Electro-membrane filtration for the selective isolation of bioactive peptides from an alpha(s2)-casein hydrolysate

For the isolation of the ingredients required for functional foods and nutraceuticals generally membrane filtration has too low a selectivity and chromatography is (too) expensive. Electro-membrane filtration (EMF) seems to be a breakthrough technology for the isolation of charged nutraceutical ingredients from natural sources. EMF combines the separation mechanisms of membrane filtration and electrophoresis. In this study, positively charged peptides with antimicrobial activity were isolated from an alpha(s2)-casein hydrolysate using batch-wise EMF. alpha(s2)-Casein f(183-207), a peptide with strong antimicrobial activity, predominated in the isolated product and was enriched from 7.5% of the total protein components in the feed to 25% in the permeate product. With conventional membrane diafiltration using the same membrane (GR60PP), isolation of this and other charged bioactive peptides could not be achieved. The economics of EMF are mainly governed by the energy costs and the capital investment, which is affected by the flux of the desired peptide. A maximum average transport rate of alpha(s2)-casein f(183-207) during batch-wise EMF of 1.2 g/m2. h was achieved. Results indicate that an increase in the hydrolysate (feed) concentration, the applied potential difference and the conductivity of the permeate and electrode solutions, and a reduction in the conductivity of the feed result in a higher transport rate of alpha(s2)-casein f(183-207). This is in line with the expectation that the transport rate is improved when the concentration, the electrical field strength, or the electrophoretic mobility is increased, provided that the electrophoretic transport predominates. The expected energy consumption of the EMF process per gram of peptide transported was reduced by approximately 50% by applying a low overall potential difference and by processing desalinated hydrolysate. Considerable improvements in transport rate, energy efficiency, and process economics seem to be attainable by additional optimization of the process parameters and the EMF module design.     

Angiotensin-I-converting enzyme inhibitory peptides from tryptic hydrolysate of bovine alphaS2-casein

Angiotensin-I-converting enzyme (ACE) inhibitory activity of a tryptic digest of bovine alpha(S2)-casein (alpha(S2)-CN) was extensively investigated. Forty-three peptide peaks were isolated and tested. Seven casokinins (i.e. CN-derived ACE inhibitory peptides) were identified and their IC50 values were determined. Four peptides exhibited an IC50 value lower than 20 microM. Peptides alpha(S2)-CN (f174-181) and alpha(S2)-CN (f174-179) had IC50 values of 4 microM. Surprisingly, deletion of the C-terminal dipeptide of two of these casokinins did not significantly alter their inhibitory activity.     

Angiotensin I-converting-enzyme-inhibitory and antibacterial peptides from Lactobacillus helveticus PR4 proteinase-hydrolyzed caseins of milk from six species

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine alpha(S1)-casein (alpha(S1)-CN) 24-47 fragment (f24-47), f169-193, and beta-CN f58-76; ovine alpha(S1)-CN f1-6 and alpha(S2)-CN f182-185 and f186-188; caprine beta-CN f58-65 and alpha(S2)-CN f182-187; buffalo beta-CN f58-66; and a mixture of three tripeptides originating from human beta-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 micro g/ml (100 micro mol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC(50)) of some of the crude peptide fractions was very low (16 to 100 micro g/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC(50)s were confirmed. An antibacterial peptide corresponding to beta-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 micro g/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.     

Identification of novel antibacterial peptides isolated from a commercially available casein hydrolysate by autofocusing technique

Autofocusing, as a simple and safe technique, was used to fractionate casein hydrolysate based on the amphoteric nature of its peptides. The antibacterial activity of casein hydrolysate and its autofocusing fractions (A1-10) was examined against Escherichia coli and Bacillus subtilis. The basic fraction A9 exhibited the highest activity with minimum inhibitory concentration (MIC) of 150 μg/mL, whereas casein hydrolysate showed MIC values ranging from 2000 to 8000 μg/mL. The antibacterial peptides in A9 were purified by using a series of size exclusion and reversed phase chromatographies. Three peptides exhibited the most potent antibacterial activity with MIC values ranging from 12.5 to 100 μg/mL. These peptides were generated from α(s2) -casein, α(s1) -casein, and κ-casein and identified as K(165) KISQRYQKFALPQYLKTVYQHQK(188) , I(6) KHQGLPQEV(15) , and T(136) EAVESTVATL(146) , respectively. Therefore, the results revealed that casein hydrolysate had potent antibacterial peptides that could be isolated by autofocusing technique. ? 2012 International Union of Biochemistry and Molecular Biology, Inc.     

