Liquid Chromatography–Mass Spectrometry Analysis of Microwave-Treated Ovalbumin Enzymatic Product: Molecular Weight Gradient Separation of Allergenic Peptide, Cellular Allergenicity Assessment, and Epitope Identification

Combining physical modification with enzymolysis reduces ovalbumin (OVA) allergenicity by disrupting conformational and linear epitopes. Yet hydrolytic-resistant, epitope-containing peptides can retain IgE-binding sites, limiting further decreases in allergenicity. We observed an inverse relation between peptide molecular weight and allergenicity. Accordingly, size-exclusion chromatography separated native OVA and hydrolysates from microwave-treated OVA (OVA-N-E, OVA-M-20-E) into three fractions (F1-F3) by molecular size to probe link molecular weight, epitope distribution, and allergenicity. Liquid chromatography demonstrated that longer-retention-time components had lower molecular weights, and in OVA-M-E-F3, components below 1 kDa accounted for as much as 97.89%. Mass spectrometry and cellular degranulation experiments revealed that as molecular weight decreased, the proportion of short peptides increased, while the proportion of peptides containing intact IgE-binding epitopes gradually decreased, leading to reduced allergenicity. Additionally, OVA's amino acid sequences (Y125-T136, A141-N154, F188-V200, V243-E248, G301-S308, I323-A332) were protease-resistant and displayed a structural profile dominated by β-folds.