Hydrolysis with Cucurbita ficifolia serine protease reduces antigenic response to bovine whey protein concentrate and αs-casein

Abstract

In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 μg/ml where the inhibition of the reaction with nWPC was ≤47 %. One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal.

Keywords: Cow’s milk allergy; Cucurbita ficifolia; Serine protease; Whey protein hydrolysates; αs-casein hydrolysates.

Fig. 1

RP-HPLC profiles of peptide fractions (black) obtained after 1, 3, 5, 24 h hydrolysis of αs-casein with serine protease isolated from Cucurbita ficifolia introduced at a dose of 150 U/mg. Undigested 1 % protein solution was used as control (red) (color figure online)

Fig. 2

RP-HPLC profiles of peptide fractions (black) obtained after 1, 3, 5, 24 h hydrolysis of WPC-80 with serine protease isolated from Cucurbita ficifolia introduced at a dose of 150 U/mg. Undigested 1 % protein solution was used as control (red) (color figure online)

Fig. 3

Effect of pre-incubation of the sera pool from children with CMA (a, b), healthy children (c), goat antiserum (d), with a native protein/hydrolysed WPC (.1) and αs-casein (.2) on the inhibition of the reactivity of IgE (a.1, a.2) or IgG (b.1, b.2, c.1, c.2, d1)