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Applied and Environmental Microbiology, July 2007, p. 4658-4667, Vol. 73, No. 14
0099-2240/07/$08.00+0     doi:10.1128/AEM.00096-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Casein Fermentate of Lactobacillus animalis DPC6134 Contains a Range of Novel Propeptide Angiotensin-Converting Enzyme Inhibitors

M. Hayes,^1,2 C. Stanton,^1,3 H. Slattery,¹ O. O'Sullivan,¹ C. Hill,^2,3 G. F. Fitzgerald,^2,3 and R. P. Ross^1,3*

Teagasc Biotechnology Centre, Moorepark Food Research, Fermoy, County Cork, Ireland,¹ Department of Microbiology, University College, Cork, Ireland,² Alimentary Pharmabiotic Centre, Cork, Ireland³

Received 15 January 2007/ Accepted 23 April 2007

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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 µl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from -, ß-, and -caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to ß-casein f(73-89); peptide IGSENSEKTTMP, corresponding to _s1-casein f(201212); peptide SQSKVLPVPQ, corresponding to ß-casein f(166-175); peptide MPFPKYPVEP, corresponding to ß-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to ß-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 µM to 790 µM. 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.

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Angiotensin-converting enzyme (ACE; also known as kininase II; EC 3.4.15.1) is a nonspecific but highly selective key multifunctional ectoenzyme, involved in the regulation of peripheral blood pressure (52). ACE catalyzes the release of the dipeptide His-Leu from the angiotensin I C terminus, which results in the octapeptide angiotensin II, a potent vasoconstrictor (45). ACE also hydrolyzes and inactivates the vasoactive bradykinin (4). Additionally, ACE is a stimulant for the release of aldosterone in the adrenal cortex (6, 7). As a result, ACE inhibitors have been shown to reduce peripheral blood pressure and exert an antihypertensive effect in vivo.

A myriad of food protein sources including fish, gelatin, maize, soy, and milk proteins have been reported to contain bioactive peptide sequences (2, 44). Casein-derived inhibitors (casokinins) (31) and whey-derived inhibitors of ACE (lactokinins) (11) have been released during enzymatic hydrolysis during fermentation.

Proteases of microbial origin potentially release antimicrobial peptides (29). During dairy fermentations, lactic acid bacteria (LAB) degrade milk proteins such as casein and whey in order to grow in milk, and subsequent utilization of the degradation products by LAB requires a complex proteolytic system. Given the proteolytic nature of LAB such as Lactococcus lactis (24, 34, 43) and Lactobacillus helveticus, their use as microbial catalysts for the generation of bioactive peptides has been investigated (36, 54). Examples of food products on the market containing ACE peptides are Ameal S (Calpis Co. Ltd., Tokyo, Japan), a sour milk tablet-form product based on the milk drink Calpis, which contains two potent ACE inhibitors, VPP [ß-casein f(84-86)] and IPP [ß-casein f(74-76)], generated from casein using the proteolytic capabilities of Lactobacillus helveticus CP790 and Saccharomyces cerevisiae (36). Furthermore, ACE-inhibitory peptides were released from whey and casein following fermentation with different strains of LAB followed by hydrolysis with digestive enzymes (41). Peptides identified were LAYFYP, corresponding to _s1-casein f(142-147); TTMPLW, corresponding to _s1-casein f(194-199); and ß-casein f(108-113), corresponding to the sequence EMPFPK, in addition to two ACE-inhibitory peptides from whey, GLDIQK, corresponding to ß-lactoglobulin (ß-Lg) f(9-14), and VAGTWY, corresponding to ß-Lg f(15-20) (41). Characterization of ACE-inhibitory peptides produced during casein degradation has been described for L. helveticus (14, 58) and to a lesser extent for Lactobacillus casei (8). Milks fermented with L. helveticus CPN4, R211, R289, and LP01; Enterococcus faecalis CECT 5827, 5727, and 5728 (35); and Lactococcus lactis subsp. cremoris LP25 have all been shown to contain ACE-inhibitory peptides and to display antihypertensive activity in vivo (12). Compared to ACE-inhibitory drugs such as captopril, food-derived ACE inhibitors have lower ACE-inhibitory activity in vitro but also display no harmful side effects such as dry cough and angioedema often associated with chemically synthesized drugs (45), and additionally, they are lower in cost (55).

