WO2007096855A2 - Inhibiteur de l'enzyme qui convertit l'angiotensine-i - Google Patents

Inhibiteur de l'enzyme qui convertit l'angiotensine-i Download PDF

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WO2007096855A2
WO2007096855A2 PCT/IE2007/000027 IE2007000027W WO2007096855A2 WO 2007096855 A2 WO2007096855 A2 WO 2007096855A2 IE 2007000027 W IE2007000027 W IE 2007000027W WO 2007096855 A2 WO2007096855 A2 WO 2007096855A2
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ace
peptides
inhibitory
lactobacillus
peptide
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PCT/IE2007/000027
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WO2007096855A3 (fr
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Paul Ross
Catherine Stanton
Colin Hill
Ger Fitzgerald
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Teagasc, The Agriculture And Food Development Authority
University College Cork
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4741—Keratin; Cytokeratin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8103—Exopeptidase (E.C. 3.4.11-19) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/245—Lactobacillus casei

Definitions

  • the present invention relates to Angiotensin-I-converting enzyme inhibitors and to methods of producing such inhibitors.
  • the invention also relates to functional foods comprising such inhibitors.
  • Angiotensin-I-converting enzyme (ACE; also known as kininase II; EC 3.4.15.1) is a non-specific but highly selective key multifunctional ectoenzyme, involved in the regulation of peripheral blood pressure (32).
  • This enzyme is a halide-activated, EDTA- sensitive, peptidase that catalyses the cleavage of the dipeptidyl residue His-Leu from the COOH terminus of the decapeptide angiotensin-I; an inactive decapeptide released from angiotensinogen by rennin, into the potent octapeptide vasoconstrictor angiotensin-II, resulting in increased blood pressure.
  • ACE-inhibitors may reduce peripheral blood pressure and exert an antihypertensive effect in vivo. Peptides occurring naturally in snake venom were the first reported ACE-I inhibitors (26).
  • milk proteins are the primary precursors for ACE-I-inhibitory peptides (21), (33), (28), (9). These peptides are present in milk proteins such as casein and whey proteins in an encrypted form, stored as propeptides or mature C-terminal peptides that are only released upon proteolysis.
  • Casein-derived inhibitors casokinins
  • lactokinins lactokinins
  • Coagulants and microbial enzymes from starter Lactic Acid Bacteria may generate ACE-inhibitory peptides during milk fermentation and secondary proteolysis during cheese ripening was shown previously to generate ACE-inhibitory peptides (22).
  • An object of this invention is to provide novel ACE inhibitors and methods of generating ACE inhibitors.
  • a further object is to provide functional foods comprising ACE inhibitors or capable of providing ACE inhibitors in vivo.
  • a further object is to provide GRAS source for such methods and inhibitors.
  • Lactobacillus animalis DPC6134 (NCIMB41355) was selected to produce a sodium caseinate hydrolysate containing ACE-inhibitory peptides.
  • the ACE-inhibitory peptides were isolated and identified, sequenced and some were chemically synthesised to confirm their ACE- inhibitory activities.
  • the potential release of further activated in vitro ACE-inhibitory peptides during digestion from the ACE-inhibitory hydrolysate was also estimated on the basis of a simulation of digestion under gastrointestinal conditions.
  • the invention provides a method of producing an ACE-inhibitor comprising culturing a Lactobacillus species in the presence of milk proteins.
  • the milk proteins may be casein, Sodium caseinate or whey.
  • the Lactobacillus strain may be Lactobacillus casei/ paracasei, Lactobacillus johnsonii, Lactobacillus reuteri and Lactobacillus animalis.
  • the invention also provides an ACE-inhibitor whenever produced by the above- described method.
  • the ACE-inhibitor may have the peptide sequence shown in any one of tables 1, 2 or 3.
  • a further aspect the invention provides Lactobacillus Animalis DPC6134 as deposited at the National Collection of Industrial and Marine Bacteria on 18 November 2005, under the accession No. 41355.
  • the invention also provides Lactobacillus johnsonii DPC6092, as deposited on 20 February 2006 in the National Collection of Industrial and Marine Bacteria under the accession No. 41355 and Lactobacillus casei SS paracasei DPC6109 as deposited on 20 February 2006 in the National Collection of Industrial and Marine Bacteria under the accession No. 41378.
  • the invention provides a functional food comprising a food-stuff and an ACE-inhibitory peptide as defined above, or a Lactobacillus strain on a combination thereof.
  • the Lactobacillus strain may be one or more of the strains Lactobacillus casei SS paracasei DPC6109, Lactobacillus johnsonii DPC6092, Lactobacillus animalis DPC6134, or the strains as deposited.
  • the invention also provides a pharmaceutical composition comprising an ACE- inhibitor as defined above.
  • Figure 1 Semi-preparative reversed-phase HPLC of the L. animalis DPC6134 Sodium caseinate hydrolysate. Fractions 10, 19 and 43 are the fractions showing the highest Angiotensin-I-converting enzyme inhibitory activity. RP-HPLC was carried out at room temperature and following the conditions shown in the materials and methods section. Solid line refers to elution of solvent B.
  • FIG. 2 A Liquid chromatography (LC) chromatogram obtained during the analysis of ACE-I-inhibitory fraction 10 obtained from a semi-preparative 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 earlier (Materials and Methods section).
