Influence of wheat flour refinement on oxidative modifications and pro‑inflammatory properties of amylase‑trypsin inhibitors

Amylase/trypsin inhibitors (ATIs), cereal proteins that confer natural resistance against pest and pathogen digestive enzymes, have been implicated in TLR4-mediated innate immune activation and wheat-related intolerances in vitro, though their causal role and genetic susceptibility factors in non-coeliac wheat sensitivity remains unclear. Given the established influence of the oxidation state of triggering proteins on TLR4 binding and activation, this study investigated how common industrial milling methods (roller versus stone milling) affect the proinflammatory potential of ATIs from a single commercial wheat variety. Integrated immunometric and targeted proteomic analyses showed that milling technology and processing parameters are critical determinants of the inflammatory potential of wheat flour, as they shape ATI redox status; notably, proinflammatory cytokine secretion paralleled oxidative modification rather than ATI abundance.