Development of a Two-Step Hydrolysis Hypoallergenic Cow’s Milk Formula and Evaluation of Residue Allergenicity by Peptidomics and Immunoreactivity Analysis

Cow's milk allergy (CMA) is an abnormal immune response that severely affects the nutritional supplementation of allergic infants. Currently, only a limited number of hypoallergenic formulas are available on the market, and these are only categorized according to their degree of hydrolysis, which still poses an allergy risk and cannot be consumed by CMA patients, especially infants. To address this issue, we developed a two-step hydrolysis hypoallergenic formula targeting destruction of allergen epitope from whey protein. Then, a comprehensive evaluation system was constructed, including peptidomics analysis, in vivo and in vitro allergenicity assessments, revealing allergic changes in the product from the epitope structure level to the immunological level. The results showed that 97.14% of hydrolyzed peptides from α-lactalbumin and β-lactoglobulin did not contain allergenic epitopes after treatment with trypsin and flavourzyme. In vitro and in vivo allergenicity assessment results confirmed that the two-step hydrolysis method effectively reduced the allergenicity of whey protein. Compared with the common milk powder, the hypoallergenic formula induced lower levels of basophil degranulation and relieved the body's anaphylactic symptoms caused by cow milk. This study provides a promising solution to the limited hypoallergenic formula problem and may benefit allergic infants who require nutritional supplements.