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WO2006125847A1 - Obtention de peptides derives de proteines de blanc d'oeuf et proprietes antihypertension de ces derniers - Google Patents

Obtention de peptides derives de proteines de blanc d'oeuf et proprietes antihypertension de ces derniers Download PDF

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Publication number
WO2006125847A1
WO2006125847A1 PCT/ES2006/070067 ES2006070067W WO2006125847A1 WO 2006125847 A1 WO2006125847 A1 WO 2006125847A1 ES 2006070067 W ES2006070067 W ES 2006070067W WO 2006125847 A1 WO2006125847 A1 WO 2006125847A1
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seq
peptides
bioactive
egg white
products
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Spanish (es)
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Marta Miguel Castro
Rosina LÓPEZ-ALONSO FANDIÑO
Mercedes Ramos González
Amaya Aleixandre de Artiñano
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Consejo Superior De Investigaciones Científicas
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/04—Animal proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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
    • C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
    • 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
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention consists in the production of peptides with antihypertensive activity, derived from egg white proteins, which can be applied in the food and pharmaceutical industry.
  • bioactive peptides can be generated in vivo, during the hydrolysis of proteins by the action of gastrointestinal enzymes, or in vitro, by the action of specific enzymes or during the processes of making certain foods. Due to their diversity and multifunctionality, bioactive peptides can be used as ingredients in functional foods to produce different beneficial effects for health, since they exert different biological activities, such as antihypertensive, antithrombotic, opioid, antioxidant, immunomodulant etc. [Gobbetti, M., Stepaniak, L., de Angelis, M., Corsetti, A., di Cagno, R. Latent bioactive peptides in milk proteins: proteolytic activation and significance in dairy processing. Crit. Rev. Food Sci. Nutr. 2002, 42: 223-239].
  • Blood pressure is defined as the force exerted by the blood against the arterial wall and, arterial hypertension can be defined as a chronic elevation of systolic blood pressure (SBP) and / or diastolic blood pressure (PAD) above normal values
  • SBP systolic blood pressure
  • PAD diastolic blood pressure
  • Captopril drugs with antihypertensive activity, such as Captopril, which act by inhibiting the angiotensin converting enzyme (ACE).
  • ACE angiotensin converting enzyme
  • ECA catalyzes the formation of angiotensin II, an octapeptide with a potent vasoconstrictor activity and, in addition, inactivates bradykinin, which produces vasodilation.
  • ACEI ACE inhibitory activity
  • ACE inhibition is not the only mechanism that can justify the antihypertensive action of peptides derived from food proteins.
  • Essential hypertension is associated with exaggerated lipoperoxidation, and with an imbalance in the antioxidant status that leads to excess formation of reactive oxygen species [Touyz, RM, Pu, Q., He, G., Chen, X., Yao, G., Neves, MF, Dahl, E. Effects of a low dietary magnesium intake on development of hypertension in stroke-prone spontaneously hypertensive rats: role of reactive oxygen species. J. Hypertens. 2002, 20: 2221-2232]. Some of these peptides have antioxidant effect. Some may also exert a direct vasodilator action.
  • JP5331190 showed relaxing activity in canine mesenteric arteries and antihypertensive activity when administered orally to SHR rats at a dose of 100 mg / kg.
  • Matoba et al. [Matoba, N., Usui, H., Fujita, H., Yoshikawa, M.
  • a novel anti-hypertensive peptide derived from ovalbumin induces nitric oxide-mediated vasorelaxation in an isolated SHR mesenteric artery. FEBS Lett.
  • ovokinin analogs (2-7) were synthesized to increase their antihypertensive activity.
  • RPFHPF and RPLKPW were synthesized to increase their antihypertensive activity.
  • RPFHPF and RPLKPW were synthesized to increase their antihypertensive activity.
  • RPFHPF and RPLKPW showed 10 and 100 times more activity than ovokinin (2-7) after oral administration to SHR (minimum effective doses of 1 and 0.1 mg / kg), which was attributed to a higher Resistance to digestive tract proteases [Matoba, N., Yamada, Y., Usui, H., Nakagiri, R., Yoshikawa, M. Designing potent derivatives of ovokinin (2-7), an anti-hypertensive peptide derived from Ovalbumin, Biosci.
