Hydrolysis of goat’s milk casein using various enzymes at different conditions and antigenicity through enzyme-linked immunosorbent assay

Goat’s milk has shown a rising demand among individuals with cows’ milk protein allergy (CMPA) due to its lower allergenic properties than cows. However, casein, the main protein in goat’s milk, showed a high homology to cow’s casein, contributing to allergic reactions in CMPA patients. To mitigate the allergenic immune responses, casein, as an important epitope responsible for this problem, can be broken down through enzymatic hydrolysis, producing varying-sized peptides. Each enzyme will have its unique condition to work optimally, and there is a lack of understanding on how enzymes can break down goat’s milk casein. Apart from hydrolysis conditions, there is limited research on detecting goat’s milk casein and its antigenicity reduction using various enzymes. Hence, this study aims to evaluate optimal hydrolysis conditions using various enzymes for reducing antigenicity in goat’s milk casein through enzyme-linked immunosorbent assay (ELISA). The hydrolysis conditions of goat’s milk casein were optimised using non-gastrointestinal digestive enzymes (bromelain and alcalase) and gastrointestinal digestive enzymes (trypsin and pepsin), at different hydrolysis conditions, which include pH, enzyme-substrate ratio, the temperature of hydrolysis, and time. This study evaluated a series of parameters, including ELISA formats, types, and antibody concentrations to evaluate antigenicity reduction. Result showed that the optimal hydrolysis condition for bromelain (1:100, pH 5.5, 50 °C, 6 h), alcalase (1:50, pH 8.0, 55 °C, 6 h), trypsin (1:10, pH 8.0, 37 °C, 6 h) and pepsin (1:10, pH 1.5, 37 °C, 6 h) produced degree of hydrolysis (DH) (40-60%) and peptide content (ca. 2 mg/mL). Indirect ELISA format produced the highest signal-to-noise (S:N) ratio (9.80) using polyclonal antibody anti-casein (1:500 v/v) and goat anti-rabbit HRP as the secondary antibody (1:15,000 v/v). In terms of antigenicity reduction, bromelain which exhibited maximal DH of 60% and peptide content of 2.86 mg/mL, had the highest reduction of antigenicity (33.60 ± 0.014%), producing peptides below 10 kDa. This study demonstrated that hydrolysis conditions with appropriate enzymes are crucial for the effective reduction of goat’s milk casein antigenicity. This discovery provides the foundational development of a hypoallergenic goat milk formula, enhancing the added value of goat milk while ensuring consumer safety.

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