The effect of enzymatic hydrolysis assisted glycosylation on the allergenicity and digestive characteristics of whey protein isolate

In this study, whey protein isolate (WPI) was moderately hydrolyzed with alkaline protease, then glycosylated with allose to yield enzyme hydrolysis-assisted glycosylated WPI. The structural changes of the modified protein were characterized by spectroscopy and chromatography. Mass spectrometry identified and compared in vitro digestion peptides to explore modified WPI's allergenicity changes and antigenic epitopes. The results indicated that the enzyme hydrolysis-assisted glycosylation could significantly reduce the allergenicity of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) in WPI. After gastrointestinal digestion, the antigenicity of α-LA and β-LG decreased up to 96.32 ± 1.41 % and 94.95 ± 1.92 %. This is attributed to the fact that the pretreatment with enzymatic hydrolysis induces structural changes in WPI, thereby increasing the glycosylation sites and reducing its allergenicity. Peptide identification in digestion products showed 37 fewer allergenic peptides in enzyme-pretreated glycosylated (E-All-WPI) compared with glycosylation-only (All-WPI) groups. Moreover, no allergenic peptides were detected in E-All-WPI's α-LA epitope library. Finally, molecular docking of peptides common to digested All-WPI and E-All-WPI explored interactions between glycosylated/non-glycosylated peptides and MHC-II in immune responses. We found that the binding energies of AA114–121 peptides in α-LA and AA59–72 peptides in β-LG were reduced by 0.4 kJ/mol and 2.4 kJ/mol, respectively, after glycosylation with MHC-II. Thus, glycosylation interferes with the binding of antigens to immune factors, thereby inhibiting allergenic expression. This study provides a novel modification method for WPI, as well as new insights into further reducing WPI allergenicity and its applications in the food industry.