  • the present invention consists in the production of bioactive peptides with antihypertensive activity by hydrolysis of egg proteins with different enzymes. These peptides would be the minimum functional peptide units that, after gastrointestinal digestion, would be able to be gastrointestinally assimilable and pass into the bloodstream. This property opens the application of these peptides to forms of administration other than oral or increases their absorption rate.
  • Bioactive peptides are produced by the hydrolysis of one or more peptides or proteins containing the amino acid sequence of said bioactive peptides (preferably ovalbumin or ovalbumin fragments), using enzymes (preferably pepsin and Corolase PP ® ) and hydrolysis conditions that allow the breakdown of the amino acid chain in the right places for release.
  • peptides can also be obtained by chemical or enzymatic synthesis or by recombinant methods etc. Said peptides can be consumed as such, or from the hydrolysates containing them, of low molecular weight concentrates or other active subfractions obtained by size separation methods or chromatographic methods. In addition to being part of food products, such peptides and hydrolysates or their fractions could also be part of pharmaceutical products. Thus, they could be used for the treatment and prevention of some diseases; in particular they could facilitate blood pressure control.
  • the invention extends the applications of egg proteins, contributing to their utilization and revaluation.
  • the invention provides a method for producing the bioactive peptides identified with the amino acid sequences shown in SEQ. ID. No. 1 and SEQ. ID. No. 2 (Table 1), which possess antihypertensive activity. Due to its structure and resistance to gastrointestinal enzymes, SEQ. ID. No. 1 and SEQ. ID. No. 2 would be the minimum functional peptide units that after gastrointestinal digestion they would be able to be gastrointestinally assimilable and pass into the bloodstream.
  • the starting material of the present invention would be any suitable substrate comprising one or more peptides or proteins, of animal, plant origin or originating from microorganisms, containing the amino acid sequence of the bioactive peptides of interest (SEQ. ID. No. 1 and SEQ ID No. 2, Table 1), preferably the YAEERYPIL SEQ ID No. 6, FRADHPFL SEQ ID No. 5, RADHPFL SEQ ID No. 3, RADHPF SEQ ID No. 4, ovalbumin or clear peptides of egg Since they all belong to the ovalbumin sequence, it is obvious that any preparation containing ovalbumin, or ovalbumin peptides or fragments of any size, alone or mixed with other proteins could be used.
  • Said starting material is dissolved or dispersed, at an appropriate concentration, in water or in a buffer solution, at a pH suitable for the action of the proteolytic enzyme.
  • Any proteolytic enzyme capable of breaking the protein present in the starting material and providing the peptides of interest can be used, but preferably pepsin at pH 2.0-3.0, and then PP ® chorolase at pH 7.0-8.0.
  • Proteolytic microorganisms that carry out fermentation of the substrate could also be used.
  • the hydrolysis conditions pH, temperature, pressure, enzyme-substrate ratio, reaction interruption etc., are optimized in order to select the most active hydrolysates.
  • the bioactive peptides are obtained using egg white as substrate and pepsin at pH 2.0, in an enzyme / substrate ratio 1 / 100-1 / 50, p / p and performing hydrolysis at 37 ° C, over a period of time between 10 min and 24 hours but, preferably, for an approximate time of 3 hours.
  • the crude hydrolyzate can then be used or the low molecular weight fraction concentrated by methods such as ultrafiltration, dialysis, electrodialysis with membranes of suitable pore size, gel filtration chromatography, etc.
  • the fraction of molecular weight less than 3000 of the hydrolysates is obtained by ultrafiltration through a suitable hydrophilic pore membrane.
  • the egg white hydrolyzate with pepsin and its fraction less than 3000 Da have ACEI, antioxidant and antihypertensive properties that, as demonstrated above, are due to the presence of bioactive peptides [Miguel, M., López-Fandi ⁇ o, R., Hard. L, Ramos, M., López -Fandi ⁇ o, R., Aleixandre, A. 2003. Bioactive peptides derived from egg white proteins by enzymatic hydrolysis. Spanish patent application 200301829].
  • the antihypertensive effect could be accelerated by subjecting them to a second hydrolysis to produce smaller peptides, likely to be directly absorbed through the gastrointestinal mucosa, which would be ultimately responsible for their properties.
