Rimozione di ocratossina A in un vino modello mediante proteasi acide
Author(s)
Fabianelli, Tiziana
Date Issued
October 3, 2008
Type
Doctoral Thesis
Abstract
This PhD thesis was aimed at studying ochratoxin A (OTA) removal in a model wine (tartaric buffer at pH 3.2 and ethanol 12%vol) by using two commercial acid proteases of GRAS microbial (aspartic protease, EC 3.4.23.18) or vegetable (cysteinic protease, EC 3.4.4.24) origin. This work reports the main results of the following two activities directed to:
Screening and characterization of the activity of acid microbial and vegetable proteases in a model wine solution;
OTA degradation by means of the two enzymes in model wine solution.
For the characterization trials, the enzymatic activity of two acid proteases was tested in function of different pH (3.2, 3.5, 3.8), and temperatures (25, 35, 40 °C), ethanol concentrations (until 12%vol.), in presence or no of potassium-metabisulfite (until 100 mg/l) and grape tannins of seeds and of skin (0, 5-2 g/l), to reproduce the physical-chemical conditions of wine.
The effect of two proteases on red wine colour was tested too, adding different concentrations (from 20 to 2000 mg/l) of two enzymes.
Aspartic protease was more stable than cysteinic one. In fact, temperature, pH, ethanol and K2S2O5, did not influence protease aspartic activity in short time such as the cysteinic protease. Both proteolytic activity were negatively influenced by tannins. The two enzymes showed a total inactivation in 24h.
For degradation trials, OTA decrement was assessed with three different analytic methods (TLC, spectrophotometric analysis and HPLC-FL), as a function of enzyme concentration, time and stirring conditions.
In TLC trials the OTA degradation, as function of reaction time with aspartic protease, was observed by the decreasing of spot fluorescence brought UV light.
With spectrophotometric analysis the OTA decrease, in presence of aspartic protease, was monitored at 330 nm, as well as the corresponding spectra in the range from 250 to 450 nm.
It was revealed a decrease in OTA of about 20% in 20 hours, without stirring conditions and of about 38% after 24 hours under stirring conditions.
In the trials lead to study OTA degradation in presence of both enzymes, with HPLC-FL, all dispersions were poured in 40-ml flasks kept at 35°C under stirring condition (400 rpm). The time course of hydrolytic activity was assessed at different enzyme and OTA concentrations.
Both the acid proteases tested were able to remove the OTA added to a model wine. Cysteinic acid protease resulted more active than the aspartic one, their corresponding OTA reduction yields being about 70 and 30%, respectively.
Additional information
Dottorato di ricerca in Biotecnologie degli alimenti
Subjects
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