were morphologically characterized and further identified by amplification and sequencing of ITS and D1-D2 regions, grouping into a total of fifteen species. One isolate of each species was randomly chosen and evaluated for its technological traits. In presence of ethanol (6 to 16% v/v) and sulfur dioxide (40 mg/L), native Saccharomyces cerevisiae 3FS presented the best growth rates and minor lag phase. Regarding non-Saccharomyces strains, Starmerella bacillaris 3MS stood out for its behavior in vinification conditions, more closely related to S. cerevisiae strains. Saccharomyces cerevisiae 3FS, Starmerella bacillaris 3MS, and Saturnispora diversa 1FS conducted a successful fermentation process reaching a final ethanol concentration ≥ 10% v/v and presenting a killer and resistant phenotype, suggesting that they could be used as pure starter cultures, as well as in mixed culture fermentations. This preliminary screening and oenological characterization of indigenous Saccharomyces cerevisiae and non-Saccharomyces yeasts might be a useful tool to identify some strains as potential candidates for wine vinification.

Influence of beta glucosidases from native yeast on the aroma of Muscat and Tannat wines.

It is now well established that ß-glucosidases (BGLs) from non-Saccharomyces yeasts are key enzymes that hydrolyze grape-derived aroma precursors enhancing the flavour of wines. This work reports on the specificity for wine glycosides and the impact on wine aroma, of three native yeast ß-glucosidases. Volatile compounds were analyzed by gas-chromatography and mass spectroscopy (GC-MS) and wine aroma was studied by sensory analysis. Issatchenkia terricola ß-glucosidase stood out from the other ß-glucosidases studied. The I. terricola BGL showed remarkable specificity for norisoprenoid aglycones such as: 3-oxo-7, 8-dihydro-alpha-ionol, 3-oxo-α-ionol, vomifoliol. This different specificity was perceived in the sensory tests. The judges described pleasant fruity, sweet, honey and raisin notes in both Tannat and Muscat wines treated with I. terricola BGL. These results are particularly remarkable for Tannat wines, since there are few reports concerning the application of ß-glucosidases to enhance its aroma of Tannat, and none with BGLs from native yeasts.

Aroma enhancement in wines using co-immobilized Aspergillus niger glycosidases.

A major fraction of monoterpenes and norisoprenoids in young wines is conjugated to sugars representing a significant reservoir of aromatic precursors. To promote their release, ß-glucosidase, α-arabinosidase, and α-rhamnosidase from a commercial Aspergillus niger preparation, were immobilized onto acrylic beads. The aim of this work was the development and application of an immobilized biocatalyst, due to the well-known advantages over soluble enzyme preparations: control of the reaction progress and preparation of enzyme-free products. In addition, the obtained derivative showed increased stability in simile wine conditions. After the treatment of Muscat wine with the biocatalyst for 20days, free monoterpenes increased significantly (from 1119 to 2132µg/L, p<0.01) with respect to the control wine. Geraniol was increased 3,4-fold over its flavor thresholds, and accordingly its impact on sensorial properties was very relevant: nine of ten judges considered treated wine more intense in fruit and floral notes.

Immobilization of enzymes: a literature survey.

The term immobilized enzymes refers to "enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously." Immobilized enzymes are currently the subject of considerable interest because of their advantages over soluble enzymes. In addition to their use in industrial processes, the immobilization techniques are the basis for making a number of biotechnology products with application in diagnostics, bioaffinity chromatography, and biosensors. At the beginning, only immobilized single enzymes were used, after 1970s more complex systems including two-enzyme reactions with cofactor regeneration and living cells were developed. The enzymes can be attached to the support by interactions ranging from reversible physical adsorption and ionic linkages to stable covalent bonds. Although the choice of the most appropriate immobilization technique depends on the nature of the enzyme and the carrier, in the last years the immobilization technology has increasingly become a matter of rational design. As a consequence of enzyme immobilization, some properties such as catalytic activity or thermal stability become altered. These effects have been demonstrated and exploited. The concept of stabilization has been an important driving force for immobilizing enzymes. Moreover, true stabilization at the molecular level has been demonstrated, e.g., proteins immobilized through multipoint covalent binding.

One-step purification and characterization of an intracellular beta-glucosidase from Metschnikowia pulcherrima.

A collection of 60 non-Saccharomyces yeasts isolated from grape musts in Uruguayan vineyards was screened for beta-glucosidase activity and Metschnikowia pulcherrima was the best source of this enzyme activity. Its major beta-glucosidase was successfully purified to homogeneity by ion-exchange chromatography on amino-agarose gel. The enzyme exhibited an optimum catalytic activity at 50 degrees C and pH 4.5 and was active against (1 --> 4)-beta and (1 --> 2)-beta glycosidic linkages. In spite of preserving 100% of its activity and stability in the presence of 12% (v/v) ethanol and 5 g glucose/l, the enzyme was unstable below pH 4. We characterized the beta-glucosidase from M. pulcherrima with a view to its potential applications in wine-making.