Chitinase and Thaumatin-like Pathogenesis-Related Proteins from grapes and their effects on wine stability.

Wine proteins play a key role in determining the quality of white wines, mainly because they are involved in haze formation during wine storage. Most of the proteins found in wine are pathogenesis-related (PR) proteins of grape origin, corresponding to chitinases and thaumatin-like proteins (TLP). Fractionation of grape proteins (cv. Incrocio Manzoni 6.0.13) by anion exchange FPLC allowed to recover of nine fractions (1-9), with fraction 5 containing the largest quantity of protein (about 30% of the total area of the chromatogram). Most of the recovered fractions contained more than one protein band, as shown by SDS-PAGE analysis. Different electrophoretic bands with chitinolytic activity were detected in all the fractions with the exception of fraction 1. Fractions 4 and 5 had the strongest activity and showed three chitinolytic bands of different SDS-PAGE mobility. Western blot analysis with an antibody raised against the Thaumatococcus daniellii thaumatin showed the presence of immuno-reactive bands (TLP) in fractions 2, 3 and 5. Similar results were obtained also when the proteins of the wine were examined. Heat tests were performed in both a model wine and in ultrafiltered wine (cut off 10 K) after addition of 150 mg/L of the total proteins purified from grape juice and wine, using BSA as the control. In all the cases, turbidity after heating was detectable only in ultrafiltered wine, confirming the key role of factors of non-protein nature in haze formation. Moreover grape and wine proteins showed similar behaviour in heat tests. The same heat test experiments were performed also with the different fractions obtained by FPLC separation of the grape proteins. Unexpectedly, the most unstable fraction (fraction 1) was that not containing chitinolityc activity or immuno-reactive TLPs, whereas the fractions richest in chitinases did not appear to be particularly unstable, in contrast to what was previously reported by others authors.