  • this is achieved by hydrolysis of the egg white hydrolyzate with pepsin, the fraction thereof less than 3000 Da, or the synthetic peptides it contains (YAEERYPIL SEQ ID No. 6, FRADHPFL SEQ ID No. 5 , RADHPFL SEQ ID N ° 3), with PP ® chorolase, at pH 7-8, in an enzyme / substrate ratio 1: 25 p / pa at 37 ° C, for a time of approximately 2.5 hours.
  • PP ® Chorolase is a proteolytic enzyme preparation of pig pancreas that contains, in addition to trypsin and chymotrypsin, amino and carboxypeptidases.
  • the identified bioactive peptides can be obtained by chemical and / or enzymatic synthesis of peptides, or by recombinant methods.
  • Egg white is a cheap and affordable protein substrate to produce bioactive peptides.
  • this process allows obtaining bioactive peptides using enzymatic preparations and conditions that mimic gastrointestinal digestion. Therefore, it is likely that the fragments obtained are the final products of hydrolysis, susceptible of being absorbed in the gastrointestinal tract, and of being directly responsible for the antihypertensive action. Do not however, further hydrolysis by plasma peptidases can be ruled out.
  • Obtaining small active fragments is advantageous because these fragments would be easier to administer by different routes, and when administered orally they would produce the effect before. It should be noted that these are natural peptides, present in frequently used foods, from which few side effects and good tolerance can be expected. Its small size, on the other hand, lowers the cost of obtaining it by chemical or enzymatic methods.
  • hydrolyzate or one or more of its constituent bioactive peptides (including its derivatives, pharmaceutically acceptable salts and mixtures thereof), could be used as therapeutic substances with antihypertensive activity.
  • Such products can be subjected to a heat treatment, such as pasteurization, or undergo drying or lyophilization etc., to be used as functional food products, additives or food ingredients, or pharmaceutical products, for the treatment and / or prevention of hypertension.
  • a heat treatment such as pasteurization, or undergo drying or lyophilization etc.
  • the amount of hydrolyzate, peptides, their derivatives or pharmaceutically acceptable salts and mixtures thereof, which would be useful for a pathology will vary depending on numerous factors, such as age, severity thereof, route of administration and dose frequency.
  • These compounds could be presented in any form of administration, solid or liquid, or encapsulated, and could be administered, in principle, by different routes (oral, respiratory, rectal, topical, etc.) although they are particularly designed for solid or liquid administration via route. Oral and topical.
  • the process of obtaining these products can be optimized, directing it to the production of the greatest possible amount of bioactive peptides, or controlling, if possible, the appearance of bitterness normally caused by a high concentration of hydrophobic peptides of intermediate or low molecular weight.
  • Esquire-LC equipment (Bruker Daltonik GMBH, Bremen, Germany) is used.
  • the HPLC equipment (1100 series) consists of a quaternary pump, an injector automatic, an eluent degassing system and a variable wavelength ultraviolet detector (Agilent Technologies, Waldbronn, Germany) coupled in-line to an Esquire 3000 ion trap mass spectrometer (Bruker Daltonik).
  • the column is a Hi-Pore C 18 column (250 x 4.6 mm ID, 5 ⁇ m particle size) (Bio-Rad Laboratories, Richmond, CA, USA).
  • Solvent A is a mixture of water and trifluoroacetic acid (1000: 0.37), and solvent B a mixture of acetonitrile and trifluoroacetic acid (1000: 0.27).
  • 50 ⁇ l of synthetic peptides are injected before and after treatment with PP ® pepsin and chorolase, 25 ⁇ l of egg white hydrolyzate with pepsin before and after treatment with PP ® chorolase, and 50 ⁇ l of the fraction less than 3000 Da before and after treatment with PP ® chorolase.
  • a flow of 0.8 ml / minute is used, with a linear gradient of solvent B in A from 2% to 10% in 15 minutes, from 10 to 20% in 35 minutes and from 20 to 30% in 20 minutes.
  • the eluent is monitored at 214 nm, and then the flow is divided by placing a T-piece (Valco, Houston, TX, USA), connected to a 75 ⁇ m internal diameter tabopeek, the length of which is adjusted to provide an injection flow from the sample to the mass spectrometer, through the electrospray nebulizer, of 20 ⁇ l / minute.
  • the equipment uses nitrogen as a nebulizer and drying gas, and operates with a helium pressure of 5 x 10 ⁇ 3 bar.