  • Figure 2 B The masses and amino acid sequences produced from LC chromatography and MS work were applied to a MASCOT database with an all species and a "no enzyme" search. Nine peptide sequences from fraction 10 were assigned to ⁇ -casein.
  • FIG. 3 A Liquid chromatography (LC) chromatogram obtained during the analysis of ACE-I-inhibitory fraction 19 obtained from a semi-preparative 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 earlier (Materials and Methods section).
  • Figure 3 B The masses and amino acid sequences produced from LC chromatography and MS work were applied to a MASCOT database with an all species and a "no enzyme" search.
  • FIG. 4A Liquid chromatography (LC) chromatogram obtained during the analysis of ACE-I-inhibitory fraction 43 obtained from a semi-preparative 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 earlier (Materials and Methods section).
  • Figure 4 B The masses and amino acid sequences produced from LC chromatography and MS work were applied to a MASCOT database with an all species and a "no enzyme" search. Eight peptide sequences from fraction 43 were assigned to ⁇ -casein (5 peptide sequences) and ⁇ sl -casein (3 peptide sequences). These peptide sequences are highlighted in bold font.
  • FIG. 5 A Electropherogram of ACE reaction mixture with Captopril used as a control at a concentration of 0.005mg/ml.
  • Enzyme reaction conditions 5mM HHL and 500 ⁇ U of ACE in 100 mM Boric acid-borate buffer (pH 8.3) with 0.5N NaCl; total volume 550 ⁇ l, enzyme reaction time, 60 mins @ 37°C. Peak A, Histidyl Leucine, migration time of 2.7 min, B, HHL, migration time of 3.89 min and C, HA, migration time of 4.98min.
  • the Capillary electrophoresis conditions are as described in the text.
  • HA is the most anionic component and eluted at 5-6 min, while less anionic His-Leu and HHL migrated at 2.5 to 3.89 min respectively.
  • 5B Electropherogram of ACE reaction mixture without Captopril or ACE-inhibitory reagent added. Enzyme reaction conditions as described for Captopril 0.005mg/ml. A, migration time of 2.5 mins, B, migration time of 3.8mins and C, migration time of 5,3 mins.
  • 5C Electropherogram of ACE reaction mixture with peptide MPFPKYPVEP (830 ⁇ M) added as ACE-inhibitor. Enzyme reaction conditions as described earlier.
  • A migration time of 3.2min, B, migration time of 3.8min and C, migration time of 5,3 min.
  • 5D Electropherogram of ACE reaction mixture with peptide IGSENSEKTTMP (77.3 l ⁇ M) used as the ACE inhibitor. A, migration time of 2.47min, B, migration time of 3.8min and C, migration time of 5.5 min.
  • 5E Electropherogram of ACE reaction mixture with the peptide NIPPLTQTPVVVPPFIQPEV (450 ⁇ M) added as the ACE-inhibitor. A, migration time of 2.7 min, B, migration time of 3.8min and C, migration time of 5.5 min.
  • 5F Electropherogram of ACE reaction mixture with the peptide SQSKVLPVPQ (92.4uM) added as the ACE inhibitor. A, migration time of 2.7 min, B, migration time of 3.8min and C, migration time of 5.5 min. 5G:
  • Electropherogram of the peptide EPVLGPVRGPFP (790.80 ⁇ M) added as the ACE inhibitory substance A, migration time of 2.7 min, B 3 migration time of 3.8 min and C, migration time of 5.5 min.
  • Figure 6 RP-HPLC chromatogram profiles of the L. animalis DPC6134 sodium caseinate hydrolysate after in vitro simulated gastrointestinal physiological digestion with
  • HPLC HPLC was carried out at room temperature and following the conditions shown in the materials and methods section.
  • Figure 7 Graphical representation of identified ACE-inhibitory peptides generated from the fermentation of sodium caseinate with Lactobacillus johnsonii DPC6092 on the structure of ⁇ -casein using the molecular visualisation program Chimera
  • Figure 8 Graphical representation of the location of peptide binding on the ACE structure predicted using the docking software ESCHER-NG (2) converted to a structural file with VEGA ZZ (16) and the results visualised with the graphical program CHIMERA
  • HHL Hippuryl-L-histidyl-L-leucine
  • ACE from rabbit lung, lyophilised powder
  • Corolase PP was from Rohm (Rohm, Enzyme GmbH,
  • Bovine sodium caseinate was from Dairygold
  • L. animalis DPC6134, L. casei subspecies paracasei DPC6109, L. johnsonii DPC6092 and L. reuteri DPC6215 were isolated from the porcine small intestine (data unpublished) and stocked in the culture collection of Teagasc Dairy Products Research Centre, Cork, Ireland. These strains were propagated in MRS broth (Oxoid Ltd, Basingstoke, United Kingdom) anaerobically for 24 h at 37 0 C. Standard cultures were prepared by inoculation of 10 ml MRS broth with 10 ⁇ l of the frozen stocks (-80 0 C) and then incubated at 37 0 C for 16-24 h. Screening fermentations.
  • the screening fermentations were performed in sodium caseinate (2.5 % w/v) with stirring at 100 revolutions per minute (rpm), temperature 37 0 C for 24 hrs with pH maintained at 7 with 0.1 M NaOH.
