Release of ACE-inhibitor Peptides during in vitro Gastrointestinal Digestion of Parmigiano Reggiano PDO Cheese and their Absorption through an in vitro Model of Intestinal Epithelium

The occurrence of eight bovine CN-derived peptides (VPP, IPP, RYLGY, RYLG, AYFYPEL, AYFYPE, LHLPLP and HLPLP) reported as Angiotensin Converting Enzyme-inhibitor (ACE-I) was investigated in the 3 kDa-UF water-soluble extract (WSE) of Parmigiano Reggiano (PR) cheese samples by UPLC/ESI-HRMS. Only VPP, IPP, LHLPLP and HLPLP were revealed in the WSEs and their total amount was in the range 8.46–21.55 mg/kg of cheese. Following in vitro static gastrointestinal digestion (GID), the same ACE-I peptides along with the newly-formed AYFYPEL and AYFYPE were found in the 3 kDa WSEs of PR digestates. Digestates presented high amounts (1,880–3,053 mg/kg) of LHLPLP, whereas the remaining peptides accounted for 69.24–82.82 mg/kg. The IC50 values decreased from 7.92±2.08 in undigested cheese to 3.20±1.69 after in vitro GID. The 3 kDa WSEs of digested cheeses were used to study the transport of the eight ACE-I peptides across the monolayers of the Caco-2 cell culture grown on a semipermeable membrane of the transwells. After 1h incubation, 649.20±148.85 mg/kg of LHLPLP remained in the apical compartment, whereas VPP, IPP, AYFYPEL, AYFYPE and HLPLP accounted in total for less than 36.78 mg/kg. On average, 0.6% of LHLPLP initially present in the digestates added to the apical compartment were transported intact to the basolateral chamber after the same incubation time. Higher transport rate (2.9%) was ascertained for the peptide HLPLP. No other intact ACE-I peptides were revealed in the basolateral compartment. For the first time, these results demonstrated that the ACE-I peptides HLPLP and LHLPLP present in the in vitro digestates of PR cheese are partially absorbed through an in vitro model of human intestinal epithelium.