Epitope-Guided Two-Step Enzymatic Hydrolysis for Targeted Control of the Immunoreactivity of the Major Sichuan Pepper Allergen Zan b 2.

Sichuan pepper (Zanthoxylum bungeanum) is widely consumed in China, but its allergenicity raises food-safety concerns. In this study, the 11S globulin allergen Zan b 2 was identified as the major allergen and characterized through structural and immunological analyses. Integrated immunoinformatics, molecular docking, and molecular dynamics simulations were used to predict B-cell epitopes and screen proteases capable of specifically cleaving these regions. Prediction results indicate that proteinase K targets the following epitopes: Glu13, Thr100, Tyr149, Glu164, Glu229, Glu231, Leu239, Glu241, Thr245, Val328, Glu368, Ala381, and thermolysin targets Arg182, Lys247, Ser273, Asn294, Gln332, and Arg380. Experimental validation using enzymatic hydrolysis, SDS-PAGE, indirect ELISA, LC-MS/MS, and proteomics showed that combined treatment with proteinase K and thermolysin markedly reduced the IgE-binding activity. These findings demonstrate that simulation-assisted targeted enzymatic hydrolysis is an effective strategy for reducing Zan b 2 immunoreactivity and supports the development of hypoallergenic Sichuan pepper products.