Chymosin pretreatment accelerated papain catalysed hydrolysis for decreasing casein antigenicity by exposing the cleavage site at tyrosine residues.

Cow milk protein allergy (CMPA) induced by casein poses major health challenges that hinders the consumption of milk-based formulas. In this study, a novel sequential enzymatic hydrolysis catalysed by chymosin and papain was proposed to reduce casein antigenicity. Its effects on reducing casein antigenicity, structural properties and peptide profiles were evaluated by ELISA, multispectral techniques and peptidome analysis. It was revealed that the sequential enzymatic hydrolysis obtained a similarly residual antigenicity level in a shorter time (60 min) compared to papain-hydrolysis for 360 min. The hydrolysis-site at Tyr residues accessibility of papain was increased to 36.84 % by the chymosin pretreatment and it was significantly higher than 26.93 % obtained by only papain for 60 min. Moreover, the sequential enzymatic hydrolysis led to decrease in the large fragment peptides from αs1 casein. These findings suggested that the proposed sequential enzymatic hydrolysis can be exploited in the development of CMPA-free formulas.