  • High Performance Liquid Chromatography using Delta-Pak C18 Column, (size; 600 mm X 7.5 cm, Varian Chromatography Systems, Walnut creek, California, USA) was then performed on the resultant fermentates.
  • the mobile phase was a binary mixture of acetonitrile and HPLC grade water (30 % v/v) containing trifluoroacetic acid (TFA) (0.1 % v/v). The flow rate was 1 ml/min.
  • Casein breakdown by both porcine intestinal isolates and infant faecal isolates was monitored by measurement of UV absorbance at 214 nm using a HPl 100 diode array detector.
  • the sodium caseinate substrate (2.5 % w/v) was inoculated with L. animalis DPC6134 (1 % w/v) and incubated at 37 0 C for 24 h with mixing at 100 rpm at constant pH 7 maintained via addition of 0.1 M NaOH.
  • the fermentation was performed in triplicate.
  • Peptides ⁇ 10 kDa in size were separated from sodium caseinate hydroly sates using an RP- HPLC reverse-phase high performance liquid chromatography system containing a narrow-bore column (Nucleosil C 18, 5 mm X 250 mm: Varian Chromatography Systems, Walnut creek, California, USA) and an UV detector operating at 214 nm. Aliquots of the freeze-dried powders were diluted in distilled HPLC-grade water and filtered through a 0.45 ⁇ m filter (Millipore) and 30 mg/ml of the fermentate loaded onto the column.
  • the mobile phase was a binary mixture of acetonitrile and HPLC grade water (100 % v/v) containing trifluoroacetic acid (0.1 % v/v).
  • 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 using a centrivap console (Labconco Corporation, Kansas City, USA). The fractions were redissolved in 1 ml of distilled water prior to subsequent assays for antimicrobial activity.
  • Lactobacillus johnsonii DPC6092 was used individually in further fermentations on the basis of its proteolytic activity determined from molecular mass distribution profiles. Three fermentations were performed in triplicate. The sodium caseinate substrate (2.5 % w/v) was inoculated with the strain (1 % w/v) and incubated at 37°C for 24 h with mixing at 100 rpm at constant pH 7 maintained via addition of 0.1 M NaOH. Fermentates were subsequently heated to 80 0 C to inactivate cultures and filtered through a size-exclusion SlYlO 3-kDa spiral cartridge filter (Millipore Ltd., Hertfordshire, UK), to separate the peptides less than 3-kDa. Fractions containing these peptides were freeze-dried and stored at -20 0 C until further use. Separation of peptides in fermentations
  • the filtrate containing peptides ⁇ 3-kDa in size was separated from the sodium caseinate (2.5 % w/v) fermentates using reverse-phase high performance liquid chromatography (RP-HPLC).
  • RP-HPLC reverse-phase high performance liquid chromatography
  • This system contained a narrow-bore column (Nucleosil Cl 8, 5mm X 250mm: Varian Chromatography Systems, Walnut creek, California, USA) and a UV detector operating at 214nm. Aliquots of the freeze-dried powdered fermentates were diluted in distilled HPLC-grade water and filtered through a 0.45 ⁇ m filter (Millipore) and 30 mg protein/ml of the fermentate loaded onto the column.
  • the mobile phase was a binary mixture of acetonitrile and HPLC grade water (100 % v/v) containing trifluoroacetic acid (0.1 % v/v).
  • the content of acetonitrile in the mobile phase was increased linearly from 0 to 100 % for 72 min at a flow rate of lml/min.
  • Peptides were detected using a detector operating at a wavelength of 214 nm. This step was repeated three times and solvents removed from the collected fractions by evaporation using a centrivap console (Labconco Corporation, Kansas City, USA). The centres of each active peak were collected, freeze-dried and used for further analysis. Fractions were redissolved in 1 ml of distilled water prior to subsequent assay for ACE-I-inhibitory activities.
  • the molecular mass distribution of the Lb. johnsonii DPC6092 fermentation was determined using High Performance Liquid Chromatography (HPLC) analysis perfonned using a Delta-Pak C18 Column, (size; 600mm X 7.5cm, Varian Chromatography Systems, Walnut creek, California, USA).
  • HPLC High Performance Liquid Chromatography
  • the mobile phase was a binary mixture of acetonitrile and HPLC grade water (30 % v/v) containing trifluoroacetic (TFA) acid (0.1 % v/v).
  • the flow rate was 1 ml/min.
  • Sodium caseinate breakdown was monitored by measurement of UV absorbance at 214nm using a HPl 100 diode array detector.
  • the protein standards used was unfermented sodium caseinate (M r unknown).
  • Molecular mass distribution was calculated by dividing the total chromatogram area into seven ranges of molecular mass (> 20-kDa, 20-10-kDa, 10-5-kDa, 5-2-kDa, 2-1-kDa, 1-0.5-kDa and ⁇ 0.5-kDa).
  • Fractions containing ACE-I-inhibitory activity were purified further using Liquid chromatography HPLC a second time. ACE-I-inhibitory fractions were further analysed by mass spectroscopy. The protein concentration of fractions was determined using the Biorad Protein Assay Method (9). Absorbance at 595 nm was determined and protein concentrations reported as mg protein/ml.
