Removal of unstable proteins from white wine by immobilized acid protease
Author(s)
Date Issued
February 17, 2011
Type
Doctoral Thesis
Abstract
This PhD thesis research project was aimed at assessing and optimizing different
immobilization procedures of pineapple stem bromelain, in order to develop an
innovative biotechnological technique, alternative to bentonite fining, useful to removal
selectively unstable proteins from white wines.
Stem bromelain activity was assessed on a suitable synthetic substrate at a reference pH
value (3.2), this being the average minimum pH value of wine. Protease was covalently
immobilized on different supports by various procedures and the best biocatalyst was
chosen measuring immobilization percentage, kinetic parameters and half-life (in model
wine buffer). Moreover, the influence on free and immobilized protease activity of
potential inhibitors naturally present in wine, such as ethanol, tannins and sulphur
dioxide (SO2) over the average range concentration of wine, was investigated. Finally a
kinetic study was carried out using 6 artisan and unrefined white wines, spiced with the
synthetic substrate, in order to compare catalytic properties of free and immobilized
bromelain. Immobilized protease activity, then, was tested in these wines both on total
proteins and on unstable ones.
Covalent immobilization reduced bromelain catalytic properties. All kinds of
procedures applied at pH 7 allowed the highest immobilization yield. Nevertheless,
biocatalysts immobilized at pH 3.2 showed the best catalytic performance. Stem
bromelain was successfully immobilized on chitosan beads without glutaraldehyde at
pH 3.2, obtaining the most interesting and food-safe biocatalyst, which was used for all
other experiments.
Inhibition study proved that all the inhibitors tested resulted to be reversible for stem
bromelain activity. Nevertheless, the immobilized enzyme was lesser affect respect to
the free one. Free sulphur dioxide was the strongest inhibitor, being a mixed-type for
free stem bromelain and an uncompetitive inhibitor for the immobilized one.
Kinetic study of free and immobilized protease in different artisan and unrefined white
wines, showed that the catalytic efficiency of immobilized one, as measured with
respect to the synthetic substrate, paralleled that on wine proteins, varied depending on
the different inhibitors content in the tested wines.
In terms of turbidity haze, bromelain immobilized on chitosan beads exerted its useful
proteolytic activity on unstable white wine proteins in almost the same way whatever
their nature and content, in a 24-h treatment.
Additional information
Dottorato di ricerca in Biotecnologia degli alimenti
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