The aim of this study was to exploit the proteolytic capabilities of LAB intestinal isolates for efficient generation of propeptide ACE inhibitors. Lactobacillus animalis DPC6134 (NCIMB 41355) was identified as a useful strain for release of propeptide ACE inhibitors from casein.

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Substrates and chemicals.
Hippuryl-L-histidyl-L-leucine (HHL), ACE (from rabbit lung; lyophilized powder), captopril, pepsin, and other chemicals were from Sigma Aldrich Chemical Co. (Sigma Aldrich Chemie, Steinheim, Germany). The commercial enzyme preparation corolase PP was from Röhm (Enzyme GmbH, Abitec Group, Darmstadt, Germany). Bovine sodium caseinate was from Dairygold (Mitchelstown, County Cork, Ireland).

Microorganisms and culture conditions.
Lactobacillus animalis DPC6134 (NCIMB deposit 41355) was isolated from the porcine small intestine and stocked in the culture collection of Teagasc Dairy Products Research Centre (DPC), Fermoy, Ireland. This strain was propagated in MRS broth (Oxoid Ltd., Basingstoke, United Kingdom) anaerobically using AnaerocultA gas packs, in accordance with the manufacturer's instructions (Merck, Germany), for 24 h at 37°C. Standard cultures were prepared by inoculation of 10 ml MRS broth with 10 µl of the frozen stocks (–80°C) followed by incubation at 37°C for 16 to 24 h.

Fermentation with L. animalis DPC6134.
The sodium caseinate substrate (2.5%, wt/vol) and glucose (0.5%, wt/vol) were inoculated with L. animalis DPC6134 (1%, wt/vol) and incubated at 37°C for 24 h with mixing at 100 rpm and a constant pH of 7, maintained by addition of 0.1 M NaOH as described previously (17).

RP-HPLC analysis of the 10-kDa sodium caseinate L. animalis DPC6134 fermentate filtrate.
Peptides within the 10-kDa filtrate were separated further using a reverse-phase high-performance liquid chromatography (RP-HPLC) system containing a narrow-bore column (Nucleosil C₁₈; 5 mm x 250 mm; Varian Chromatography Systems, Walnut Creek, CA) and a UV detector operating at 214 nm. Aliquots of the freeze-dried powders were diluted in distilled HPLC-grade water and filtered through an 0.45-µm filter (Millipore), and 30 mg/ml of the fermentate was loaded onto the column. The mobile phase was a binary mixture of acetonitrile and HPLC-grade water (100%, vol/vol) containing trifluoroacetic acid (0.1%, vol/vol). The content of acetonitrile in the mobile phase was increased linearly from 0 to 100% for 72 min at a flow rate of 1 ml/min. Peptides were detected using a detector operating at a wavelength of 214 nm. Solvents were removed from the collected fractions by evaporation. Fractions were redissolved in 1 ml of distilled water prior to subsequent assays for ACE-inhibitory activity.

Determination of ACE-inhibitory activity.
ACE-inhibitory activity was assayed using the modified method of Roy et al. (49). Briefly, 200 µl of HHL buffer (5 mM HHL in 0.1 M sodium borate buffer containing 0.3 M NaCl, pH 8.3) was mixed with 80 µl of inhibitory solution for 3 min at 37°C. The reaction was initiated by adding 20 µl of ACE (0.05 units/ml), and the mixture was incubated for 1 h at 37°C. The reaction was stopped by adding 250 µl of 1 M HCl, and the reaction mixture was mixed with 1.7 ml of ethyl acetate. Solvents were removed from the test fractions by evaporation. Fractions were redissolved in 1 ml of distilled water, and the absorbance was measured at 228 nm. The extent of inhibition was calculated as follows: ACE-inhibitory activity (%) = 100 – [100 x (C – D)/(A – B)], where A is absorbance in the presence of ACE without the ACE-inhibitory component, B is absorbance without ACE and the ACE-inhibitory component, C is absorbance with ACE and the ACE-inhibitory component, and D is absorbance without ACE and with the ACE-inhibitory component. The capillary electrophoresis method of Zhang et al. (60) was also performed using a Beckman P/ACE System MDQ. Separations were carried out using an uncoated fused-silica capillary with an internal diameter of 50 µm. The capillary temperature was 22°C, and 17 kV was used. Percent inhibition was calculated based on a standard curve prepared from several dilutions of the rabbit lung acetone extract. The ACE inhibitor captopril (50% inhibitory concentration [IC₅₀], 6 µM) was used as a reference ACE-inhibitory substance at a concentration of 0.005 mg/ml. Resulting values are the averages for three separate assays.

