T-cell epitope-containing hypoallergenic β-lactoglobulin for oral immunotherapy in milk allergy

Optimally hydrolyzed β-Lactoglobulin (βLg) is a promising milk oral immunotherapy (OIT) candidate with respect to showing reduced B-cell reactivity but retaining the T-cell epitope. To demonstrate that an edible hypoallergenic βLg hydrolysate containing the T-cell epitope is suitable for OIT. We tested how chymotrypsin affected the retention of the T-cell epitope of βLg when preparing βLg hydrolysates using food-grade trypsin.

We investigated the effect of chymotrypsin activity on the formation of the T-cell epitope-containing peptide of βLg (βLg_102-124 ) and prepared an edible βLg hydrolysate containing βLg_102-124 using screened food-grade trypsins. B-cell reactivity was determined using immunoassays in which ELISA was performed with anti-βLg rabbit IgG and Western blotting was performed with a milk-specific IgE antiserum.

In βLg hydrolysis performed by varying the activity of trypsin and chymotrypsin, chymotrypsin activity inhibited the formation of βLg_102-124 with an increase in hydrolysis time in a dose-dependent manner. βLg_102-124 was generated by two of five food-grade trypsins used at a ratio of 1:50 (w/w, enzyme/substrate) for 20 h at 40°C. The edible βLg hydrolysate retained βLg_102-124 and showed a reduction in molecular weight distribution and antigenicity against IgG and IgE.

Chymotrypsin activity inhibited the formation of βLg_102-124 in the trypsin hydrolysate of βLg. This βLg trypsin hydrolysate is a novel candidate for peptide-based OIT in cow's milk allergy for safely inducing desensitization.