  • Mass spectra are acquired in a range of 100-1500 m / z and at a speed of 13000 Da / second. The interpretation of the MS spectra in tandem for the identification of the peptide sequences is carried out with the Biotools 2.1 program (Bruker Daltonik GmbH, Bremen, Germany).
  • Angiotensin-converting enzyme (ACEI) inhibitory activity is measured in vitro according to the method of Cushman and Cheung [Cushman, DW and Cheung, HS Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem Pharmacol 1971, 20: 1637-1648], subsequently modified by Kim et al. [Kim, Y. K., Yoon, S., Yu., D. Y., Lónnerdal, B., Chung, B. H. Novel angiotensin-I-converting enzyme inhibitory peptides derived from recombinant human ⁇ sl-casein expressed in Escherichia coli. J. Dairy Res. 1999, 66: 431-439].
  • the histidyl leucine hippyl substrate (HHL, Sigma, Chemicals Co, St. Louis, MO, USA) is dissolved in 0.1 M borate buffer with 0.3 M NaCl, pH 8.3, to obtain a 5 mM final concentration.
  • To 100 ⁇ l of substrate are added 40 ⁇ l of each of the samples whose IECA activity is to be determined. ECA is added (EC 3.4.15.1,
  • the reaction is carried out at 37 ° C, for 30 minutes in a water bath.
  • the enzyme is inactivated by lowering the pH with 150 ⁇ l of HCl IN.
  • the formed hippuric acid is extracted with 1000 ⁇ l of ethyl acetate. After vortexing for 20 seconds, it is centrifuged at 4000 rpm for 10 minutes and at room temperature. 750 ⁇ l of the organic phase is evaporated by heating at 95 ° C for 10 minutes.
  • the hippuric acid residue is redissolved in 800 ⁇ l of double distilled water and, after stirring for 20 seconds, the absorbance at 228 nm is measured on a Dur-70 spectrophotometer from Beckman Instruments, Inc., Fullerton, USA.
  • White is used to correct background absorbance. This contains substrate, enzyme and 20 ⁇ l of double distilled water instead of sample, and the reaction is stopped at zero time.
  • the control assumes one hundred percent of the enzymatic action on the substrate in the absence of inhibitors, and contains 20 ⁇ l of water instead of the sample and is incubated at the same time as the sample.
  • the results are presented as the IC 50 or protein concentration ( ⁇ g / ml) that inhibits 50% of the enzyme activity.
  • the protein concentration is determined by the bicinconinic acid (BCA) assay (Pierce, Rockford, IL, USA) using bovine serum albumin as the standard.
  • SHR spontaneously hypertensive rats
  • a tail cuff device To perform systolic blood pressure (PAS) measurements, a tail cuff device is used [Bu ⁇ ag, RD Validation in awake rats of a tail-cuff meted for measuring systolic pressure. J. Appl Physiol. 1973, 34: 279-282] (Le5001, Letica) that provides a digital value of the PAS automatically, and records and facilitates the heart rate of the animals Before placing the cuff and the transducer on the tail of the animals.
  • PAS systolic blood pressure
  • PAS measurements are taken in animals , before administration, and then periodically, every 2 hours, up to 8 hours post-administration. Additionally, a measure of PAS is taken 24 hours after administration.
  • a negative control to establish the circadian variation of the SBP in probed rats
  • the SBP measurements obtained in rats that are administered by intragastric tube 1 ml of water are used.
  • the SBP measurements obtained in rats that are administered by intragastric tube 50 mg / kg of Captopril (prototype IECA drug) are used.
  • the amount of drug administered is always dissolved in 1 ml of distilled water.
  • Figure 1 is a chromatogram obtained using reverse phase high efficiency liquid chromatography (RP-HPLC) of the synthetic peptide YAEERYPIL SEQ ID NO: 1
  • Figure 2 is a chromatogram obtained using reverse phase high efficiency liquid chromatography (RP-HPLC) of the synthetic peptide RADHPFL SEQ ID No.
  • Figure 3 is a chromatogram obtained using reverse phase high efficiency liquid chromatography (RP-HPLC) of the synthetic peptide RADHPF SEQ ID No. 4, before and after sequential hydrolysis with pepsin and PP ® chorolase.
  • the absorbance at 214 nm is represented on the ordinate axis and the time in minutes is represented on the abscissa axis.