Structure-Activity Relationship of Synthetic Variants of the Milk-Derived Antimicrobial Peptide αs2-Casein f(183–207)

Template-based studies on antimicrobial peptide (AMP) derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties or residues that are important for biological activity. The present study sheds light on the importance of specific amino acids of the milk-derived α_s2-casein f(183–207) peptide to its antibacterial activity against the food-borne pathogens Listeria monocytogenes and Cronobacter sakazakii. Trimming of the peptide revealed that residues at the C-terminal end of the peptide are important for activity. Removal of the last 5 amino acids at the C-terminal end and replacement of the Arg at position 23 of the peptide sequence by an Ala residue significantly decreased activity. These findings suggest that Arg23 is very important for optimal activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the α_s2-casein f(183–207) peptide also caused a significant reduction of the effectiveness against C. sakazakii, which points toward the importance of the positive charge of the peptide for its biological activity. Indeed, simultaneous replacement of various positively charged amino acids was linked to a loss of bactericidal activity. On the other hand, replacement of Pro residues at positions 14 and 20 resulted in a significantly increased antibacterial potency, and hydrophobic end tagging of α_s2-casein f(193–203) and α_s2-casein f(197–207) peptides with multiple Trp or Phe residues significantly increased their potency against L. monocytogenes. Finally, the effect of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the activity of the 15-amino-acid α_s2-casein f(193–207) peptide was also determined, and its biological activity was shown to be completely abolished in high-saline environments.     

Identification of the initial binding sites of αs2-casein f(183-207) and effect on bacterial membranes and cell morphology

The aim of this work was to identify the initial binding sites to the bacterial membranes of the antimicrobial peptide α_s2-casein f(183-207) and also to acquire further insight into membrane permeabilization of this peptide. Furthermore, cell morphology was studied by transmission electron microscopy. In all the experiments, bovine LFcin was employed as a comparison. Results showed that initial binding sites of α_s2-casein f(183-207) peptide were lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative. The peptide was able to permeabilize the outer and inner membranes. Moreover, the α_s2-casein peptide f(183-207) generated pores in the outer membrane of Gram-negative bacteria and in the cell wall of Gram-positive bacteria. In the Gram-negative bacteria, f(183-207) originated cytoplasm condensation, and in the Gram-positive bacteria the cytoplasmic content leaked into the extracellular medium. Furthermore, the experiments of inner and outer membrane permeabilization performed with LFcin-B showed that this peptide also has the ability to permeabilize both the inner and outer membranes.     

Identification of the initial binding sites of alphas2-casein f(183-207) and effect on bacterial membranes and cell morphology

The aim of this work was to identify the initial binding sites to the bacterial membranes of the antimicrobial peptide alphas2-casein f(183-207) and also to acquire further insight into membrane permeabilization of this peptide. Furthermore, cell morphology was studied by transmission electron microscopy. In all the experiments, bovine LFcin was employed as a comparison. Results showed that initial binding sites of alphas2-casein f(183-207) peptide were lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative. The peptide was able to permeabilize the outer and inner membranes. Moreover, the alphas2-casein peptide f(183-207) generated pores in the outer membrane of Gram-negative bacteria and in the cell wall of Gram-positive bacteria. In the Gram-negative bacteria, f(183-207) originated cytoplasm condensation, and in the Gram-positive bacteria the cytoplasmic content leaked into the extracellular medium. Furthermore, the experiments of inner and outer membrane permeabilization performed with LFcin-B showed that this peptide also has the ability to permeabilize both the inner and outer membranes.     

Hydrolysis of casein-derived peptides alpha(S1)-casein(f1-9) and beta-casein(f193-209) by Lactobacillus helveticus peptidase deletion mutants indicates the presence of a previously undetected endopeptidase

Peptides derived from hydrolysis of alpha(S1)-casein(f1-9) [alpha(S1)-CN(f1-9)] and beta-CN(f193-209) with cell extracts of Lactobacillus helveticus CNRZ32 and single-peptidase mutants (Delta pepC, Delta pepE, Delta pepN, Delta pepO, and Delta pepX) were isolated by using reverse-phase high-performance liquid chromatography and were characterized by mass spectrometry. The peptides identified suggest that there was activity of an endopeptidase, distinct from previously identified endopeptidases (PepE and PepO), with specificity for peptide bonds C terminal to Pro residues. Identification of hydrolysis products derived from a carboxyl-blocked form of beta-CN(f193-209) confirmed that the peptides were derived from the activity of an endopeptidase.     