  • ACE inhibitory activity is usually analysed in vitro and implies the determination of inhibitory activity by means of a synthetic substrate with amino di- and tri- substituted peptides, such as Hippuryl-L-Histidyl-L-Leucine (HHL) (Sigma), as used in this study.
  • HHL Hippuryl-L-Histidyl-L-Leucine
  • the spectrophotometric method used in this study corresponded to a modified version of the method of Roy et al. (2000)(31). Briefly, 200 ⁇ l of HHL buffer (5mM Hip-His-Leu in 0.1 M Sodium Borate buffer containing 0.3 M NaCl, pH 8.3) was mixed with 80 ⁇ l of inhibitory solution for 3 mins at 37 0 C.
  • the reaction was initiated by adding 20 ⁇ l of ACE (0.05 units/ml), and the mixture was incubated for 1 hr at 37 "C. The reaction was stopped by adding 250 ⁇ l of IM HCL and mixed with 1.7 ml of ethyl acetate. Solvents were removed from the test fractions by evaporation using a centrivap console (Labconco Corporation, Kansas City, USA). The fractions were redissolved in 1 ml of distilled water and absorbance measured at 228 nm.
  • 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 ACE inhibitor component and D is absorbance without ACE and with the ACE-inhibitory component.
  • Bioactive fractions were collected and the peptide composition analysed by Mass spectrometry (MS) using a matrix assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectrophotometer (PE Biosystems Voyager-DE STR Biospectrometry Workstation, Aberdeen Proteome Facility) with a laser operating at 337 nni and an acceleration voltage of 2OkV.
  • MALDI-TOF matrix assisted laser desorption ionisation time of flight
  • MALDI-TOF matrix assisted laser desorption ionisation time of flight
  • ACE inhibitory activity is usually analysed in vitro and implies the determination of inhibitory activity by means of a synthetic substrate with amino di- and tri- substituted peptides, such as Hippuryl-L-Histidyl-L-Leucine (HHL) (Sigma), as used in this study.
  • HHL Hippuryl-L-Histidyl-L-Leucine
  • the spectrophotometric method used in this study corresponded to a modified version of the method of Roy et al. (2000)(26). Determination of ACE inhibitory activity.
  • ACE inhibitory activity was determined by two methods. The first corresponds to a modified version of the method of Roy etal (31). Briefly, 200 ⁇ l of HHL buffer (5mM Hip-His-Leu in 0.1 M Sodium Borate buffer containing 0.3 M NaCl, pH 8.3) was mixed with 80 ⁇ l of inhibitory solution for 3 mins at 37 0 C. The reaction was initiated by adding 20 ⁇ l of ACE (0.05 units/ml), and the mixture was incubated for 1 hr at 37 0 C. The reaction was stopped by adding 250 ⁇ l of IM HCL and mixed with 1.7 ml of ethyl acetate.
  • ACE-inhibitory activity 100- [100 X (C-D) / (A-B)] Where A is the absorbance in the presence of ACE without the ACE-inhibitory component, B is the absorbance without ACE and the ACE-inhibitory component, C is the absorbance with ACE and ACE inhibitor component and D is the absorbance without ACE and with the ACE-inhibitory component.
  • ACE inhibition was expressed in terms of IC5 C defined as the sample concentration needed to inhibit 50 % of the ACE activity.
  • the second corresponds to the method of Zhang et ⁇ l (40) based on Capillary electrophoresis with modifications to the instrumentation.
  • Capillary electrophoresis was performed using a Beckman P/ACE System MDQ. All separations were carried out using an uncoated fused-silica capillary with an internal diameter of 50 ⁇ m. Data was collected with Beckman System MDQ 1.6 version software. Capillary temperature was 22°C and 17kV 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 was used as a reference ACE-inhibitory substance at a concentration of 0.05mg/ml. Values of the percentage of ACE inhibition and ICs 0 are the average for three separate assays.
  • ACE-inhibitory peptides were superimposed onto the 3D structure of ⁇ -casein obtained by Kumosinski et al. (1993) (41) using SWISS MODEL and PDB viewer (42).
  • the resulting superposition of ⁇ -casein with the 4 identified ACE-inhibitory peptides was visualised using the molecular viewer CHIMERA (http://www.cgl.ucsf.edu/chimera) (43).
  • the strain L. animalis DPC 6134 (NCIMB41355) was chosen to produce an ACE- inhibitory sodium caseinate hydrolysate, as firstly it is a food grade bacterium with GRAS status that exhibited the greatest proteolytic activity of the LAB used in this study, and hydrolysed sodium caseinate to oligopeptides of between 1-2 kDa corresponding to peptides of between 7-24 amino acids, the size of which are similar to bioactive peptides discovered to date.
  • HPLC the powder had an ACE-inhibitory activity of 85.51 % (+/-15.62) and an IC 50 value of 0.8 mg protein/ml calculated using the two ACE-inhibition assays described above. Seventy- two fractions were collected by RP-HPLC and assayed for ACE-inhibitory activity. The casein hydrolysate subjected to semi-preparative RP-HPLC and eluted as described above gave the UV absorbance profile shown in Fig 1. To isolate peptides with relatively high ACE-inhibitory activities from the hydrolysate, eluted fractions were concentrated by repeated RP-HPLC of the 10 kDa hydrolysate and ACE-inhibitory activities measured.
