Modulation of structure, functionality, allergenicity and in vitro digestibility of ovalbumin through cold plasma-induced conjugation with rosemary-derived phenolic acids

Abstract

Cold plasma (CP)-induced covalent conjugation between ovalbumin (OVA) and three rosemary-derived phenolic acids (RDPAs), namely chlorogenic acid (CGA), caffeic acid (CA), and rosmarinic acid (RA), was investigated to elucidate how phenolic acid molecular architecture governs the structure, functionality, in vitro digestibility, and allergenicity of OVA. After 45 s of CP treatment (40 kV, 13 kHz), the covalent grafting efficiency of OVA-RDPAs conjugates showed a clear structure dependence, following the order OVA-RA (86.55 ± 2.39 μmol/g) > OVA-CA (79.27 ± 1.62 μmol/g) > OVA-CGA (53.07 ± 2.16 μmol/g). This distinct hierarchy is attributed to RA's dual aromatic rings and four evenly distributed hydroxyl groups, which provide more accessible reactive sites, in contrast to the substantial steric hindrance imposed by CGA's bulky cyclohexane moiety. Multi-spectroscopic and SDS-PAGE analyses revealed that RDPAs grafting induced pronounced conformational unfolding and intermolecular cross-linking in OVA. Consequently, these structure-driven changes translated into remarkably augmented emulsifying and antioxidant properties and lower allergenicity of OVA. Among the conjugates, OVA-RA exhibited superior improvements, achieving 5.1-fold and 14.6-fold increases in DPPH and ABTS radical scavenging capacities, respectively. Notably, RDPAs incorporation effectively masked antigenic epitopes, and OVA-RA exhibited the greatest reduction in IgE-binding capacity (65.81 ± 0.74%). Furthermore, covalent grafting of RDPAs, particularly RA, effectively promoted gastrointestinal digestive trajectories of OVA, and the resulting digests substantially reduced IgE-binding affinity. These findings suggest that CP-mediated covalent RDPAs conjugation is a mild and effective, structure-guided approach that holds potential for the development of egg protein ingredients with enhanced functionality and reduced allergenicity.