Identification of Angiotensin-I-Converting Enzyme Inhibitory Peptides Derived from Sodium Caseinate Hydrolysates Produced byLactobacillus helveticusNCC 2765

Angiotensin-I-converting enzyme (ACE) inhibitory activity was identified in milk proteins fermented with Lactobacillus (Lb.) helveticus NCC 2765 (Nestlé Culture Collection, Vers-chez-les-Blanc, Switzerland). Hydrolyzing sodium caseinate for 1 and 2 h inhibited ACE activity, as measured by an in vitro ACE inhibition test. The hydrolysates with the highest ACE inhibitory potential were fractionated by gel permeation chromatography and their low molecular weight fractions collected. These fractions were subsequently subfractionated by reverse-phase high-pressure liquid chromatography. Several hydrophobic subfractions showed high ACE inhibitory potential, and their peptide composition was determined using an ion trap mass spectrometer equipped with an elctrospray ionization source. Analysis of the low molecular weight fraction identified 14 peptides with known antihypertensive activity and 1 with previously described opioid activity. On the basis of the peptide composition of active subfractions, two potentially active novel sequences were defined, and the following synthetic peptides were synthesized: FVAPFPEVFG (αS1 39−48), ENLLRFFVAPFPEVFG (αS1 33−48), NENLLRFFVAPFPEVFG (αS1 32−48), LNENLLRFFVAPFPEVFG (αS1 31−48), NLHLPLPLL (β 147−155), ENLHLPLPLL (β 146−155), and VENLHLPLPLL (β 145−155). The ACE inhibitory potential of these synthetic peptides was assessed, and IC50 values were determined. NLHLPLPLL (β 147−155), which was the only synthetic peptide also present in the sodium caseinate hydrolysates, and NENLLRFFVAPFPEVFG (αS1 32−48) showed the highest inhibition of ACE activity, with IC50 values of 15 and 55 μM, respectively. Furthermore, the stability of all synthetic peptides was assessed using an in vitro model simulating gastric digestion. The β-casein-derived peptides remained intact following the successive hydrolysis by pepsin and pancreatin, whereas αS1-casein-derived peptides were degraded by pepsin. Keywords: ACE inhibitory peptides; milk; fermented caseins