Iolanda Rosi

Study of β‐glucosidase production by wine‐related yeasts during alcoholic fermentation. A new rapid fluorimetric method to determine enzymatic activity

Aims:  The β‐glucosidase activity is involved in the hydrolysis of several important compounds for the development of varietal wine flavour. The aim of the present study was to investigate the production of β‐glucosidase in a number of wine‐related yeast strains and to measure and identify this activity over the course of grape juice fermentation.

Extraction and immobilization in one step of two β-glucosidases released from a yeast strain of Debaryomyces hansenii

Abstract An extracellular, constitutive, and nonglucose repressed β-glucosidase from a yeast strain of Debaryomyces hansenii was purified and immobilized using a one-step procedure on hydroxyapatite (HTP). Analysis of purified enzyme gave two bands both on SDS gel electrophoresis, native gel electrophoresis, and capillary electrophoresis. The two bands on SDS gels were positive for carbohydrate staining. Their apparent molecular mass was estimated to be 122 and 96 kDa with carbohydrates, and 109 and 81 kDa after carbohydrate removal, respectively. Amino acid analysis of electroblotted bands revealed that the n -terminus was blocked in both cases. Gel slices corresponding to the two bands, as obtained after native gel electrophoresis, were found to be reactive when incubated separately with p-nitro-phenyl-β- d -glucopyranoside (pNPG) as substrate. The Km of the two forms coeluted from HTP in the same fractions was 3.68 ± 0.06 m m . The optimum pH was 5. The immobilized enzyme exhibited a lower activity than the purified free enzymes, but both were much more stable than the enzymes in cell-free supernatant. The two enzyme isoforms in the mixture were only active against few glycosides with β-linkage configuration. Since the HTP-bound enzyme was found to be active, stable, easily separable from the substrate, and reusable, it could be potentially used in its immobilized form for the release of specific-bound aroma in wine and fruit juices.

Effect of yeast strain and fermentation conditions on the release of cell wall polysaccharides

To improve our understanding of the factors involved in polysaccharide release during alcoholic fermentation, we investigated three Saccharomyces cerevisiae strains in fermentation trials conducted at two temperatures (25 degrees C and 32 degrees C) and three sugar concentrations (20%, 23.5%, and 27%), with or without supplementation of grape juice with diammonium phosphate (DAP) or microcrystalline cellulose. In two yeast strains, the release of cell wall polysaccharides increased significantly with an increase in fermentation temperature and sugar concentration of the grape juice; the polysaccharide release was greater in stressed conditions, in which the cells were less viable and less metabolically active. In the third strain, the average amount of polysaccharides released into the medium decreased significantly at 32 degrees C with 27% sugar, and increased in grape juice supplemented with DAP. Thus, this strain released more polysaccharides when conditions were nearer to optimal and the yeast cells were more viable and metabolically active. Our results suggest that the yeast strains released cell wall polysaccharides via different mechanisms, and that the cell wall integrity pathway may account for some of the differences in polysaccharide release among the strains.

Effect of Oenotannin Addition on the Composition of Sangiovese Wines from Grapes with Different Characteristics

Different types of oenotannins were added to Vitis vinifera cv. Sangiovese grapes of various composition and the resulting wine color parameters were measured, including intensity, hue, total phenol index, monomer anthocyanin content, and colored polymeric pigment content. Oenotannins were also tested during pre- and postfermentation on grapes harvested in 2003 from two growing areas. Grapes from the 2004 harvest were tested at different ripeness using only oenotannins exhibiting the best wine color stabilization in 2003. The response of different oenotannins varied according to grape characteristics, with gallnut and grape seed tannins most reliably stabilizing and increasing color intensity even after six months of storage. The same tannins positively affected wine color stabilization, an effect further enhanced in wine produced from less ripe grapes. Timing of oenotannin addition and grape characteristics had a significant effect on color intensity and stabilization. The estimation of grape phenolic maturity may allow for improved wine color characteristics by tailoring the use of oenotannins.