  • the three active fractions collected at 10, 19 and 43 minutes were further fractionated and purified by HPLC and peptides within identified and sequenced. Some peptides were chemically synthesised to confirm their ACE-inhibitory activities.
  • the peptide mixtures in fractions 10, 19, and 43 were analysed on-line by HCT-ion trap mass spectrometry (Bremen, Germany) and using Data Analysis TM (version 3.0, Bruker Daltoniks, Bremen, Germany), the voJz spectral data was processed and transformed to spectra representing mass values. 2.1, Bruker Daltoniks) to process the MS (n) spectra and tentative sequence assignments could be carried out.
  • fraction 63 Fermentation of sodium caseinate with the proteolytic strain Lactobacillus johnsonii DPC6092, 3-kDa membrane filtration, and RP-HPLC analysis of the peptide fractions, yielded fraction 63, which displayed an ACE-I-inhibitory percentage of 95.45 % (+/-7.5) at a concentration of 0.05 mg protein/ml using the spectrometry method of Roy et al. (2000)(31).
  • This fraction was purified further using liquid chromatography. Further liquid chromatography produced 6 fractions (2-7).
  • Peptides within the fractions were identified using MALDI-TOF analysis (Aberdeen, Scotland) and corresponded to a region of Bos taurus beta casein B corresponding to Pubmed accession number CAC37028.
  • Fractions 2, 3 and 4 were sequenced from a LC-HPLC trace and 4 peptides identified within them collectively (Table 3). Each fraction contained the same peptide and only the mass of the fractions differed. In fraction 2, three sequences were found. These were L [AV] [YI] PFPGPIH ([], are possible insertion amino acids and were not found on the beta-casein B sequence) which corresponds to mass 1099 and LVYPFPGPIHNS corresponding to f (11-22) which corresponds to mass 1340.
  • Fraction 3 contained a peptide with molecular mass of 1793 which produced a 10 residue sequence LVYPFPGPIH corresponding to bovine beta-casein B f (11-20).
  • LVYPFPGPIHNS was found to have an IC 50 value of 116.77 ⁇ M, LVYPFPGPIHNSLPQN; an IC 50 value of 65.0 ⁇ M and LVYPFPGPIHN an IC 50 value of 16.1 ⁇ M.
  • IC50 values are based on the C-terminal tetrapeptide sequence of each peptide.
  • the identified ACE-inhibitory peptides were superposed onto the 3D structure of ⁇ - casein obtained by Kumosinski etal. (1993) (41) using SWISS MODEL and PDB viewer (6) and visualised using the molecular viewer CHIMERA
  • ACE-inhibitory activities of the crude fractions and chemically synthesised peptides were calculated as 67.532 %, (+ 15.5), 83.71 %, ( ⁇ 19.38), 84.73 %, ( ⁇ 11.35) respectively, at peptide concentrations of 0.554 mg/ml, 0.5 mg/ml and 1.24 mg/ml, respectively.
  • the IC5 0 values of the chemically synthesised peptides and the concentrations used for assay conditions are shown in Table 2.
  • the ICs 0 values of these peptides were determined using Captopril as a positive control. Captopril inhibited ACE at a concentration of 0.005mg/ml.
  • the ACE-inhibitory electropherograms of each chemically synthesised peptide are shown in Figure 5 (a) to 5 (g). Many of the peptides identified are partly homologous with previously described ACE-inhibitory peptides and peptides with other bioactivities. These homologies are shown in Table 3.
  • L. animalis DPC6134 was chosen in this study based on initial assays demonstrating its proteolytic activity against casein and therefore its potential to generate a large number of peptides ⁇ 10 kDa in addition to its GRAS status. It has been shown that proteases of LAB can hydrolyse more than 40 % of the peptide bonds of ⁇ -casein resulting in the formation of hundreds of oligopeptides, many of which may be bioactive in nature. Additionally, 16 peptidases responsible for the conversion of the released peptides into free amino acids have been characterised from LAB (17).
  • ACE-inhibitory peptides Two potent ACE-inhibitory peptides, IPP and VPP, derived from ⁇ - and ⁇ -casein during milk fermentation with Lactobacillus helveticus and Saccharomyces cerevisiae are the causative agents for antihypertensive activity in Calpis TM sour milk (34).
  • PvP-HPLC fraction eluted at time 10 min was found to contain 9 peptides, 5 of which share homology with previously identified ACE-inhibitory peptides.
  • peptide SQSKVLPVPQ corresponding to ⁇ -casein f (166-175) and shown to have an IC5 0 value of 92 ⁇ M has not been reported previously as possessing ACE-inhibitory activity.
  • KVLPVPQ corresponding to ⁇ -casein f (169-175) reported to have an IC 50 value of 39 ⁇ M has been identified as an ACE-I-inhibitor previously (19),(14).
  • Peptide SKVLPVPQ which shares 8 amino acids with SQSKVLPVPQ, has also demonstrated antihypertensive activity in vivo (38).
  • Peptide EMPFPKYPVEP corresponding to ⁇ -casein f (123-133) and identified in fraction 10 and 19 (Table 1) also shares homology with the previously identified ACE-inhibitory (28) and Bradykinin-potentiating (27) peptide EMPFPK corresponding to ⁇ -casein f (123-128) and gamma casein f (108-113).
