High hydrostatic pressure pretreatment enhances quercetin-mediated attenuation of wheat gliadin immunoreactivity and FcεRI signaling

Gliadin is a major trigger of allergic responses to wheat-based products. Although previous studies have shown that polyphenol intervention or physical modification can attenuate its immunoreactivity, the combined effects of polyphenols and high hydrostatic pressure (HHP) on this property remain poorly understood, and the optimal processing order is unclear. In this study, quercetin (Que) and 500 MPa HHP were initially selected as the most effective polyphenol and pressure conditions based on IgE-binding assays. Notably, HHP pretreatment followed by Que supplementation led to a significantly greater reduction in IgE-binding capacity than either treatment alone or the reverse sequence, while also sustaining stronger inhibitory effects following simulated gastrointestinal digestion. Structural characterization and molecular dynamics simulations further revealed that HHP pretreatment facilitated Que binding to gliadin through conformational relaxation and surface remodeling, resulting in lower conformational flexibility, a more robust interaction network, and the most favorable binding free energy (− 20.18 ± 3.17 kcal/mol). Additionally, peptide-level mass spectrometry analysis identified 27 putative linear B-cell epitope-containing peptides, 21 of which exhibited reduced abundance in the Gli-HHP-Que group, while several responsive peptides mapped near the predicted Que-binding region. Consistently, Gli-HHP-Que most effectively suppressed Ca²⁺ mobilization, degranulation, and inflammatory mediator release in serum-sensitized KU812 cells, together with reduced activation of the FcεRI-associated Lyn/Syk/PLCγ1 axis and downstream MAPK/NF-κB signaling. Collectively, these findings elucidate the mechanistic basis by which combined HHP and polyphenol treatment modulates gliadin immunoreactivity, and propose a feasible combinatorial processing strategy for reducing the allergenicity of wheat-based products.