Design and synthesis of novel antimicrobial peptides on the basis of alpha helical domain of Tenecin 1, an insect defensin protein, and structure-activity relationship study

Tenecin 1, a peptide consisting of 43 amino acids, exhibits a potent bactericidal activity against various Gram-positive bacteria and shares a common structural feature of insect defensin family corresponding to cysteine stabilized alpha/beta motif. Our previous research indicated that an active fragment was successfully extracted from C-terminal beta sheet domain of Tenecin 1, whereas the fragment corresponding to the alpha helical region of the protein had no antibacterial activity. We chose this inactive fragment corresponding to alpha helical region of Tenecin 1 and synthesized derivatives with a different net positive charge by using rational design. Interestingly, we successfully endowed antibacterial activity as well as antifungal activity to the inactive alpha helical fragment by single or double amino acid replacement(s) without an increase of hemolytic activity. The leakage of dye from vesicles induced by the active peptides suggested that these peptides act on the membranes of pathogen as a primary mode of action. Structure-activity relationship study of a series of the active derivatives revealed that amphiphilic structure and high net positive charge were prerequisite factors for the activity and that there was a relationship between the antibacterial activity and the isoelectric point of the active peptides. In this work, we showed an efficient method to endow the antibacterial activity as well as antifungal activity to the inactive fragment derived from a cyclic insect defensin protein and suggested a facile method to screen for active fragments from cyclic host defense peptides.     

Effect of products of alphaS1-casein proteolysis on the activity of angiotensin-converting enzyme

The influence of alpha s1-casein proteolysis products of cow milk on the activity of angiotensin-converting enzyme (ACE) has been investigated. The peptide fraction has been obtained after incubation of alpha s1-casein with different strains Lactococcus lactis ssp. lactis and pepsin. The peptides with low molecular weight has been obtained with the help of a gel filtration. It is shown, that such peptides received with the help of some strains of Lactococcus lactis ssp. lactis and pepsin are capable to inhibit the activity of ACE. A conclusion about possibility of appearing antihypertensive peptides as a result of proteolitic processes in milk products has been made.     

Purification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61. A metalloendopeptidase that recognizes the size of its substrate

An endopeptidase (LEP-II), which has a unique substrate specificity, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme was a metalloendopeptidase since it was inhibited by EDTA and 1,10-phenanthroline; the metal-depleted enzyme could be fully reactivated by micromolar levels of Zn2+ and was not inhibited by specific inhibitors for serine or thiol protease. The molecular mass of the enzyme was estimated to be 80 kDa by Sephacryl S-300 gel filtration and high-performance liquid chromatography with a TSK-G3000SW column. The enzyme consisted of two identical subunits and the N-terminal sequence of LEP-II was determined up to the 19th residue. Although the enzyme had a broad substrate specificity it specifically hydrolyzed the peptide bonds involving the amino groups of hydrophobic amino acid residues. Various small polypeptides, such as alpha s1-CN(f1-23), alpha s1-CN(f91-100), oxidized insulin B chain, glucagon and some biologically active peptides were hydrolyzed. However, a variety of larger polypeptides or proteins, such as alpha s1-CN(f1-54), alpha s1-CN(f61-123), alpha s1-CN(f136-196), alpha s1-casein, beta-casein, and kappa-casein were not hydrolyzed. LEP-II recognized the size of its substrates, which were limited below a molecular mass of about 3.5 kDa.     

