Biofunctional properties of ultrafiltrate fraction of Kankrej milk casein hydrolysate

Bioactive peptides derived from milk proteins have attracted considerable interest due to their antioxidant and antihypertensive potential. This study focused on the production, isolation, and characterization of biofunctional peptides from Kankrej cow milk casein using trypsin-mediated enzymatic hydrolysis followed by ultrafiltration. Casein was isolated by acid precipitation and hydrolysed using trypsin at an enzyme-to-substrate ratio of 1:25 under optimized conditions (pH 8.0, 37 °C) for 8 h. The casein hydrolysates were then sequentially ultrafiltered using 10, 5 and 3 kDa molecular weight cut-off membranes. The permeate and retentate fractions were analysed for peptide content, antioxidant activity (ABTS, DPPH, and FRAP assays), and ACE inhibitory activity. Peptide content decreased with decreasing molecular weight cut-off, with the 3 kDa permeate showing the lowest peptide concentration (10.10 mg/ml). Despite this, the 3 kDa permeate exhibited the highest biofunctional activity, showing ABTS radical scavenging activity of 5524.79 µM TE/mg peptide, DPPH radical scavenging activity of 67.57%, FRAP value of 510.35 µM Fe²⁺/mg peptide, and ACE inhibitory activity of 46.98%. Tricine SDS-PAGE confirmed the presence of low molecular weight peptides in the ultrafiltrate fractions.The results demonstrate that low molecular weight peptides (