  • Peptide sequence TEDELQDKIHP corresponding to ⁇ -casein f (56-66) may also contribute to the ACE- inhibitory activity displayed by this fraction as it has 9 C-terminal amino acids in common with the previously identified ACE-inhibitor
  • Peptide IGSENSEKTTMP identified in fraction 43 and corresponding to ⁇ s i -casein f (201-212) displayed an IC50 value of 773.10 ⁇ M and shares homologies with previously identified ACE-inhibitory peptides TTMPL W(20). It also has 5 C-terminal homologous amino acids with peptide KTTMP identified previously as an ACE-inhibitory peptide (28).
  • ACE-I-inhibitors often display other bioactivities.
  • the ACE-I- inhibitor and commercially available antihypertensive drug Captopril which was used in this study as a positive control, has been shown previously to exhibit antioxidant properties (12).
  • the structure of some of the peptide sequences identified in this study may predict the presence of other biologically active peptides.
  • peptide SQSKVLPVPQ identified in fraction 10, may also possess antioxidant activity as it shares 6 C-terminal amino acids with the previously reported antioxidant peptide VLPVPQK (30).
  • the O 81 -casein derived peptide IGSENSEKTTMP shares homologies with the previously identified ACE-I-inhibitory peptide TTMPLW (IC50 value 51 ⁇ M). As TTMPLW also displays immunomodulatory activity (25), it is believed to suggest that IGSENSEKTTMP may also share this immunomodulatory activity.
  • Peptide EMPFPKYPVEP shares homology with previously identified Bradykinin-potentiating peptides YPVEPFTE and EMPFPK and may also display this activity along with ACE-I-inhibition due to these shared amino acid sequence homologies.
  • Previously described bioactive peptides that share structure homologies with sequences identified in fractions 10, 19 and 43 are shown in Table 3.
  • ACE inhibitory activity is usually analysed in vitro and implies the determination of inhibitory activity by means of a synthetic substrate with amino di- and tri- substituted peptides, such as Hippuryl-L-Histidyl-L-Leucine (HHL) (Sigma) used in this study.
  • HHL Hippuryl-L-Histidyl-L-Leucine
  • the most commonly used ACE-inhibitory assay method measures angiotensin-converting enzyme activity in terms of the rate of release of Hippuric acid [HA] from Hippuryl-L- histidyl-L-leucine [HHL] based on the method of Cushman & Cheung (4).
  • the ACE enzyme is an exopeptidase and is therefore highly specific for the terminus, especially the C-terminus of peptide substrates or inhibitory peptides.
  • the importance of specific amino acid sequences on ACE-inhibitory activity has been suggested frequently (3).
  • the C-terminal tripeptides sequence is the primary structural feature governing this inhibitory response, and reports indicate that ACE appears to prefer substrates and inhibitors containing hydrophobic amino acid residues in the three C- terminal positions.
  • aliphatic (V, I, A), basic (R), and aromatic (Y, F) residues are preferred in the penultimate positions and aromatic (W, Y, F) proline (P) and aliphatic (I, A, L, M) residues being preferred in ultimate positions (3).
  • Arginine (R) at the C-terminus has also been shown to contribute to the ACE-I-inhibitory potency of several peptides (10). Also, a C-terminal lysine (K) with a positive charge on the ⁇ -amino group contributes substantially to inhibitory potency (36). Many of the ACE- I-inhibitory peptides identified in this study comply with the above governing features for an inhibitory response.
  • peptide EPVLGPVRGPFP corresponding to ⁇ - casein f (210-221), identified in fractions 10 and 19 and shown to have an IC 50 value of 790 ⁇ M contains at its C-terminus an aromatic phenylalanine (F) in the penultimate position and a proline (P) residue in the antepenultimate position.
  • ACE-inhibition is a biological marker for an antihypertensive effect
  • the demonstration of an antihypertensive effect is an endpoint marker for cardiovascular disease (7).
  • 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 (16).
  • To exert an antihypertensive effect in vivo peptides must survive gastrointestinal transit and reach the cardiovascular system intact and active. To evaluate whether the L.
  • Pepsin and Corolase PP an enzyme from porcine pancreatic glands that, contains trypsin, chymotrypsin and other amino- and carboxylpeptidases were used to mimic the enzymatic cleavage pattern of ACE-I- inhibitory peptides in the gastrointestinal tract.
  • Digestion of the hydrolysate with pepsin and subsequent analysis of ACE-inhibitory activity revealed that the pepsin treated hydrolysate maintained a similar ACE-inhibitory activity as the unhydrolyed L. animalis DPC6134 hydrolysate (85.51 % and 84.96 %).
  • the RP-HPLC chromatogram of the pepsin digested hydrolysate is depicted in Figure 6A.
  • the separation by RP-HPLC is based on molecular weight and hydrophobicity, with larger, more hydrophobic molecules eluting at later times.
  • the RP-HPLC chromatograms of the undigested hydrolysate (Fig. 1), the pepsin digest (Fig. 6A) and the Corolase PP digests at times 120 min (Fig. 6 B) and 240 min Fig 6 C) are compared, very minor transitions occur for some peaks eluting after 20 min in the non-hydrolysed hydrolysate, to more peaks eluting earlier on the solvent gradient as digestion with Pepsin and Corolase PP progressed.
