The Preparation of Hypoallergenic Goat Milk Protein Hydrolysates via Targeted Hydrolysis of Cross-Reactive Linear Epitopes Between Cow and Goat Milk

Cow’s milk allergy (CMA) is the most common food allergy in infants. Goat milk exhibits relatively low allergenicity and is widely regarded as a potential substitute for cow milk. However, the high sequence homology between cow and goat milk proteins may trigger cross-reactivity, significantly restricting the practical application of goat milk. This study aims to disrupt cross-linear epitopes of cow and goat milk proteins through enzymatic hydrolysis. We prepare hypoallergenic goat milk protein hydrolysates and systematically evaluate their desensitization effects. Bioinformatics methods were employed to predict the B-cell linear epitopes of six major allergens (αS1-casein, αS2-casein, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin) from cow, goat, and sheep milk, and sequence homology was analyzed through protein-protein Basic Local Alignment Search Tool (BLASTP). The results showed that the sequence similarity of homologous allergens among the three milk sources all exceeded 30%. Subsequently, six proteases (protamex, alcalase, pepsin, trypsin, papain and bromelain) were used to hydrolyze whole goat milk protein. Indirect enzyme-linked immunosorbent assay (ELISA) results indicated that all six hydrolysates significantly reduced immunoreactivity with antibodies against the five major cow’s milk allergens. Among them, alcalase exhibited significant efficacy against all five allergens; papain and protamex were particularly effective against β-casein; trypsin showed pronounced efficacy against α-lactalbumin and β-lactoglobulin; and bromelain and pepsin also significantly reduced immunoreactivity against certain allergens. Mass spectrometry revealed the peptide composition and epitope coverage of the hydrolysates: bromelain yielded two overlapping peptides (FAWPQY, LKDLKDY), protamex one (LAMAAS), Alcalase three (PPQSVLS, IPIQYVLS, LPYPYY), trypsin two (YLGYLEQL, AMAASDISLL), papain three (NEINQFYQK, FQSEEQQQTEDELQDK, AMAASDISL); and no matching peptide was detected for pepsin. These results confirmed at the molecular level that enzymatic hydrolysis can effectively disrupt cross-reactive epitopes.