In Vitro Intestinal Transepithelial Transport, Safety, and Bioactivity Evaluation of Milk Peptides

Abstract

This study aimed to evaluate the bioaccessibility, stability to brush border peptidases, bioavailability, intestinal safety, and bioactivity of low molecular weight (LMW)-milk protein hydrolysate on intestinal Caco-2 and STC-1 cellular models. Milk proteins were first subjected to simulated gastrointestinal digestion, and the resulting peptide mixture was analyzed for intestinal absorption using differentiated human Caco-2 cells. Using high-performance liquid chromatography-mass spectrometry (HPLC-MS), 82 peptides from casein and 16 peptides from β-lactoglobulin were identified as bioaccessible and stable, with some peptides already known to circulate in human plasma. Notably, 47% of apical peptides successfully crossed the epithelial barrier to the basolateral side. Importantly, the peptide mixtures preserved the intestinal monolayer integrity as shown by unchanged transepithelial electrical resistance (TEER) values at 5 mg/mL and demonstrated the intestinal safety through the absence of cytotoxicity in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability experiment in differentiated Caco-2 cells. Moreover, the bioactivity of the LMW-milk protein hydrolysate was assessed through in vitro and cell-based assays. Antioxidant potential was assessed using the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and ferric reducing antioxidant power (FRAP) assays, revealing a strong radical scavenging effect (up to 72.6%) and a FRAP increase of 3864% at 2.5 mg/mL. In addition, LMW hydrolysate significantly inhibited dipeptidyl peptidase-IV (DPP-IV) activity by 70.1% in vitro and 20.9% in Caco-2 cells at 10 mg/mL and stimulated glucagon-like peptide-1 (GLP-1) secretion in STC-1 cells by up to 122.4%. Finally, angiotensin-converting enzyme (ACE) inhibition reached 23% at 6.67 mg/mL in a dose-dependent manner. These findings demonstrate that LMW-milk peptides are safe, bioavailable, and exert multifunctional biological activities antioxidant, hypoglycemic, and hypotensive, supporting their potential application in functional food development.