Model and real wines detoxification by free and immobilised acid urease in stirred bioreactors
Author(s)
Andrich, Lucia
Date Issued
February 16, 2010
Type
Doctoral Thesis
Abstract
This PhD thesis research project was aimed at selecting the most appropriate
immobilisation carrier (i.e., Eupergit® C 250 L and chitosan beads, named Chitopearls
BCW-3001, BCW-3003, BCW-3010, and HP-3020) for acid urease in terms of the specific activity of immobilised enzyme under different operating conditions, such as
immobilization time, enzyme- or activating reagent-to-carrier ratios, pH of the
immobilization buffer, and storage stability for as long as 30-170 days. The performance of immobilised acid urease in model wine solutions with or without grape seed tannins and real wines was assessed in bench-top scale stirred bioreactors and mathematically described by resorting to a conventional diffusion-reaction model. In particular, at the optimal glutaraldehyde-to-chitosan bead ratio (YGA/CHI) of 0.625 g g-1 , the specific activity (ABi) of acid urease immobilised covalently onto porous
chitosan beads decreased from circa 300 to 70 IU g-1 wet support as the bead average
diameter (dP) increased from 0.14 to 2.2 mm. Generally, ABi reduced less than 5% after
preservation in the wet form at 4°C for 150-170 days. Only the biocatalyst prepared
using the Chitopearl BCW-3001 lost about 40% of its initial activity. The activity of
acid urease immobilised onto Eupergit® C 250L was smaller (19±3 IU g-1 ws) and
unstable. In fact, in the same storage conditions ABi reduced to 68±15% of the initial activity after about 34 days. The kinetics of urea degradation in the model wines using these biocatalysts was of the pseudo-first order with respect to the urea concentration in the liquid bulk, the apparent pseudo-first order kinetic rate constant (kIi) ranging from about two thirds to one fifth of that (kIf) pertaining to free acid urease. In the operating conditions tested, the reaction kinetics was estimated as unaffected by the contribution of the external film and intraparticle diffusion mass transfer resistances. When the model wine solution was enriched with the high-inhibitory tannins extracted from grape seeds, at the maximum level tested (374±2 g GAE m-3) kIi reduced to no more than (58±9)% of kIf, this proving
quite a higher protective action against such compounds for the chitosan-based
biocatalysts towards free or Eupergit(R) C 250 L-immobilised acid urease.The kinetics of urea degradation in two target Italian white (i.e., Grechetto and Sauvignon Blanc) wines by using acid urease immobilised onto Eupergit® C 250 L or glutaraldehyde-crosslinked chitosan-derivatives beads (that is, Chitopearls BCW-3003 and BCW-3010), was confirmed to be of the pseudo-first order with respect to the urea concentration in the liquid bulk, and not limited by urea mass transfer. In Grechetto and Sauvignon Blanc wines the apparent pseudo-first order kinetic rate constants, when using Eupergit® C 250 L-, BCW-3003- or BCW-3010-based biocatalyst, were quite similar, even if they reduced to about 7, 18 or 17% of that observed for free enzyme in the model wine solution, respectively.The chitosan-based biocatalysts resulted to be more than twice less sensitive to the phenolic content of the wines tested than the Eupergit® C 250 L-based ones, this making the former potentially employable in the make up of packed-bed cartridges to remove continuously urea from commercial wines.
Additional information
Dottorato di ricerca in Biotecnologia degli alimenti
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