Phenolic Acids Inhibiting Lysinoalanine Cross-Linking in Soybean Protein Isolate via Covalent Site Blocking and Conformational Stabilization

Abstract

Alkali-heat treatment is widely used to functionalize protein-based food materials but inevitably induces lysinoalanine (LAL), a harmful nonnatural cross-linked amino acid. Herein, p-hydroxybenzoic acid, 3,5-dihydroxybenzoic acid, and gallic acid were covalently grafted onto alkali-heat-treated soy protein isolate (SPI) models to elucidate the topology-dependent inhibition of LAL formation. The inhibitory effect increased with the hydroxyl number, with gallic acid showing the highest inhibition rate (68.25%). Covalent conjugation stabilized SPI conformation by reducing reactive-site exposure, promoting ordered β-sheet structures, and enhancing thermal stability, thereby creating conditions unfavorable for LAL cross-linking. In addition, covalent bonding captured ε–NH2 and competitively blocked the LAL pathway. The catechol structure of gallic acid enabled both Michael addition and Schiff base reactions, resulting in the strongest structural stabilization and LAL inhibition. These findings provide molecular-level insight into topology-dependent strategies by which phenolic acids inhibit LAL formation, thereby mitigating adverse effects associated with the alkali-heat processing of food proteins.