Design and synthesis of cyclic disulfide-bonded antibacterial peptides on the basis of the alpha helical domain of Tenecin 1, an insect defensin

We synthesized cyclic disulfide-bonded (i, i+4) peptides with various net positive charges (+2-+5) from linear peptides derived from the alpha helical domain of Tenecin 1, an insect defensin, and investigated the effect of the intradisulfide bridge (i, i+4) on hydrophobicity, secondary structure, leakage activity and binding activity for large unilamellar vesicles, antimicrobial activity, and hemolytic activity. Intradisulfide bridge formation of the peptides resulted in the increase of amphiphilicity and hydrophobicity. Cyclic forms of the peptides did not deeply penetrate into PG/PC (1:1, mole ratio) large unilamellar vesicles and had a decreased lipid membrane perturbation activity for PG/PC LUVs. When the peptides interacted with PG/CL (2:1, mole ratio) LUVs, cyclic peptides with a high net positive charge (+4-+5) showed similar binding affinities and leakage activities for vesicles to those of linear forms, whereas cyclic peptides with a low net positive charge (+2-+3) exhibited lower leakage activity than their linear forms. CD spectra indicate that the intradisulfide bridge (i, i+4) provided little conformational constraint to linear peptides in buffer solution but resulted in the decrease of alpha helicity of the peptides in lipid membrane mimic conditions. The cyclic peptide with the highest net positive charge had a similar antibacterial activity to that of the linear peptide, whereas the cyclic peptides with a low net positive charge (+3-+4) exhibited lower antibacterial activity than their linear forms. The cyclic peptides of an appropriate net charge showed more potent activities against some bacteria than those of linear forms under high salt conditions.     

Casein-derived antimicrobial peptides generated by Lactobacillus acidophilus DPC6026

Three peptides produced by a Lactobacillus acidophilus DPC6026 fermentation of sodium caseinate and showing antibacterial activity against pathogenic strains Enterobacter sakazakii ATCC 12868 and Escherichia coli DPC5063 were characterized. These peptides were all generated from bovine alpha(s1)-casein and identified as IKHQGLPQE, VLNENLLR, and SDIPNPIGSENSEK. These peptides may have bioprotective applicability and potential use in milk-based formula, which has been linked to E. sakazakii infection in neonates.     

Isolation and characterization of four antibacterial peptides from bovine hemoglobin

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields several intermediate peptide fractions after separation by reversed phase HPLC exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli, and Salmonella enteritidis. From these fractions, four new antibacterial peptides were isolated and analyzed by ESI-MS/MS. Three of these peptides correspond to fragments of the alpha-chain of bovine hemoglobin: alpha107-141, alpha137-141, and alpha133-141, and one peptide to the beta-chain: beta126-145. The minimum inhibitory concentrations (MIC) of these peptides towards the four strains and their hemolytic activity towards bovine erythrocytes were determined.     

Design and synthesis of novel antimicrobial pseudopeptides with selective membrane-perturbation activity

By incorporating carbamate bond(s) into a cytolytic peptide, novel pseudopeptides with potent antibacterial activity and low hemolytic activity were synthesized. Circular dichroism spectra suggested that the incorporation of carbamate bond(s) decrease the alpha helical conformation of the peptide in lipid membrane circumstances, which must be regarded as a major factor for the separation of antibacterial activity from cytotoxic activity for mammalian cell. Experiments in which dye was released from vesicles indicated that the potent antibacterial activity and low hemolytic activity of the pseudopeptides must be due to their great lipid membrane selectivity. The present result suggest that backbone modifications can be a great tool for developing pseudopeptides with improved biological activity and bioavailability from cytolytic peptides.     

Amyloid fibril formation by bovine milk alpha s2-casein occurs under physiological conditions yet is prevented by its natural counterpart, alpha s1-casein

The calcified proteinaceous deposits, or corpora amylacea, of bovine mammary tissue often comprise a network of amyloid fibrils, the origins of which have not been fully elucidated. Here, we demonstrate by transmission electron microscopy, dye binding assays, and X-ray fiber diffraction that bovine milk alpha s2-casein, a protein synthesized and secreted by mammary epithelial cells, readily forms fibrils in vitro. As a component of whole alpha s-casein, alpha s2-casein was separated from alpha s1-casein under nonreducing conditions via cation-exchange chromatography. Upon incubation at neutral pH and 37 degrees C, the spherical particles typical of alpha s2-casein rapidly converted to twisted, ribbon-like fibrils approximately 12 nm in diameter, which occasionally formed loop structures. Despite their irregular morphology, these fibrils possessed a beta-sheet core structure and the ability to bind amyloidophilic dyes such as thioflavin T. Fibril formation was optimal at pH 6.5-6.7 and was promoted by higher incubation temperatures. Interestingly, the protein appeared to be less prone to fibril formation upon disulfide bond reduction with dithiothreitol. Thus, alpha s2-casein is particularly susceptible to fibril formation under physiological conditions. However, our findings indicate that alpha s2-casein fibril formation is potently inhibited by its natural counterpart, alpha s1-casein, while is only partially inhibited by beta-casein. These findings highlight the inherent propensity of casein proteins to form amyloid fibrils and the importance of casein-casein interactions in preventing such fibril formation in vivo.     