  • Figure 4 is a chromatogram obtained using reverse phase high efficiency liquid chromatography (RP-HPLC) of the synthetic peptide RADHP SEQ ID No. 2, before and after sequential hydrolysis with pepsin and PP ® chorolase.
  • the absorbance at 214 nm is represented on the ordinate axis and the time in minutes is represented on the abscissa axis.
  • Figure 5 is a chromatogram obtained using reverse phase high efficiency liquid chromatography (RP-HPLC) of the synthetic peptide FRADHPFL SEQ ID No. 5, before and after sequential hydrolysis with pepsin and PP ® chorolase. On the axis of The absorbance at 214 nm is plotted and the time in minutes is represented on the abscissa axis.
  • RP-HPLC reverse phase high efficiency liquid chromatography
  • Figure 6 represents the decrease in systolic blood pressure (SBP), obtained in spontaneously hypertensive rats, after administration by intragastric tube of 1 ml of water (o), 50 mg / kg of Captopril (D), 2 mg / kg of the peptide
  • SBP systolic blood pressure
  • T (h) represents the time elapsed since the administration expressed in hours.
  • Data represent the mean ⁇ ESM for a minimum of 5 animals. at P ⁇ 0.05 vs water; b P ⁇ 0.05 vs Captopril; ° P ⁇ 0.05 vs RADHP
  • Figure 7 represents the decrease in systolic blood pressure (SBP), obtained in spontaneously hypertensive rats, after administration by intragastric tube of 1 ml of water (o), 50 mg / kg of Captopril (D), 100 mg / kg of the fraction less than 3000 Da of the egg white hydrolyzate with pepsin (A), and 100 mg / kg of the fraction less than 3000 Da of the egg white hydrolyzate with pepsin treated with PP ® chorolase (4).
  • Data represent the mean ⁇ ESM for a minimum of 5 animals. at P ⁇ 0.05 vs water; b P ⁇ 0.05 vs Captopril; ° P ⁇ 0.05 vs the fraction less than 3000 Da of the egg white hydrolyzate with pepsin.
  • Figure 8 depicts the chromatograms obtained using reverse phase high efficiency liquid chromatography (RP-HPLC) of the egg white hydrolyzate with pepsin before and after treating with PP ® chorolase and the fraction less than 3000 Da of the hydrolyzate of Egg white with pepsin before and after treating with PP ® chorolase.
  • RP-HPLC reverse phase high efficiency liquid chromatography
  • the peaks corresponding to the identified peptide sequences are indicated, which are listed in Table 3.
  • the absorbance at 214 nm is represented on the ordinate axis and the time in minutes is represented on the abscissa axis.
  • Example 1 ACEI and antihipcrtcnsive activity of the peptides obtained after simulating the gastrointestinal digestion of the fragments of the ovalbumin YAEERYPIL SEQ ID No. 6 and RADHPFL SEQ ID No. 3
  • the YAEERYPIL SEQ ID No. 6 and RADHPFL SEQ ID No. 3 peptides which had previously been identified in the fraction less than 3,000 Da of an egg white hydrolyzate with pepsin and which possessed in vitro ACEI activity and antihypertensive activity in SHR rats [Miguel, M., López-Fandi ⁇ o, R., Recio. I., Ramos, M., López-Fandi ⁇ o, R., Aleixandre, A. 2003. Bioactive peptides derived from egg white proteins by enzymatic hydrolysis.
  • Spanish Application 200301829 are chemically synthesized by the solid phase Fmoc method with a 43 IA model synthesizer from Applied Biosystems Inc. (Überlingen, Germany). The purity of the synthetic peptides is verified by RP-HPLC-MS / MS. They then undergo a two-stage hydrolysis process that simulates gastrointestinal digestion [Alting, AC, Meijer, RJGM, Van Beresteijn, ECH Incomplete elimination of the ABBOS epitope of bovine serum albumin under simulated gastrointestinal conditions of infants. Diabetes Care, 1997, 20: 875-880].
  • aqueous solutions of the synthetic peptides (2 mg / ml) are hydrolyzed, first with pepsin (EC 3.4.4.1; 1: 60,000, 3,400 U / mg) (Sigma) (enzyme: substrate ratio, 1:50, p / p) at pH 2.0 and 37 ° C for 90 minutes and subsequently with PP ® chorolase (enzyme ratio: substrate 1:25, p / p) (Rohm, Darmstadt, Germany) at pH 7-8 and 37 ° C, for 2.5 hours. The reaction is interrupted by heating at 95 ° C for 10 minutes in a water bath.