Determination of protein concentration and A_w.
The protein concentration of the fractions was determined using the Bio-Rad protein assay (26). The protein concentration of the entire sodium caseinate L. animalis DPC6134 hydrolysate was determined using the Kjeldahl method. Water activity (A_w) was measured at 25°C using the water activity (equilibrium relative humidity) monitor (Labcell, Ltd., Hampshire, England).

Purification, sequencing, and synthesis of propeptide ACE inhibitors.
A liquid chromatography (LC) separation of the collected fractions was performed using an LC Packings nano-LC system (Bremen, Germany). A 75-µm PepMap column was used, into which a 5-µl volume was injected. Solvent A was a mixture of methanol and water (0.1% HCOOH in water), and solvent B contained methanol and acetonitrile (0.1% HCOOH in acetonitrile). Peptides were eluted with a linear gradient of solvent B in solvent A going from 2 to 90% over 40 min. The peptide mixtures in fractions exhibiting ACE-I-inhibitory activity were analyzed online by high-capacity ion-trap (HCT) mass spectrometry (Bremen, Germany), and using Data Analysis (version 3.0; Bruker Daltoniks, Bremen, Germany), the m/z spectral data were processed and transformed to spectra representing mass values. The molecular mass values were compared with known sequences of bovine _s1-casein, ß-casein, and -casein using BioTools (version 2.1; Bruker Daltoniks) to process the mass spectrometry (n) spectra, and subsequently, tentative sequence assignments could be carried out. The amino acid composition was also determined by sequencing (Bruker Daltoniks, Bremen, Germany).

Predicted ACE-inhibitory activity.
The five peptides chosen for chemical synthesis in this study comply with the governing features indicative of an ACE-inhibitory peptide sequence as outlined previously (57), as shown in Fig. 1. Predictive IC₅₀ values were also calculated for the peptides using quantitative structure-activity relationship modeling based on the C-terminal tetrapeptide residues of the peptides (57), and subsequently five peptides were chemically synthesized to confirm their ACE-inhibitory activities (Peptide Protein Research Ltd., Fareham, United Kingdom). The purity of the synthesized peptides was greater than 95%. Chemically synthesized peptides and captopril (positive control) were tested for susceptibility to proteinase K (Sigma) by incubation of proteinase K (2,050 U/ml) as described previously (17). Cleavage analysis of the chemically synthesized peptide was performed using Expasy Peptide Cutter (http://ca.expasy.org/cgi-bin/peptidecutter/peptidecutter.pl).

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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 -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.

Simulated gastrointestinal digestion.
The 10-kDa L. animalis DPC6134 fermentate was proteolytically degraded with pepsin and corolase PP to simulate gastrointestinal digestion according to the method of Alting et al. with modifications (1). Briefly, samples were hydrolyzed with pepsin (EC 3.4.4.1; 1:6,000; 3,400 units/mg) (Sigma) (20 mg/gram protein) for 90 min at 37°C at a pH of 2.0 followed by hydrolysis with corolase PP (40 mg/gram protein) (Röhm, Germany) at pH 7.5. Hydrolysis was carried out at 37°C in a water bath, under temperature-controlled conditions with constant stirring. Enzyme activity was inactivated by heating at 95°C for 10 min, followed by cooling to room temperature. Samples were taken after 90 min of hydrolysis with pepsin and at 30, 120, and 240 min of hydrolysis with corolase PP. Sample aliquots were centrifuged at 10,000 x g for 30 min, and supernatants were filtered through a 0.45-µm filter. Fermentate breakdown into peptides was analyzed using RP-HPLC as previously described, and the ACE-inhibitory activity of each aliquot was confirmed using the capillary electrophoresis method as described previously (60).