  • the non-hydrolysed L Compared to the non-hydrolysed L.
  • ACE-inhibitory hydrolysates and peptides should be resistant to inactivation with gastrointestinal enzymes.
  • the L animalis DPC6134 hydrolysate did not lose its ACE-I-inhibitory activity in a simulated gastrointestinal digestion and therefore offers the possibility of use as an additional treatment to commercially available ACE-inhibitory drugs such as Captopril in the treatment of hypertension.
  • lactobacilli strains were identified as Lactobacillus casei/paracasei DPC 6109, L. johnsonii DPC 6092, L. reuteri DPC 6215 and L. animalis DPC 6134 (NCIMB deposit 41355).
  • Bovine sodium caseinate was hydrolysed in four separate small-scale fermentations performed in triplicate, by the four GRAS lactobacilli strains, in order to release ACE-inhibitory peptides from the latent casein proteins. Only Lactobacillus animalis DPC 6134 was chosen for further large-scale fermentation.
  • Reverse-Phase-High-Performance (RP-HPLC) was used to separate peptides ⁇ 10KDa into 72 fractions, and the ACE-inhibitory activity of each peptidic fraction analysed using Capillary Electrophoresis and Spectrophotometric methods.
  • the 10 kDa hydrolysate prior to RP-HPLC separation had an ACE-inhibitory percentage value of 85.51 % (+/- 15.62) and an IC 50 value (the 50 % inhibitory concentration) of 0.8 mg protein / ml.
  • Fraction 3 contained a peptide with molecular mass of 1793 which produced a 10 residue sequence LVYPFPGPIH corresponding to bovine beta-casein B f (11-20). The correct matched the sequence NSLPQN corresponding to beta casein B f (22-26). The same sequence was found in the other two fractions (2 and 3). Subfraction 3 from fraction 63 displayed a mass of 1254 with the sequence LVYPFPGPIHN corresponding to bovine beta casein B f (11-21) identified by MALDI-TOF analysis. Fractions 5, 6 and 7 produced no meaningful sequence information.
  • ACE-I-inhibitory peptides identified in this study share amino acid sequence homologies with previously identified bioactive peptides, however the complete sequences have not been identified previously.
  • Peptides LVYPFPGPIHNS predictive IC 50 value 116.77 ⁇ M
  • LVYPFPGPIHNSLPQN predictive IC 50 value 65.0 ⁇ M
  • LVYPFPGPIHN predictive IC 50 value 16.1 ⁇ M
  • johnsonii DPC6092 share homology with the ACE-I-inhibitory peptide LVYPFPGPIPNSLPQNIPP corresponding to bovine Beta- casein f (58-76) generated through hydrolysis of sodium caseinate with trypsin (49).
  • the IC50 values calculated for this peptide was 19.00 ⁇ M (29).
  • the peptides identified within fraction 63 share homologies with the chemically synthesised ACE- inhibitor LVYP with a calculated IC 50 of 170.00 ⁇ M.
  • ACE-inhibitory peptides which have been found to have an antihypertensive effect in spontaneously hypertensive rats have IC50 values within the range 5- 150.00 ⁇ M (19) and although these peptides have higher IC 50 values compared to the ACE-I-inhibitory peptides Ue-Pro-Pro (IC 50 5 ⁇ M) and Val-Pro-Pro (IC 50 9 ⁇ M), they compare favourably with other food derived ACE-I- inhibitors identified in various fermented milks and cheeses such as AVPYPQR, generated using 'Ropy milk starters' and corresponding to ⁇ -casein f (177-183) with a corresponding ICso value of 274.0OuM (3).
  • AVPYPQR generated using 'Ropy milk starters' and corresponding to ⁇ -casein f (177-183) with a corresponding ICso value of 274.0OuM (3).
  • Proteolysis under gastrointestinal conditions was also simulated for the 10 kDa L. animalis DPC6134 hydrolysate to assess whether gastrointestinal enzymes may further activate and therefore increase, or deactivate the ACE-inhibitory activity of the hydrolysate.
  • Corolase PP catalysed Digestion pH7-8 (240 min) 45.7 (+/- 1.6)
  • Captopril (0.005 mg/ml) 94.2 (+/-4.5)

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Abstract

La présente invention concerne des inhibiteurs de l'enzyme qui convertit l'angiotensine-I et des méthodes pour produire de tels inhibiteurs comprenant la culture d'une souche de Lactobacillus en présence de protéines de lait. L'invention concerne en outre les souches utiles pour la production de ces inhibiteurs. L'invention concerne aussi des nourritures fonctionnelles qui comprennent de tels inhibiteurs.
