Digestion supplemented with commercial proteases: Evaluation of the fate of gluten immunogenic peptides in pizza

Enzymatic supplements designed to aid in gluten digestion can be found in the market, also in combination with enzymes targeting other macronutrients. Their impact on digestion of gluten sequences that are immunogenic and/or toxic for individuals with Celiac Disease (CeD), remains uncertain, especially within a complex food matrix. This study aims at applying a biochemical and immune-based approach for understanding the effect of such supplements on the degradation of gluten immunogenic peptides in pizza. Plane-pizza was digested using the INFOGEST model, with/without the addition of three over the counter gluten enzyme supplements (S1, S2 and S3). Proteins digestion was monitored in the gastric (G) and gastroduodenal (GD) phases using electrophoresis and primary amines detection. Residual immune-toxic epitopes were quantified by R5- and G12-based ELISA. Immunogenicity of resistant, deamidated peptides, was assayed on CeD-derived intestinal-T cell lines (iTCLs). The highest content of primary amines was determined in presence of S1 and S2 supplements, containing starch-degrading enzymes in addition to dipeptidyl-peptidase-IV (DPP-IV) and other proteases. The most rapid degradation of R5- and G12-immune-toxic epitopes was observed in presence of S3 supplements, containing prolyl-endopeptidase (An-PEP). Consistently, iTCLs showed significant IFN-γ decrease toward S3-treated peptides. Nevertheless, none of the supplements was able to abolish iTCLs response, particularly at the end of gastric phase, thus allowing to conclude that they work to different, but potentially limited extent. The developed approach, combining simulated gastric and gastroduodenal digestion, biochemical/immunochemical characterization and functional bioassays, has proven to be a robust tool to assess residual immunoreactivity of enzyme-treated foods.