Development of Hypoallergenic Cow’s Milk Protein Peptides via Enzymatic Hydrolysis Coupled with Ultrafiltration and Analysis of Their Allergenicity

To address cow's milk protein allergy, we developed a process coupling protease hydrolysis with sequential ultrafiltration to reduce the allergenicity of cow's milk allergens. Among three proteases compliant with infant formula standards, Alcalase conc BG most effectively degraded milk allergens, whereas Neutrase conc BG and Protamex produced high-molecular-weight whey protein peptide aggregates. Sequential ultrafiltration (10-3 kDa) of Alcalase-hydrolyzed casein and whey proteins enriched peptides <1 kDa (95.19% for casein; 88.22% for whey) and removed fragments >3 kDa, significantly reducing antibody binding capacity. In a BALB/c mouse model, these hydrolysates alleviated allergic symptoms, suppressed allergen-specific IgE/IgG levels, and reduced Th2 cytokine secretion. These results demonstrate that Alcalase hydrolysis coupled with ultrafiltration effectively produces hypoallergenic peptide fractions, offering a viable strategy for developing safe dairy ingredients for allergic individuals.