Sequential high hydrostatic pressure and polyphenol oxidase treatments modulate the structure, digestion behavior, and antibody-binding immunoreactivity of amandin

BACKGROUND Amandin (Pru du 6), a major almond allergen, exhibits immunochemical recognition that is influenced by protein conformation and the accessibility of antibody-recognized regions. This study investigated whether processing sequence governs the effects of high hydrostatic pressure (HHP) and polyphenol oxidase (PPO) on the structural, digestive, and commercial antibody-based immunoreactivity of amandin. RESULTS HHP and PPO conditions were optimized before individual and sequential treatments were compared. Among the tested pressures, 400 MPa produced the lowest commercial antibody-based relative immunoreactivity and was selected for the sequential experiments. HHP pretreatment followed by PPO treatment produced the greatest reduction in antibody-binding immunoreactivity and the most pronounced accumulation of high-molecular-weight material. Spectroscopic and physicochemical analyses showed altered secondary structure, fluorescence behavior, surface hydrophobicity, and sulfhydryl accessibility. Molecular docking supported a potential non-covalent PPO–amandin interaction but was not interpreted as evidence of aggregate formation. HHP-PPO treatment also generated a more interconnected aggregate morphology, increased radical-scavenging activity, and maintained comparatively low antibody-binding immunoreactivity during simulated gastrointestinal digestion despite a lower degree of hydrolysis. CONCLUSION Processing sequence was a key determinant of amandin structural remodeling and antibody-binding immunoreactivity. The observed structural changes are consistent with HHP pretreatment influencing subsequent PPO-mediated association, although the exact intermolecular linkages and antibody-recognized regions were not determined. Because the enzyme-linked immunosorbent assay used commercial anti-almond-protein antibodies rather than patient immunoglobulin E, further validation with allergic sera and biological models is required before conclusions regarding allergenicity can be made. © 2026 Society of Chemical Industry.