  • pepsin EC 3.4.4.1; 1: 60,000, 3,400 U / mg
  • PP ® chorolase enzyme ratio: substrate 1:25, p / p
  • the YAEERYPIL SEQ ID No. 6 peptide is fully hydrolyzed after incubation with pepsin and the pancreatic extract.
  • the main resulting fragments, identified by tandem mass spectrometry, are the YAEER SEQ ID No. 8 pentapeptide and the YPI SEQ ID No. 0 tripeptide.
  • a small proportion of YAEERYPI SEQ ID No. 7 is also found, suggesting that it is an intermediate fragment after the hydrolysis of the C-terminal amino acid.
  • the ACEI activity of YAEERYPIL SEQ ID No. 6 decreases approximately 100 times after the simulation of digestion (Table 2).
  • none of their degradation products YAEER SEQ ID No. 8 and YPI SEQ ID No.
  • RADHPFL SEQ ID No. 3 The second sequence studied, RADHPFL SEQ ID No. 3, is hydrolyzed to give RADHPF SEQ ID No. 4 and RADHP SEQ ID No. 2 ( Figure 2). Both fragments can be chemically synthesized and, in turn, subjected to sequential hydrolysis with pepsin and PP ® chorolase. RADHPF SEQ ID No. 4 partially resists hydrolysis with pepsin and pancreatic extract, but also gives RADHP SEQ ID No. 2 as the main degradation product ( Figure 3). The RADHP SEQ ID No. 2 peptide, in turn, is not susceptible to the action of gastrointestinal enzymes, which suggests that it is the end product of proteolysis ( Figure 4).
  • RADHPFL SEQ ID N ° 3 decreases considerably after digestion.
  • the determination of the ACEI activity of its proteolysis products: RADHP SEQ ID No. 2 and RADHPF SEQ ID No. 4 shows that they are only weak inhibitors (Table T).
  • the sequence RADHPF SEQ ID No. 4 had already been described by Matoba et al. [Matoba, N., Usui, H., Fujita, H., Yoshikawa, M. A novel anti-hypertensive peptide derived from ovalbumin induces nitric oxide-mediated vasorelaxation in an isolated SHR mesenteric artery.
  • Ovokinin (2-7) had antihypertensive properties in SHR rats [Matoba, N., Yamada, Y., Usui, H., Nakagiri, R., Yoshikawa, M. Designing potent derivatives of ovokinin (2-7), an anti-hypertensive peptide derived from ovalbumin, Biosci.
  • RADHPFL SEQ ID No. 3 sequence is very similar to the ovokinin sequence (FRADHPFL SEQ ID No. 5), a vascular relaxing octapeptide and ACE inhibitor [Yoshikawa, M., Hasegawa, M., Fujita, H. 1993. New peptide and its production.
  • FRADHPF SEQ ID N ° 9 and ADHPF SEQ ID N ° 10 also appeared to a lesser extent. This would indicate that FRADHPFL SEQ ID No. 5, RADHPFL SEQ ID No. 3 and RADHPF SEQ ID No. 4 peptides would be digested in the tract gastrointestinal giving as final product RADHP SEQ ID N ° 2, which has a moderate ACEI activity (Table 2).
  • YAEERYPIL SEQ ID No. 6 the YAEER SEQ ID No. 8 peptide and SEQ. ID No. 1, YPI
  • FRADHPFL SEQ ID No. 5 RADHPFL SEQ ID No. 3 and RADHPF SEQ ID No. 4 (SEQ. ID. No. 2, RADHP).
  • SHR rats the sequential hydrolysis of YAEERYPIL SEQ ID No. 6
  • FRADHPFL SEQ ID No. 5 the RADHPFL SEQ ID No. 3
  • RADHPF SEQ ID No. 4 SEQ. ID. No. 2, RADHP
  • Figure 6 shows the decreases in SBP obtained in SHR rats at different times, after administration of the YAEER SEQ ID N ° 8, SEQ peptides. ID. No. 1 and SEQ. ID. No. 2. It can be seen that the administration of SEQ peptides. ID. No. 1 and SEQ. ID. No. 2 causes a significant decrease in SBP in these animals.
