Fifty yeast strains previously isolated from grapes and wines were investigated for extracellular protease activity. Saccharomyces yeasts showed no detectable protease activity, while most of the non-Saccharomyces yeasts exhibited proteolytic activity. Nine of the fifty yeast strains were cultivated in grape juice and were analysed for the production of extracellular proteases. The proteolytic activity of Metschnikowia pulcherrima (M 004) was as high as that of Rhodotolura sp. (R3). Very low or undetected activity was found for in the other yeasts. In a synthetic grape juice supplemented with grape proteins, the yeasts exhibiting protease activity did not show considerably higher growth than the yeasts exhibiting undetectable protease activity.
The characterization of eleven non-Saccharomyces yeasts during fermentation of pasteurized Riesling juice revealed differences in the concentrations of non-volatile and volatile metabolites. There was a great variation in the ability of yeasts to consume sugars and to influence the composition of odouriferous compounds of the fermented must. The analysis of reducing sugars in the fermented musts at the end of fermentation permitted the classification of yeasts into 3 groups according to their ability to ferment. The concentrations of other volatile and non volatile metabolites varied within genera, species and strains.
To find out whether yeasts exhibiting protease activity could support growth of Saccharomyces yeasts, the sequential inoculation of Non-Saccharomyces and Saccharomyces was performed in grape juice fermentation. 500 mg/l of DMDC and 250 mg/l of lysozyme were added to grape juice in order to suppress the natural microflora. The target concentration of residual sugars (less than 2 g/l) was achieved in the wine obtained from the sequential inoculation of Hanseniaspora uvarum (H 045) prior to S. cerevisiae (S-EC). The concentrations of assimilable nitrogen slightly increased in wines with sequential inoculation of H. uvarum (H 045, H 097) prior to S. cerevisiae (S-EC, S-CM).
The effect of the inoculation protocols of non-Saccharomyces and Saccharomyces yeasts on the formation of metabolites during the fermentation of clarified and unclarified Rielsing juice was investigated. The three inoculation strategies studied were: pure strain inoculation, where only S. cerevisiae was added to the grape juice; co-inoculation, where the yeast strains were simultaneously added to grape juice; and sequential inoculation, where the non-Saccharomyces species were added 4 days prior to S. cerevisiae. The fermentation kinetics were influenced by the inoculation protocols and by the turbidity level of musts. The concentrations of reducing sugars were lower and the concentrations of ethanol were slightly higher in wines obtained from the fermentation of clarified must where sequential inoculation of non-Saccharomyces yeast prior to S. cerevisiae was used. This effect was not observed in wines obtained from fermentation of unclarified must. The analyses of nitrogen and aroma compounds in the resultant wines have revealed that a wide variability in the production and transformation of these compounds during fermentation existed, depending on inoculation protocols and turbidity level of musts. Seven yeasts were cultivated in defined medium and cell free supernatants were used to determine the concentrations of soluble proteins. The content of soluble proteins in the medium after cultivation ranged from 3600 to 12100 mug/l. When the cultivation of yeasts in grape juice was performed under microaerobic conditions, the peptides and proteins in the resultant fermented juices ranged from 14 to 1500 mug/l and 1490 to 2301 mug/l, respectively. Wines were also chosen to examine the content of peptides and proteins. The concentrations of soluble peptides and proteins of those wines ranged from 86 to 1516 g/l and 40 to 699 mug/l, respectively. The variability of proteins in grape juices and wines was studied by SDS-PAGE. Results showed that various sizes of proteins with a molecular weight of lesser than 14.4 kDa to greater than 99 kDa were present in grape juices and wines.Deliberate use of lysozyme in must resulted in traces of this enzyme in the wines and musts during fermentation.
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