Endogenous polyphenol-glutelin interactions in black highland barley: Effects of noncovalent and covalent binding on thermally induced structural changes and in vitro gastrointestinal digestive behavior.

Endogenous polyphenols are abundant in black highland barley and may interact with proteins during processing, but how binding mode affects protein structure and digestive fate remains unclear. In this study, noncovalent (NPC) and covalent (CPC) polyphenol-glutelin complexes were prepared before and after heating. Amino acid side-chain reactivity assays and SDS-PAGE revealed alterations in amino and sulfhydryl groups and reduction-resistant glutelin assemblies in CPC. Structural and interaction analyses showed that hydrogen bonds and hydrophobic interactions in NPC disrupted disulfide-mediated stabilization, while CPC formed a more constrained structure. Heating further amplified the structural divergence between the two systems. Consequently, NPC enhanced gastric proteolytic accessibility and shifted polyphenol release toward the intestinal phase. In contrast, CPC suppressed gastric hydrolysis and early-stage polyphenol release but allowed partial compensatory recovery during intestinal digestion. These findings reveal that endogenous polyphenols reshape the temporal coordination between protein disassembly and polyphenol liberation in a binding mode-dependent manner.