Predicting potential peptide binding sites on somatic ACE.
Using the PDB (1o8a.pdb) three-dimensional (3D) structure of testicular ACE, which corresponds to the C-terminal domain of somatic ACE (45), potential binding sites of the identified ACE-inhibitory peptides were estimated using the program Q-SiteFinder (25). Binding sites were displayed using the graphical program Chime (Elsevier MDL).

Location of identified ACE-inhibitory peptides on the 3D structure of ß-casein.
The ACE-inhibitory peptides were superimposed onto the 3D structure of ß-casein obtained by Kumosinski et al. (23) using SWISS MODEL and PDB Viewer (15). The resulting superposition of ß-casein and the five identified ACE-inhibitory peptides was visualized using the molecular viewer CHIMERA (http://www.cgl.ucsf.edu/chimera) (40).

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Production of a sodium caseinate hydrolysate with ACE-inhibitory activity.
The strain L. animalis DPC6134 (NCIMB 41355) was chosen for release of ACE-inhibitory peptides from sodium caseinate due to its proteolytic activity, which was analyzed using HPLC and a skim milk agar diffusion assay as described previously (37). This strain was found to hydrolyze sodium caseinate to oligopeptides 0.5 to 3kDa in size corresponding to peptides of between 7 and 24 amino acids. Additionally, the crude casein fermentate of L. animalis DPC6134 displayed a high ACE-inhibitory activity of 85.51% (±15%) and an IC₅₀ value of 0.8 mg protein/ml, calculated using the two ACE inhibition assays described above.

Isolation and detection of ACE-inhibitory peptides.
The sodium caseinate fermentate produced by L. animalis DPC6134 was filtered through a size-exclusion S1Y10 10-kDa spiral cartridge filter to obtain permeate containing peptides of 10 kDa and freeze-dried. The freeze-dried powder had a final protein concentration of 75% (±0.1%) protein/gram of sample as measured by the Kjeldahl method, and the A_w value of the powder was 0.19 (±0.03).

Seventy-two fractions were collected by RP-HPLC (Fig. 2A) and assayed for ACE-inhibitory activity, and the resultant peptides were identified. Three fractions, 10, 19, and 43, exhibited the highest ACE-inhibitory activities, of 67.5% (±15%), 83.7% (±19%), and 42.36% (±11%), respectively, where ACE inhibition was determined with 80 µl of the fractions which had protein concentrations of 0.5 mg/ml.

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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.

Purification, sequencing, and characterization of peptides.
The three fractions exhibiting potent ACE-inhibitory activity were further fractionated and purified by LC, and some of the peptides within were identified and sequenced as shown in Fig. 2B. These peptide mixtures were then analyzed by HCT-ion trap mass spectrometry (Bruker Daltoniks, Bremen, Germany), and using Data Analysis (version 3.0; Bruker Daltoniks, Bremen, Germany), the m/z spectral data were processed and transformed to spectra representing mass values. The masses and amino acid sequences produced were applied to MASCOT databases with an all species and a "no-enzyme" search.

A total of 25 peptides were identified in the three analyzed fractions. The amino acid composition and observed masses for each peptide are shown in Fig. 2C. In all, 16 peptides derived from ß-casein were identified, three peptides from -casein were identified, and five from _s1-casein were identified. One peptide, TTMLIQDEDDLEMA, with an observed molecular weight of 1,639.63, was not matched to any casein.