  • the sequence YAEER SEQ ID N ° 8 causes a slight and sustained decrease during 8 hours, but without showing significant differences with the water.
  • the decrease in SBP originating from SEQ. ID. No. 1 and SEQ. ID. No. 2 is maximum at 2-4 hours after administration of these peptides, and the initial value of this variable is recovered at 24 hours.
  • SEQ. ID. No. 2 cause effects similar to Captopril at 2 hours, which is a compound with potent antihypertensive activity.
  • YAEERYPIL SEQ ID No. 6 and RADHPFL SEQ ID No. 3 the maximum reductions in SBP (-31.6 ⁇ 2.6 and -34.0 ⁇ 1.6 mm Hg, respectively) had been achieved 6 hours after oral administration of a dose of 2 mg / ml [Miguel, M., López-Fandi ⁇ o, R., Recio. L, Ramos, M., López-Fandi ⁇ o, R., Aleixandre, A. 2003.
  • the hydrolyzate is obtained using as a clear substrate of commercial pasteurized chicken egg.
  • pepsin EC 3.4.23.1. Type A, 10000 U / mg protein
  • the pH is adjusted to 2.0 by adding HCl IN and pepsin is added (enzyme / substrate ratio 1/100, w / w).
  • Hydrolysis is carried out at a temperature of 37 ° C for 3 hours.
  • Pepsin inactivation is achieved by raising the pH to 7.0 with NaOH IN. It is then treated with PP ® chorolase (Rohm, Darmstadt, Germany) (enzyme / substrate ratio 1/25, w / w) at 37 ° C for 2.5 hours.
  • the inactivation of the PP ® chorolase is carried out by heating at 95 ° C for 10 min and subsequently cooling to room temperature.
  • the measurement of ACEI activity before and after hydrolyzing with PP ® chorolase shows that the IC 50 value of egg white hydrolyzate with pepsin for 3 hours slightly increases from 44.00 ⁇ 1.18 ⁇ g / ml to 67.98 ⁇ 2.08 ⁇ g / ml after PP ® chorolase treatment, although both values are indicative of an important inhibition.
  • the fraction less than 3000 Da of the egg white hydrolyzate with pepsin for 3 hours is obtained by ultrafiltration through a hydrophilic membrane of 3000 Da (Centriprep, Amicon, Inc., Beverly, MA, USA), centrifuging at 1900 g for 40 minutes.
  • the permeate (fraction less than 3000 Da) is treated with PP ® chorolase, acting in the same way as with the pepsin hydrolyzate from which it comes, described above.
  • the ACEI activity in the permeate was measured before and after hydrolyzing with PP ® chorolase.
  • the IC 50 values are, respectively, 20.48 ⁇ 0.75 ⁇ g / ml and 74.40 ⁇ 1.16 ⁇ g / ml mi.
  • Antihypertensive activity was tested in SHR rats of the fraction less than 3000 Da of the egg white hydrolyzate with pepsin, before and after hydrolyzing with PP ® chorolase. The same concentration of both products was administered to the animals (approximately 100 mg / kg).
  • Figure 7 shows the decrease in SBP obtained in SHR rats at different times after administration. The fraction less than 3000 Da of the egg white hydrolyzate with pepsin produces clear antihypertensive effects in these animals before and after their hydrolysis with PP ® chorolase.
  • the egg white hydrolyzate with pepsin and the fraction thereof of less than 3000 Da, before and after treatment with PP ® chorolase were analyzed using RP-HPLC -MS / MS as shown in Figure 8.
  • the peptides identified in each of the products are shown in Table 3. The sequences were identified by extracting the corresponding molecular ion and taking into account the retention time at which it elutes. the pure peptide.
  • the egg white hydrolyzate with pepsin and the fraction thereof less than 3000 Da contained the YAEERYPIL SEQ ID No. 6, FRADHPFL SEQ ID No. 5 and RADHPFL SEQ antihypertensive sequences. ID No. 3. It should be noted that after the hydrolysis of both products with PP ® chorolase, none of these sequences were found. However, sequences with SEQ antihypertensive properties are found. ID. No. 1 and SEQ. ID. No. 2, which are the subject of the present invention. Neither were the peptides found: YAEERYPI SEQ ID No. 7, YAEER SEQ ID No. 8, FRADHPF SEQ ID No.

