Distinct Assemblies of Native and Auto-Oxidized EGCG with Undigested Gliadin Peptides Exhibit Limited Mucus Penetration Linked to Enhanced Mucin Association

The intestinal mucus layer represents a critical yet underexplored physiological interface that regulates epithelial exposure to dietary immunotoxic peptides. Here, we systematically investigated the assembly behavior of native and auto-oxidized (−)-epigallocatechin-3-gallate (EGCG) with undigested gliadin peptides (UGPs) under physiologically relevant conditions, and evaluated how these interactions modulate the transport of UGPs across intestinal mucus. Native EGCG promoted the formation of large, highly hydrated, soft droplet-like assemblies through reversible noncovalent interactions, whereas auto-oxidized EGCG formed smaller, compact nanoparticles with stronger apparent UGP association, likely involving both noncovalent interactions and quinone-mediated coupling. Compared with free UGPs, both assembly types exhibited limited penetration through ex vivo intestinal mucus, while quartz crystal microbalance with dissipation monitoring revealed their enhanced association with mucin. In mucin-producing Caco-2/HT29-MTX cocultures, exposure to both assemblies was associated with lower extracellular interleukin-8 levels than exposure to UGPs alone. These findings highlight the mucus layer as a functional pre-epithelial interface where polyphenol-peptide interactions can restrict the bioaccessibility of immunotoxic gliadin peptides to the underlying epithelium, offering a mechanistic basis for polyphenol-based dietary interventions against gluten-related disorders.