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

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.     

Induction, purification and characterization of an antibacterial peptide scolopendrin I from the venom of centipede Scolopendra subspinipes mutilans

The crude venom of the centipede Scolopendra subspinipes mutilans, injected with Escherichia coli K12D31 for 3-4 days showed broad-spectrum antimicrobial activity against Gram-positive. Gram-negative bacteria and fungi. It showed good antibacterial activity against E. coli K12D31 at different temperatures, pH, and ionic strengths. The crude venom was heated at 100 degrees C for 30 min, centrifuged at 10,000 rpm for 30 min at 4 degrees C and the supernatants were obtained, from which an antibacterial fraction having a molecular mass of 3000-5000 Da, was further separated by ultrafiltration. A homogeneous antibacterial peptide named scolopendrin I, having a molecular mass of 4,498 Da, was isolated using cation-exchange chromatography and two steps of reverse-phase high performance liquid chromatography (RP-HPLC). Scolopendrin I did not show any hemolytic and agglutination activities at the concentration below 30 microM.     

Structure-antibacterial, antitumor and hemolytic activity relationships of cecropin A-magainin 2 and cecropin A-melittin hybrid peptides.

In order to elucidate the structure-antibiotic activity relationship of cecropin A-magainin 2 and cecropin A-melittin hybrid peptides, several truncated peptides and the analogues with amino acid substitutions were synthesized and their antibacterial, antitumor and hemolytic activities of were examined. Cecropin A-magainin 2 hybrid analog, L16-CA(1-8)-MA(1-12) (termed as L-CA-MA in this study: KWKLFKKIGIGKFLHLAKKF-NH2), is known to have potent antibacterial and antitumor activity with less hemolytic activity. We found that the C-terminal region of L-CA-MA is more involved in the alpha-helical structure on cell membrane-like environment than N-terminal one by circular dichroism analysis. Deletion of the Gly-Ile-Gly sequence, the central hinge region of L-CA-MA, produced a considerable reduction in antitumor and hemolytic activity rather than an antibacterial one. The insertion of Pro, Gly-Ile or Gly-Pro in this hinge region of L-CA-MA caused retention of both antibacterial and antitumor activity while causing a significant decrease in hemolytic activity. However, the substitution with Gly-Pro-Gly instead of the Gly-Ile-Gly in CA(1-8)-MA(1-12), CA(1-8)-ME(1-12), CA(1-13)-MA(1-13) and CA(1-13)-ME(1-13) hybrids resulted in a drastic decrease in antibacterial, antitumor and hemolytic activity. The increase of hydrophobicity at position 16 in CA(1-8)-MA(1-12) by substituting Trp or Phe induced a significant increase in hemolytic activity without a considerable change in either antibacterial or antitumor activity. Therefore, these results suggested that the appropriate flexibility in the hinge region of CA-MA and CA-ME hybrid peptides and the appropriate hydrophobicity at position 16 in the hydrophobic region of CA (1-8)-MA(1-12) are important in potent antibacterial and antitumor activity with no hemolytic effect.     

Angiotensin I-Converting-Enzyme-Inhibitory and Antibacterial Peptides from Lactobacillus helveticus PR4 Proteinase-Hydrolyzed Caseins of Milk from Six Species

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine α_S1-casein (α_S1-CN) 24-47 fragment (f24-47), f169-193, and β-CN f58-76; ovine α_S1-CN f1-6 and α_S2-CN f182-185 and f186-188; caprine β-CN f58-65 and α_S2-CN f182-187; buffalo β-CN f58-66; and a mixture of three tripeptides originating from human β-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 μg/ml (100 μmol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC₅₀) of some of the crude peptide fractions was very low (16 to 100 μg/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC₅₀s were confirmed. An antibacterial peptide corresponding to β-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 μg/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.