Chemical synthesis of peptides.
To ensure that the peptide sequences that had been identified by Bruker Daltoniks HCT Plus electrospray ionization-tandem mass spectrometry were responsible for ACE-inhibitory activity within the three active fractions, peptide NIPPLTQTPVVVPPFIQ [corresponding to ß-casein f(73-89)], peptide IGSENSEKTTMP [corresponding to _s1-casein f(201-212)], peptide SQSKVLPVPQ [corresponding to ß-casein f(166-175)], peptide MPFPKYPVEP [corresponding to ß-casein f(124-133)], and peptide EPVLGPVRGPFP [corresponding to ß-casein f(210-221)], as shown in Table 1, were chosen for chemical synthesis based on in silico predictions of ACE inhibition. Bioinformatic analysis of the five chosen peptides demonstrated that these peptides have the potential to bind to 1 of the 10 binding sites estimated on the structure of testicular ACE, which corresponds to the C-terminal domain of somatic ACE (using the program Q-SiteFinder [25]), with the most likely binding sites highlighted by darker coloring as shown in Fig. 3.

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TABLE 1. IC50 values and percent ACE-inhibitory values reported for the chemically synthesized peptides identified in fractions 10, 19, and 43 eluted from the L. animalis DPC6134 sodium caseinate fermentatea

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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.

ACE-inhibitory activities of the crude fractions and chemically synthesized peptides.
The ACE-inhibitory values for the fractions eluted at 10, 19, and 43 min by RP-HPLC from the 10-kDa permeate dried powder were calculated as 67.5% (±15%), 83.71% (±19%), and 42.76% (±11%), respectively, at a protein concentration of 0.5 mg/ml. The IC₅₀ values of the chemically synthesized peptides and the concentrations used for assay conditions are shown in Table 1. The IC₅₀ values of these peptides were determined using captopril, with documented IC₅₀ values in the range of 6 to 17.50 µM, as a positive control which inhibited ACE (approximately 100%) at a concentration of 0.005 mg/ml captopril. The ACE-inhibitory electrophoregrams of each chemically synthesized peptide are shown in Fig. 4. Capillary electrophoresis was used to separate hippuric acid (HA) and the dipeptide His-Leu from the substrate HHL, and the concentration of HA [HA] (as peak area) liberated was used to quantify the enzyme activity. The HA peak area (Fig. 4, peak iii) was obtained from the electrophoretogram using the CE system software, and from this the concentration of HA was calculated and subsequently the ACE activity was determined following the method of Zhang et al. (60). Under assay conditions, all the chemically synthesized peptides and crude fractions lost their ACE-inhibitory activity when treated with proteinase K (data not shown).

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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.

Predicted ACE-inhibitory activities.
By using the tetrapeptide model of Wu et al. (57), NIPPLTQTPVVRPPFIQ was found to have an IC₅₀ value of 45.03 µM, IGSENSEKTTMP was found to have an IC₅₀ value of 51.37 µM, SQSKVLPVPQ was found to have an IC₅₀ value of 37.17 µM, MPFPKYPVEP was found to have an IC₅₀ value of 59.96 µM, and EPVLGPVRGPFP was found to have an IC₅₀ value of 137.43 µM. IC₅₀ values are based on the C-terminal tetrapeptide sequence of each peptide (57). The identified ACE-inhibitory peptides were superimposed onto the 3D structure of ß-casein obtained by Kumosinski et al. (23) using SWISS MODEL and PDB Viewer (15) and visualized using the molecular viewer CHIMERA (http://www.cgl.ucsf.edu/chimera) as shown in Fig. 5.

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FIG. 5. (a) Graphical representation of the five peptides on the structure of ß-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).

Simulated gastrointestinal stability of ACE-inhibitory activity.
Simulated gastrointestinal digestion of the casein fermentate subjected to semipreparative RP-HPLC at different stages in the digestion process and eluted as described above gave the UV absorbance profiles and percent ACE-inhibitory activities shown in Fig. 6. Following digestion with pepsin alone the ACE-inhibitory percentage of the total sodium caseinate fermentate declined to 61.0% (standard deviation [SD], ±4.8%) compared to captopril, which had an ACE-inhibitory activity of 94.2% (SD, ±4%) at pH 2. When the pH was increased to 7 to 8 for corolase PP digestion, the percent ACE-inhibitory activity of the fractions increased to 81% (SD, ±7%), 92.0% (SD, ±9%), and 95.0% (SD, ±1%) at 120, 240, and 360 min, respectively. These results suggest that the ACE-inhibitory activity of the L. animalis DPC6134 10-kDa fermentate may be resistant to digestion by the gastrointestinal enzymes pepsin and corolase PP in vitro.

