Identification and evaluation of antioxidant activity of bioactive casein-derived peptides during in vitro digestion and transepithelial transport in Caco-2 cells

Abstract

Dairy products are important sources of bioactive (antibacterial, antithrombotic, antihypertensive, immunomodulatory, and antioxidative) peptides, that need to be released from the target proteins. In the present study, bovine milk casein protein was hydrolyzed by pancreatin and trypsin (4 and 24 h) to evaluate the hydrolyses efficiency (ninhydrin reaction); to elucidate the sequence of casein-derived peptides (SDS-PAGE, LC-ESI-TOF-MS); and to assess the peptides antioxidant activity (DPPH and ORAC), bioactivity in silico, bioaccessibility after in vitro digestion, and bioavailablility by transepithelial transport in Caco-2 cells. The hydrolysis degree increased with the incubation time, with evident degradation of casein-derived proteins into low molecular weight peptides (<5 kDa) after 4 and 24 h incubation. Pancreatin showed higher hydrolysis degree (91%) than trypsin (21%). An increase in antioxidant activity was observed with increasing incubation time, with higher antioxidant effect observed for trypsin derived peptides (47% reduction of free radicals; 32112 μM Trolox equivalents) than pancreatin derived peptides (15% reduction; 2862 μM Trolox equivalents). A total of 69 caseins derived peptides were identified after trypsin (33 peptides) and pancreatin (36 peptides) hydrolysis for 24 h; with 26 peptides released from β-casein, 21 from α-s1-casein, 15 from α-s2-casein, and 7 from k-casein hydrolysis. Some of these peptides were described as antioxidant, anti-inflammatory, and antihypertensive. After in vitro digestion 123 peptides were found in gastric (56 peptides), duodenal (43 peptides) and colonic (24 peptides) fraction; while five peptides were bioavailable by crossing the intestinal barrier, with increased peptides’ abundance after trypsin hydrolysis treatment.