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Abstract

L'invention concerne la production d'unités peptidiques minimales présentant une activité biologique en tant qu'agents antihypertenseurs à partir de blanc d'oeuf soumis à un traitement enzymatique. Ces ovoproduits : les hydrolysats complets, les fractions de ces derniers à faible masse moléculaire ou leurs peptides constitutifs, peuvent être utilisés en tant que substances thérapeutiques avec une activité antihypertension, soit en tant que produits alimentaires fonctionnels, additifs ou ingrédients alimentaires, soit en tant que produits pharmaceutiques, pour le traitement et/ou la prévention de l'hypertension artérielle dans toutes ses formes chez un être humain ou un animal et pour le traitement et/ou la prévention de tout trouble associé à l'hypertension artérielle chez un être humain ou un animal.
PCT/ES2006/070067 2005-05-23 2006-05-23 Obtention de peptides derives de proteines de blanc d'oeuf et proprietes antihypertension de ces derniers WO2006125847A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018201759A1 (fr) * 2017-05-02 2018-11-08 西北农林科技大学 Procédé efficace et doux pour réduire l'allergénicité des blancs d'œufs
CN109485696A (zh) * 2018-11-06 2019-03-19 渤海大学 一种克服肠道降解的膜肽酶抑制肽

Families Citing this family (4)

* Cited by examiner, † Cited by third party
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CN114365792A (zh) 2014-11-11 2022-04-19 克莱拉食品公司 用于生成卵清蛋白的方法和组合物
MX2022000374A (es) 2019-07-11 2022-03-25 Clara Foods Co Composiciones de proteina y productos consumibles de las mismas.
US12096784B2 (en) 2019-07-11 2024-09-24 Clara Foods Co. Protein compositions and consumable products thereof
US10927360B1 (en) 2019-08-07 2021-02-23 Clara Foods Co. Compositions comprising digestive enzymes

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005012355A1 (fr) * 2003-07-31 2005-02-10 Consejo Superior De Investigaciones Científicas Peptides bioactifs derives de proteines de blanc d'oeuf par hydrolyse enzymatique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005012355A1 (fr) * 2003-07-31 2005-02-10 Consejo Superior De Investigaciones Científicas Peptides bioactifs derives de proteines de blanc d'oeuf par hydrolyse enzymatique

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DAVALOS A. ET AL: "Antioxidant activity of peptides derived from egg white proteins by enzymatic hydrolysis", JOURNAL OF FOOD PROTECTION, vol. 67, no. 9, September 2004 (2004-09-01), pages 1939 - 1944, XP008073906 *
MATOBA N. ET AL.: "A novel anti-hypertensive peptide derived from ovalbumin induces nitric oxide-mediated vasorelaxation in an isolated SHR mesenteric artery", FEBS LETTERS, vol. 452, 1999, pages 181 - 184, XP004259746 *
MIGUEL M. ET AL.: "Effect of simulated gastrointestinal digestion on the antihypertensive properties of ACE-inhibitory peptides derived from ovalbumin", J. AGRIC. FOOD. CHEM., vol. 54, no. 3, 8 February 2006 (2006-02-08), pages 726 - 731, XP003004274 *
MIGUEL M. ET AL: "Angiotensin I-converting enzyme inhibitory activity of peptides derived from egg white proteins by enzymatic hydrolysis", JOURNAL OF FOOD PROTECTION, vol. 67, no. 9, September 2004 (2004-09-01), pages 1914 - 1920, XP008073905 *
RAHFELD J. ET AL.: "Are diprotin A (Ile-Pro-Ile) and diprotin B (Val-Pro-Leu) inhibitors or substrates of dipeptidyl peptidase IV?", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1076, 1991, pages 314 - 316, XP002975268 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018201759A1 (fr) * 2017-05-02 2018-11-08 西北农林科技大学 Procédé efficace et doux pour réduire l'allergénicité des blancs d'œufs
US10779555B2 (en) 2017-05-02 2020-09-22 Northwest A&F University Method for efficiently and mildly reducing ovalbumin allergenicity
CN109485696A (zh) * 2018-11-06 2019-03-19 渤海大学 一种克服肠道降解的膜肽酶抑制肽
CN109485696B (zh) * 2018-11-06 2020-10-23 渤海大学 一种克服肠道降解的膜肽酶抑制肽

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