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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.

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Studies on the release of bioactive peptides from milk proteins by proteolytic enzymes have been reported elsewhere (30, 41), but few consider the use of LAB other than Lactobacillus helveticus and Lactobacillus delbrueckii strains in the fermentation of milk proteins for the generation of these peptides. LAB ascribed with the ability to liberate bioactive peptides include strains such as Lactobacillus helveticus CP790, Lactobacillus rhamnosus GG, Lactobacillus bulgaricus SS1, and Lactococcus lactis subsp. cremoris FT4 (50). However, Lactobacillus animalis strains have not been studied extensively, and the generation of ACE-inhibitory peptides by this strain has not been reported previously.

In this study, sodium caseinate was subjected to proteolysis using L. animalis DPC6134 (NCIMB deposit 41355), and ACE-inhibitory peptides from _s1- and ß-casein were identified. L. animalis DPC6134 was chosen based on initial assays demonstrating its proteolytic potential to generate a large number of peptides of <10 kDa in addition to its "generally recognized as safe" status. Twenty-four peptidases responsible for the conversion of oligopeptides released from milk proteins into smaller peptides with organoleptic and potential health benefits have been characterized from LAB (50).

Although the peptides produced in this study share amino acid sequence homologies with previously identified bioactive peptides, the complete sequences have not been identified before (10, 56). The RP-HPLC fraction eluted at 10 min was found to contain 10 peptides, five of which share homology with previously identified ACE-inhibitory peptides. Peptide SQSKVLPVPQ, corresponding to ß-casein f(166-175) (IC₅₀ value, 92 µM), has not been reported previously as possessing ACE-inhibitory activity. However, a fragment, KVLPVPQ, corresponding to ß-casein f(169-175) and reported to have an IC₅₀ value of 39 µM has been previously identified as an ACE inhibitor (18, 27). Peptide SKVLPVPQ, which shares eight amino acids with SQSKVLPVPQ, has also demonstrated antihypertensive activity in vivo (58). Peptide EMPFPKYPVEP, corresponding to ß-casein f(123-133) and identified in fractions 10 and 19 (Table 1), also shares homology with the previously identified ACE-inhibitory and bradykinin-potentiating (39) peptide EMPFPK, corresponding to ß-casein f(123-128) and gamma casein f(108-113). Peptide sequence TEDELQDKIHP, corresponding to ß-casein f(56-66), may also contribute to the ACE-inhibitory activity displayed by this fraction as it has nine C-terminal amino acids in common with the previously identified ACE inhibitor DELQDKIHPFAQSLVYPFPGPIPNS, isolated from dried bonito (41, 59). Within fraction 19, seven peptides all derived from ß-casein were identified as shown in Fig. 2C. The peptide sequence corresponding to ß-casein f(210-221) and with the amino acid sequence EPVLGPVRGPFP displays homologies with previously identified ACE-inhibitory peptides YQQPVLGPVR (31), VLGPVRGPFP (19), and YQEPVLGPV (21). Fraction 43 was found to contain eight peptides derived from both _s1- and -casein. Peptide IGSENSEKTTMP, identified in fraction 43 and corresponding to _s1-casein f(201-212), displayed an IC₅₀ value of 773.10 µM and shares homologies with previously identified ACE-inhibitory peptides TTMPLW and KTTMP (28, 41).

