Ace from porcine kidney

Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, fetal bovine serum (FBS), phosphate buffered saline (PBS), penicillin/streptomycin and 96-well plates were purchased from Euroclone (Milan, Italy). Acetonitrile (ACN), formic acid, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sitagliptin, Gly-Pro-amido-4-methylcoumarin hydrobromide (Gly-Pro-AMC) and ACE from porcine kidney were from Sigma-Aldrich (St. Louis, MO, United States). ACE1 Activity Assay Kits come from Biovision (Milpitas Blvd., Milpitias, CA, United States). The DPP-IV inhibitor screening assay kit was bought from Cayman Chemical (Michigan, United States). Caco-2 cells were obtained from INSERM (Paris, France). Casein hydrolysates (CH), which is spray dried samples from the production process, was supplied by A. Costantino S.R.L. (Italy). (Casein hydrolysate sport/health batch 218F0004, 100 g). Casein hydrolysates (CH), which is spray dried samples from the production process, was supplied by A. Costantino S.R.L. (Italy). (Casein hydrolysate sport/health batch 218F0004, 100 g).

The ACE-inhibitory activity of the CH was evaluated following previously reported methods (51 (link), 52 (link)) and tested by measuring with HPLC the formation of hippuric acid (HA) from hippuryl-histidyl-leucine (HHL), used as mimic substrate for ACE. Tests were performed in 100 mM Tris-HCOOH, 300 mM NaCl pH 8.3 buffer, and using ACE from porcine kidney (Sigma-Aldrich, Milan, Italy). CH hydrolysate was tested at 0.08, 0.17, 0.35, 0.7 and 1.0 mg/mL.

ACE inhibitory activity was proven by measuring, with HPLC, the development of hippuric acid (HA) using hippuryl-histidyl-leucine (HHL) as a mimic substrate for ACE I. FPH was tested at 1 mg/mL in 100 mM Tris-HCOOH, 300 mM NaCl pH 8.3 buffer, and using ACE from porcine kidney (Sigma-Aldrich, Milan, Italy). All experimental details of sample preparations and analyses conditions have been published elsewhere [30 (link),31 (link)].