Abstract: Objective: To optimize the tryptic (PTN 6.0S) hydrolysis conditions of sodium caseinate for preparing peptides with high-immune activity. Methods: One-factor-at-a-time experimental design combined with response surface methodology was employed systematically to develop optimal hydrolysis conditions of sodium caseinate. The effect of hydrolyzed sodium caseinate on proliferation degree (stimulation index, SI) and ammonia nitrogen (AN) of mouse spleen cells was measured by tetrazolium salt-based colorimetric (MTT) assay. Results: The optimal hydrolysis conditions were substrate concentration of 3.5 g/100 mL, enzyme/substrate ratio of 3.8:1000, hydrolysis time of 15 h, hydrolysis temperature of 55.0 ℃ and hydrolysis pH of 7.0. Under the optimal hydrolysis conditions, the maximum SI was achieved to be 0.184±0.002 and the ammonia nitrogen content was up to (0.795±0.012) g/L. Conclusion: Enzymatic hydrolysis is a better way to prepare high-immune activity peptides from sodium caseinate

Key words: sodium caseinate, immune peptides, tetrazolium salt-based colorimetric (MTT) assay, response surface methodology