As shown in Table 1, NIPPLTQTPVVVPPFIQ displayed an IC₅₀ value of 450 µM when assayed in vitro, whereas the previously identified ACE-inhibitory peptide NIPPLTQTPV had an IC₅₀ value of 173.3 µM. The IC₅₀ value increase is likely due to the extra sequence VVPPFIQ, as increased hydrophobicity in the C-terminal position enhances the ACE-inhibitory potential of peptides (42). NIPPLTQTPVVVPPFIQ, identified in this study, has a C-terminal amino acid glutamine (Q) with a hydrophobicity descriptor value of –3.5, whereas the C-terminal amino acid valine (V) of peptide NIPPLTQTPV has a hydrophobicity descriptor value of 4.2 (42). Additionally, the potency of ACE inhibitors administered in vivo decreases as the chain length increases (48). However, in vivo the ACE-inhibitory IC₅₀ values of peptide NIPPLTQTPVVVPPFIQ should decrease, due to the presence of the sequences IPP and VPP. Likewise, the increase in IC₅₀ values for SQSKVLPVPQ (IC₅₀, 92.00 µM) and IGSENSEKTTMP (IC₅₀, 773.10 µM) is likely due to increased chain lengths compared to the previously identified ACE-inhibitory peptides VLPVPQ (IC₅₀, 39 µM) and TTMP (IC₅₀, 51 µM). Peptide MPFPKYPVEP (IC₅₀, 83.00 µM) displays a lower IC₅₀ value than does EMPFPK (IC₅₀ value, 423 µg/ml^–1). Peptide MPFPKYPVEP has proline (P) with a hydrophobicity descriptor value of –1.6 in the C-terminal position whereas EMPFPK has lysine (K) with a hydrophobicity descriptor value of –3.9, and additionally proline has a higher molecular weight (155.1) than does lysine (146.2) (42). Peptide EPVLGPVRGPFP displayed an IC₅₀ value of 790 µM when assayed in vitro, and the IC₅₀ value for peptide GPVRGPFPIIV (Table 1) was not reported previously.

Peptides identified in this study have higher IC₅₀ values than either IPP or VPP but compare favorably with other food-derived ACE-inhibitory peptides identified in various fermented milks and cheeses such as AVPYPQR, generated using "ropy milk starters" and corresponding to ß-casein f(177-183) (IC₅₀ value of 274.00 µM) (12); GLDIQK, isolated from yoghurt with "ropy milk starters" and corresponding to ß-Lg f(9-14) (IC₅₀ value of 580.00 µM); YQEPVL, isolated from milk fermented with "ropy milk starters" and corresponding to ß-casein f(193-198) (IC₅₀ value of 280.00 µM); VRGPFP, isolated from Manchego cheese and corresponding to ß-casein f(199-204) (IC₅₀ value of 592.00 µM); and YP, corresponding to _s1-casein f(146-147) and isolated from milk fermented with Lactobacillus helveticus CPN4 (IC₅₀ value of 720.00 µM) (12).

As shown in Table 1, ACE-inhibitory peptides have displayed other bioactivities. The ACE inhibitor and commercially available antihypertensive drug captopril, used in this study as a positive control, has been shown previously to exhibit antioxidant properties (16). Peptide SQSKVLPVPQ may also possess antioxidant activity as it shares six C-terminal amino acids with the antioxidant peptide VLPVPQK (46). IGSENSEKTTMP shares homologies with the previously identified peptide TTMPLW (IC₅₀ value, 51 µM), which also displays immunomodulatory activity (32); IGSENSEKTTMP may also share this property. EMPFPKYPVEP shares homology with the bradykinin-potentiating peptides YPVEPFTE and EMPFPK and may also display this activity.

