Thermal and pressure processing switch lactose from a protein stabilizer to a glycation accelerator in α-lactalbumin antigenicity modulation

This study investigated how heat, mild pressurization, and lactose jointly regulate the structure and IgG-recognized antigenicity of α-lactalbumin (α-LA). An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed to quantitatively monitor α-LA immunoreactivity after thermal, pressure-assisted, and lactose treatments. Structural changes were characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, intrinsic fluorescence spectroscopy, free sulfhydryl analysis, scanning electron microscopy, OPA-reactive amino-group analysis, Maillard-related optical indices, and molecular docking. Heat and mild pressurization synergistically reduced α-LA antibody recognition under the tested conditions. Lactose showed a condition-dependent dual effect: under mild heating, it helped preserve α-LA immunoreactivity, whereas under high-temperature and pressure-assisted conditions, this protective effect was lost. The decreased OPA-reactive amino-group levels and increased A294/A420 values in the α-LA/lactose system confirmed enhanced Maillard reaction progression under severe processing conditions. Molecular docking, used as a pre-reaction non-covalent interaction model, suggested that lactose could initially contact epitope-adjacent regions of native α-LA, including residues around Arg10 and Lys13. In commercial samples, pasteurized milk retained higher α-LA immunoreactivity than ultra-high-temperature and reconstituted milk, and acceptable recovery was obtained in a spiked soymilk matrix. These findings reveal a condition-dependent switch in the role of lactose from a conformational stabilizer to a Maillard-associated contributor to α-LA antigenicity modulation. The proposed icELISA provides a practical complementary immunochemical tool for evaluating processing-induced differences in dairy products.