Casein fermentate of Lactobacillus animalis DPC6134 contains a range of novel propeptide angiotensin-converting enzyme inhibitors

Abstract

This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate Lactobacillus animalis DPC6134 (NCIMB deposit 41355). The crude 10-kDa L. animalis DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (+/-15%) and had a 50% inhibitory concentration (IC50) of 0.8 mg protein/ml compared to captopril, which had an IC50 value of 0.005 mg/ml. Fractionation of the crude L. animalis DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions. Fractions 10, 19, and 43 displayed ACE-inhibitory activity percentages of 67.53 (+/-15), 83.71 (+/-19), and 42.36 (+/-11), respectively, where ACE inhibition was determined with 80 microl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from alpha-, beta-, and kappa-caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to beta-casein f(73-89); peptide IGSENSEKTTMP, corresponding to alpha(s1)-casein f(201212); peptide SQSKVLPVPQ, corresponding to beta-casein f(166-175); peptide MPFPKYPVEP, corresponding to beta-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to beta-casein f(210-221), contained ACE-inhibitory activities. These peptides were chosen for chemical synthesis to confirm the ACE-inhibitory activity of the fractions. Chemically synthesized peptides displayed IC50 values in the range of 92 microM to 790 microM. Additionally, a simulated gastrointestinal digestion confirmed that the ACE-inhibitory 10-kDa L. animalis DPC6134 fermentation was resistant to a cocktail of digestive enzymes found in the gastrointestinal tract.

FIG. 1.

Structure-activity correlation between the C-terminal tripeptide sequences of different ACE-inhibitory peptides and ACE. (A) Binding to ACE is influenced by the hydrophobicity of the three C-terminal amino acid residues, with aromatic or branched side chain residues being preferred. As shown, aliphatic (V, I, and A), basic (R), and aromatic (Y and F) residues are preferred in the penultimate positions and aromatic (W, Y, and F), proline (P), and aliphatic (I, A, L, and M) residues are preferred in ultimate positions. The positive charge of arginine (R) at the C terminus has also been shown to contribute to the ACE-I-inhibitory potency of several peptides. A C-terminal lysine (K) with a positive charge on the ɛ-amino group contributes to inhibitory potency. (B) Observed trends in hydrophobicity for the l-amino acids are shown. Phenylalanine (F) is the most hydrophobic of the l-amino acids and is preferred as one of the C-terminal amino acid residues. The least hydrophobic amino acid residues, i.e., the branched aliphatic amino acid residues, are preferred at the N-terminal end of the ACE-inhibitory peptide, with the exception of arginine (R) (due to its positive charge), which has been shown to contribute to ACE inhibition. Underlined sequences of peptides have previously been identified as ACE-inhibitory peptides.

FIG. 2.

(A) RP-HPLC chromatogram of sodium caseinate at pH 7 incubated with L. animalis DPC6134 for 24 h. Arrows indicate positions of peptide fractions 10, 19, and 43. RP-HPLC was carried out at room temperature and according to the conditions described in Materials and Methods. (B) LC chromatograms obtained during the analysis of ACE-inhibitory fractions 10, 19, and 43 obtained from a semipreparative RP-HPLC separation of the Lactobacillus animalis DPC6134 hydrolysate of sodium caseinate. The LC chromatograms were obtained using an LC Packings nano-LC system (Bruker Daltonics Limited, Bremen, Germany). The procedure for LC analysis was as described in Materials and Methods. (C) Twenty-five peptides identified by HCT-ion trap mass spectrometry (Bruker Daltoniks, Bremen, Germany) and their corresponding sequences identified using Data Analysis (version 3.0; Bruker Daltoniks, Bremen, Germany). Percent total hydrophobicity for each peptide sequence and observed masses are shown.

FIG. 3.

Results of predicting the binding sites of ACE with Q-SiteFinder visualized using Chime. The predicted binding site selection is color coded according to the likelihood of being an actual binding site. Green is the most likely, followed by blue, purple, and orange/brown.

FIG. 4.

Electrophoregrams of the ACE reaction mixture, where peak (i) corresponds to histidyl leucine (migration time of 2.7 min), (ii) corresponds to HHL (migration time of 3.89 min), and (iii) corresponds to HA (migration time of 4.98 min). (A) Electrophoregrams with captopril used as a control at a concentration of 0.005 mg/ml. The enzyme reaction conditions were 5 mM HHL and 500 μU of ACE in 100 mM boric acid-borate buffer (pH 8.3) with 0.5 N NaCl; total volume was 550 μl, and enzyme reaction time was 60 min at 37°C. The capillary electrophoresis conditions are as described in the text. (B) Electrophoregram of ACE reaction mixture with the peptide NIPPLTQTPVVVPPFIQPEV (450 μM) added as an ACE inhibitor. (C) Electrophoregram of ACE reaction mixture with the peptide MPFPKYPVEP (83.00 μM) added as an ACE inhibitor. (D) Electrophoregram of ACE reaction mixture with the peptide EPVLGPVRGPFP (790 μM) added as an ACE inhibitor. (E) Electrophoregram of ACE reaction mixture with the peptide IGSENSEKTTMP (773.10 μM) added as an ACE inhibitor. (F) Electrophoregram of ACE reaction mixture with the peptide SQSKVLPVPQ (92.00 μM) added as an ACE inhibitor. (G) Control: electrophoregram of ACE reaction mixture without captopril or ACE-inhibitory reagent added.

FIG. 5.

(a) Graphical representation of the five peptides on the structure of β-casein using the molecular visualization program CHIMERA (http://www.cgl.ucsf.edu/chimera). (b) The actual location of binding of the peptides. The ACE structure was then predicted using the docking software ESCHER-NG (3). The output of this was converted to a structural file with VEGA ZZ (38), and the results were visualized with the graphical program CHIMERA (40).

FIG. 6.

(A) Graph of the L. animalis DPC6134 hydrolysate following physiological digestion with pepsin and corolase PP. (B) RP-HPLC chromatogram profile of the L. animalis DPC6134 fermentate before simulated digestion with pepsin and corolase PP. RP-HPLC chromatogram profiles of the L. animalis DPC6134 sodium caseinate hydrolysate after in vitro-simulated gastrointestinal physiological digestion with pepsin (C) and corolase PP digestion after 30 min (D), 120 min (E), and 240 min (F). RP-HPLC was carried out at room temperature and according to the conditions described in Materials and Methods. The percent ACE inhibition values given are the average percentages of each assay carried out in triplicate.