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WO2009130349A1 (fr) * 2008-04-22 2009-10-29 Corporación Alimentaria Peñasanta (Capsa) Obtention d'une nouvelle souche de bifidobacterium bifidum active contre l'infection par helicobacter pylori
WO2009155711A1 (fr) * 2008-06-27 2009-12-30 University Of Guelph Analyse des molécules d'interférence « signal » la-5 de lactobacillus acidophilus
US20140154217A1 (en) * 2011-09-29 2014-06-05 Belinda Vallejo Galland Lactococcus lactis strains, and bacterial preparations thereof, for the production of bioactive peptides having anti-hypertensive and cholesterol-lowering effects in mammals; nutritional and therapeutic products produced therefrom
US10716817B2 (en) 2013-08-12 2020-07-21 University Of Guelph Antiviral methods and compositions comprising probiotic bacterial molecules
FR3093923A1 (fr) * 2020-05-04 2020-09-25 Vf Bioscience Nouvelles souches de bacteries lactiques favorisant l’absorption du calcium – peptides et produits associes
CN114847347A (zh) * 2022-06-09 2022-08-05 陕西科技大学 一种含活性益生菌的发酵牛乳及其制备方法
CN114903083A (zh) * 2022-06-09 2022-08-16 陕西科技大学 一种生物活性肽益生菌发酵绵羊乳及其制备方法

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EP2036922A1 (fr) * 2007-09-17 2009-03-18 Dayeh University Nouveau peptide anti-hypertensif et son utilisation
CN101508722B (zh) * 2007-09-17 2012-07-25 大叶大学 新颖抗高血压肽及其用途
WO2009130349A1 (fr) * 2008-04-22 2009-10-29 Corporación Alimentaria Peñasanta (Capsa) Obtention d'une nouvelle souche de bifidobacterium bifidum active contre l'infection par helicobacter pylori
EP2270133A1 (fr) * 2008-04-22 2011-01-05 Corporación Alimentaria Peñasanta (Capsa) Obtention d'une nouvelle souche de bifidobacterium bifidum active contre l'infection par helicobacter pylori
EP2270133A4 (fr) * 2008-04-22 2012-11-07 Corporacion Alimentaria Penasanta Capsa Obtention d'une nouvelle souche de bifidobacterium bifidum active contre l'infection par helicobacter pylori
WO2009155711A1 (fr) * 2008-06-27 2009-12-30 University Of Guelph Analyse des molécules d'interférence « signal » la-5 de lactobacillus acidophilus
US10687543B2 (en) 2008-06-27 2020-06-23 University Of Guelph Probiotic bacterial molecules and their use in methods to treat/prevent infection by harmful bacteria and to provide nutritional health
US9533016B2 (en) 2011-09-29 2017-01-03 Centro De Investiagción En Alimentación Y Desarrollo, A.C. (CIAD) Lactococcus lactis strains
US20140154217A1 (en) * 2011-09-29 2014-06-05 Belinda Vallejo Galland Lactococcus lactis strains, and bacterial preparations thereof, for the production of bioactive peptides having anti-hypertensive and cholesterol-lowering effects in mammals; nutritional and therapeutic products produced therefrom
US9533015B2 (en) 2011-09-29 2017-01-03 Centro De Investigación En Alimentación Y Desarrollo, A.C. (CIAD) Lactococcus lactis strains
US8865155B2 (en) * 2011-09-29 2014-10-21 Centro De Investigacion En Alimentacion Y Desarrollo, A.C. (Ciad) Lactococcus lactis strains, and bacterial preparations thereof, for the production of bioactive peptides having anti-hypertensive and cholesterol-lowering effects in mammals; nutritional and therapeutic products produced therefrom
US9717773B2 (en) 2011-09-29 2017-08-01 Centro De Investigacion En Alimentacion Y Desarrollo, A.C. (Ciad) Lactococcus lactis strains for producing bioactive peptides having anti-hypertensive and cholesterol-lowering effects
US10092619B2 (en) 2011-09-29 2018-10-09 Centro De Investigación En Alimentacion Y Desarrollo, A.C. (CIAD) Composition comprising peptides made by Lactococcus lactis strains
US10092618B2 (en) 2011-09-29 2018-10-09 Centro De Investigación En Alimentacion Y Desarrollo, A.C. (CIAD) Composition comprising peptides made by lactococcus lactis strains
US9295701B2 (en) 2011-09-29 2016-03-29 Centro De Investigacion En Alimentacion Y Desarrollo, A.C. (Ciad) Lactococcus lactis strains for the production of bioactive peptides having anti-hypertensive and cholesterol-lowering effects
US10716817B2 (en) 2013-08-12 2020-07-21 University Of Guelph Antiviral methods and compositions comprising probiotic bacterial molecules
US11857581B2 (en) 2013-08-12 2024-01-02 Microsintesis Inc. Antiviral methods and compositions comprising probiotic bacterial molecules
FR3093923A1 (fr) * 2020-05-04 2020-09-25 Vf Bioscience Nouvelles souches de bacteries lactiques favorisant l’absorption du calcium – peptides et produits associes
CN114847347A (zh) * 2022-06-09 2022-08-05 陕西科技大学 一种含活性益生菌的发酵牛乳及其制备方法
CN114903083A (zh) * 2022-06-09 2022-08-16 陕西科技大学 一种生物活性肽益生菌发酵绵羊乳及其制备方法
CN114903083B (zh) * 2022-06-09 2023-10-20 西安百跃羊乳集团有限公司 一种生物活性肽益生菌发酵绵羊乳及其制备方法
CN114847347B (zh) * 2022-06-09 2023-11-03 陕西科技大学 一种含活性益生菌的发酵牛乳及其制备方法

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