Simultaneous production of multi-functional peptides by pancreatic hydrolysis of bovine casein in an enzymatic membrane reactor via combinational chromatography

Three bioactive peptides, angiotensin-converting enzyme-inhibitory peptides (ACEIPs), casein phosphopeptides (CPPs) and antimicrobial peptides (AMPs), were simultaneously prepared from casein by pancreatic hydrolysis via an enzymatic membrane reactor (EMR) and combinational chromatography. The reaction was performed at 37 °C and pH 8.0 for 3 h followed by ultrafiltration. ACEIPs were purified by size exclusion chromatography (SEC) from permeate fractions with molecular weight (MW) below 1 kDa. Concurrently, strong cation exchange high-performance liquid chromatography (SCE-HPLC) was used to isolate CPPs and AMPs from retentate fractions ranging from 1 kDa and 5 kDa. Following reverse-phase high performance liquid chromatography–electrospray ionization tandem mass spectrometry (RP-HPLC–ESI-MS/MS) analysis, potential ACEIPs and a total of 34 CPPs were identified (18 corresponded to αs1-casein, 2 to αs2-casein, 10 to β-casein and 4 to κ-casein). Additionally, the ACEIPs in fraction B from SEC had the highest ACE-inhibiting-activity (73.5% in 1.0 mg/ml), while total concentrations of AMPs were directly proportional to overall antibacterial activity. Uniquely, fraction 5 from SCE exhibited the highest activity against Staphylococcus aureus (97.49% in 1.0 mg/ml).