While ACE inhibition is a biological marker for an antihypertensive effect, the demonstration of an antihypertensive effect is an endpoint marker for cardiovascular disease (9). Several ACE-inhibitory peptides produce a strong antihypertensive effect in vivo while others lose their activity. In vivo, activation or loss of activity is perhaps due to further endogenous enzymatic cleavage (22). Small di- and tripeptides such as IPP and VPP are passively absorbed in the small intestine and can reach the cardiovascular system intact, and some evidence exists that this may hold true for larger peptides as well (5, 48). The effect of a 4-week oral administration of C12 Peption containing 1 to 6% of the ACE-I-inhibitory peptide FFVAPFPEVFGK on blood pressure resulted in reduced systolic and diastolic blood pressure (51). In addition, a randomized, double-blind, placebo-controlled trial of C12 Peption found that a single administration of 3.5 g C12 Peption reduced diastolic and systolic blood pressure by 6 mm Hg and 9 mm Hg, respectively (53), suggesting that the C₁₂ peptide FFVAPFPEVFGK either is resistant to peptidase degradation, enabling intestinal absorption and an antihypertensive response after administration in vivo, or behaves as a propeptide which is further cleaved by gastrointestinal enzymes, releasing smaller di- and tripeptides with ACE-inhibitory activities, such as VAP (IC₅₀, 4 µM).

L. animalis DPC6134 sodium caseinate 10-kDa fermentate was resistant to a cocktail of digestive enzymes, normally present in the gastrointestinal tract. Corolase PP in combination with pepsin was used to mimic the enzymatic cleavage pattern of ACE-inhibitory peptides in the gastrointestinal tract. Digestion of the 10-kDa fermentate with pepsin resulted in a 61% reduction in ACE-inhibitory activity. Separation by RP-HPLC is based on molecular weight and hydrophobicity, with larger, more hydrophobic molecules having a longer retention time. When the RP-HPLC chromatograms of the undigested fermentate (Fig. 6B), the pepsin digest (Fig. 6C), and the corolase PP digests at 120 min and 240 min (Fig. 6E and 6F, respectively) were compared, transitions occurred for some peaks eluting after 20 min in the nonhydrolyzed fermentate to more peaks eluting earlier on the solvent gradient as digestion with pepsin and corolase PP progressed. Compared to the nonhydrolyzed L. animalis DPC6134 fermentate and despite the different gradient programs, it was shown that larger peptides were no longer present, while the proportion of low-molecular-weight compounds had risen after corolase PP digestion. As shown earlier, the ACE-inhibitory activity of the fermentate was maintained and increased following simulated gastrointestinal digestion with pepsin and corolase PP in vitro, suggesting that the peptides IPP (IC₅₀, 5 µM), VPP (IC₅₀, 9 µM), and NIPPLTQTPV (IC₅₀, 173.3 µM), found within the peptide sequence NIPPLTQTPVVVPPFIQ (IC₅₀, 450 µM), may have been released, increasing the ACE-inhibitory percentage. It is plausible that pepsin and corolase PP may have generated additionally ACE-inhibitory peptides from the 10-kDa sodium caseinate fermentate.

Conclusion.
The strain L. animalis DPC6134 is capable of degrading casein to a range of peptides, some of which have ACE-inhibitory activities; the generation of ACE-inhibitory peptides by using L. animalis DPC6134 has not been reported previously. Five ACE-inhibitory propeptides with IC₅₀ values in the range of 83 µM to 790 µM were generated. While these are less efficient than captopril, they do compare favorably with other ACE-inhibitory peptides isolated from food sources. In silico studies and in vitro digestion of the ACE-inhibitory peptides and fermentate demonstrated that in some cases cleavage of the peptides resulted in further ACE-inhibitory activity. However, more extensive proteolytic activity probably inactivates the anti-ACE activity.

 
Maria Hayes is in receipt of a Teagasc Walsh Fellowship. This work was funded by the Irish Government under the National Development Plan, 2000-2006, the European Research and Development Fund, and Science Foundation Ireland (SFI).

Predictive modeling of the IC₅₀ values was carried out by Rotimi Aluko at the University of Manitoba, Winnipeg, Canada. We acknowledge the help of Ian Davidson and Laura Main in mass spectrometry analysis.

 
* Corresponding author. Mailing address: Teagasc, Biotechnology Centre, Moorepark, Fermoy, County Cork, Ireland. Phone: 353 (0) 2542229. Fax: 353 (0) 2542340. E-mail: paul.ross{at}teagasc.ie

Published ahead of print on 4 May 2007.

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Applied and Environmental Microbiology, July 2007, p. 4658-4667, Vol. 73, No. 14
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