The aim of this study was to explore the effects of an emerging and efficient heating technology, superheated steam (SS), on the allergenicity and molecular structure of ovomucoid (OVM). OVM was treated with 120–200 °C of SS for 2 to 10 min. The allergenicity (IgG/IgE binding abilities and cell degranulation assay) and molecular structure (main functional groups and amino acids modification) changes were investigated. The IgG-binding ability of OVM decreased and the releases of β-hex and TNF-γ were inhibited after SS treatment, indicating that the protein allergenicity was reduced. Significant increases in oxidation degree, free SH content and surface hydrophobicity were observed in SS-treated OVM. The protein dimer and trimer appeared after SS treatment. Meanwhile, obvious changes occurred in the primary structure. Specifically, serine can be readily modified by obtaining functional groups from other modification sites during SS treatment. Moreover, the natural OVM structure which showed resistance to trypsin digestion was disrupted, leading to increased protein digestibility. In conclusion, SS-induced OVM aggregation, functional groups and amino acids modifications as well as protein structure alteration led to reduced allergenicity and increased digestibility.

β-lactoglobulin (β-Lg), the major whey protein containing nine lysine residues, serves as an ideal model for studying protein glycation and thermal processing safety in dairy products. This study systematically compared three different high-temperature treatment methods, namely superheated steam (SS), hot air (HA), and oil bath (OB), to investigate their effects on the spatial conformation and glycation product formation of proteins in the β-Lg-glucose system. The results show that compared with OB and HA, SS has a lower degree of glycation, lower consumption of free amino groups, and less unfolding of the protein’s three-dimensional structure. It leads to a lower proportion of α-helix transformation into β-sheet and random coil in the protein. SS resulted in the least browning and produced less 5-hydroxymethylfurfural, pentosidine, fluorescent advanced glycation end products, and melanogenin, yet produced the highest amount of Carboxymethyllysine. Mass spectrometry analysis shows that lysine residues were the primary glycation sites. Therefore, this work provides molecular-level insights into how different heating techniques modulate protein glycation and structural stability, supporting the potential of superheated steam as a gentler alternative to control glycation for β-Lg in food thermal processing.