WO2001068115A1 - Procede de production de sequences peptidiques a activite hypotensive - Google Patents

Procede de production de sequences peptidiques a activite hypotensive Download PDF

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WO2001068115A1
WO2001068115A1 PCT/US2001/007531 US0107531W WO0168115A1 WO 2001068115 A1 WO2001068115 A1 WO 2001068115A1 US 0107531 W US0107531 W US 0107531W WO 0168115 A1 WO0168115 A1 WO 0168115A1
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pro
mixture
muscle
cleaving agent
protein
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PCT/US2001/007531
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English (en)
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Zhonghong Guan
Michael Kellogg
Foe Siong Tjoeng
Qi Wang
Jian-Min Zhao
Kathy Mandrell
Wei Li
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Monsanto Technology Llc
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Priority to AU2001245547A priority Critical patent/AU2001245547A1/en
Publication of WO2001068115A1 publication Critical patent/WO2001068115A1/fr

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    • 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
    • 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
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06043—Leu-amino acid
    • 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
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • 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
    • C07K5/06—Dipeptides
    • C07K5/06104—Dipeptides with the first amino acid being acidic
    • C07K5/06113—Asp- or Asn-amino acid
    • 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
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06147—Dipeptides with the first amino acid being heterocyclic and His-amino acid; Derivatives thereof
    • 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
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
    • 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
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • 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
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • 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
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • 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
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
    • C07K5/0823—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp and Pro-amino acid; Derivatives thereof
    • 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
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • PROCESS FOR PRODUCING PEPTIDE SEQUENCES POSSESSING ANTI-HYPERTENSION ACTIVITY
  • the present invention generally relates to a process for producing peptide sequences that produce anti- hypertensive activity from a variety of protein sources such as milk, soybeans, corn, wheat, rice, peanuts, beef muscle, lamb muscle, pork muscle, turkey muscle, chicken muscle, fish muscle, yeast, bacteria, or products thereof that contain the peptide sequence Leu-Lys-Pro-Asn-Met (LKPNM)
  • LKPNM Leu-Lys-Pro-Asn-Met
  • Hypertension is a medical condition of persistently elevated arterial blood pressure. There are approximately 50 million people in the United States and 170 million people throughout the world that have elevated systolic blood pressures greater than 140 mm Hg and diastolic blood pressures between 90 to 95 mm Hg that may be considered to be experiencing hypertension.
  • Hypertension may be caused by a number of different physiological mechanisms.
  • One of the most well-known and potent of these is the renin-angiotensin vasoconstrictor mechanism for control of arterial pressure.
  • the enzyme renin is produced by the kidneys when a decrease in blood flow is detected. Renin cleaves the polypeptide angiotensinogen (renin substrate) that is secreted by the liver into the blood to form the biologically inactive decapeptide angiotensin I.
  • Another enzyme, angiotensin converting enzyme (ACE) cleaves the last two residues (His-Leu) from angiotensin I to form active angiotensin II.
  • Angiotensin II is the most potent vasoconstrictor known that raises blood pressure by causing contraction of vascular smooth muscle. Angiotensin II also promotes the increase of blood pressure by causing aldosterone to be released from the adrenal cortex which results in sodium and water retention. ACE further acts to increase blood pressure by also degrading the peptide bradykinin, a potent vasodilator. Thus, the presence of ACE in the bloodstream can cause the elevation of blood pressure by acting on angiotensin I and bradykinin, thereby elevating blood pressure.
  • ACE inhibitors such as Captopril and Enalapril
  • calcium channel blockers beta blockers
  • beta blockers diuretics
  • alpha blockers central alpha agonists
  • angiotensin II antagonists Dietary supplements have also been studied as an alternative means to reduce blood pressure.
  • Dietary supplements have also been studied as an alternative means to reduce blood pressure.
  • the pharmaceutical products are useful in decreasing elevated blood pressure, they often have negative side effects associated with their use. Some common side effects include altering normal blood pressure, overreacting to produce low blood pressure, producing a dry cough, and other side effects.
  • Nippon Synthetic Chemical Antihypertensive Material Found in Japanese Traditional Food, page 14. Another characteristic of anti-hypertensive pharmaceuticals is their immediate effect of lowering blood pressure.
  • ACE inhibitory substances from natural sources such as milk protein, soybean protein or fish meat protein are proposed for practical use as anti-hypertensive agents having low toxicity and great safety.
  • Proteins from natural resources have the primary and secondary functions of supplying nutrients and energy; however, many studies have shown that proteins perform tertiary functions relating to physiological regulation.
  • Peptides from natural sources would be able to modulate blood pressure in addition to providing other beneficial effects such as promoting calcium absorption and regulating serum cholesterol. Thus, it would be beneficial to treat hypertension with anti -hypertensive products obtained from natural sources.
  • VPP and IPP tripeptides having strong ACE inhibiting activity
  • SHR spontaneously hypertensive rats
  • the tripeptides are produced by proteinase, which is produced by lactic acid bacteria as lactic acid fermentation proceeds in milk, the resulting amounts of VPP and IPP tends to vary depending on the conditions of fermentation. It has thus been difficult to consistently produce the tripeptides in predictable quantities .
  • LKPNM One oligopeptide, LKPNM, has been discovered to have significantly greater anti-hypertensive activity in potency and duration than Enalapril (1- [N- [1- (ethoxycarbonyl) -3- phenyl-propyl] -L-alanyl] -L-proline) , an ACE inhibitor. LKPNM has also been determined to result in a milder response in decreasing high blood pressure. While anti- hypertensive pharmaceutical products rapidly decrease blood pressure when taken and quickly cause the elevation of blood pressure once the product is discontinued, LKPNM decreases blood pressure more gradually and slowly returns to the elevated blood pressure when its use is discontinued. Nippon Synthetic Chemical, Antihypertensive Material Found in Japanese Traditional Food, page 8, 15.
  • LKPNM has been isolated from dried bonito digested in thermolysin.
  • Dried bonito is a traditional Japanese seasoning made of skipjack tuna (bonito) muscle.
  • Yokoyama, K. et al . Biosci. Biotech. Biochem., 56(10), 1541-1545, 1992.
  • bonito meat is treated with fungi. From its research on bonito fish, Nippon determined that the fungi treatment pre-step is a critical step in obtaining the LKPNM oligopeptides .
  • thermolysin digests of non-dried bonito protein do not produce the LKPNM oligopeptide .
  • Nippon suggests that the proteases secreted from the fungi used to treat the bonito fish likely contribute to the ability for LKPNM to be isolated. Nippon Synthetic Chemical, Antihypertensive Material Found in Japanese Traditional Food, pg 6.
  • LKPNM may be produced through thermolysin digestion of dried-bonito, the raw material costs of dried-bonito and the resultant quantity of LKPNM produced makes dried-bonito a costly source of the oligopeptide.
  • LKPNM may be converted to different fragments which also demonstrate anti-hypertensive activities. After LKPNM enters the body, it is believed to be further converted into the peptide LKP and NM in the digestive organs or blood. The LKP peptide exhibits the greatest anti-hypertensive properties of LKPNM and its fragment products. LKP has been isolated from digestion of fish muscle, corn protein, soybeans, and milk products.
  • LKPN Leu-Lys-Pro- Asn
  • the present invention is directed to a process for producing the anti-hypertensive peptides Val-Pro-Pro (VPP) , Leu-Lys-Pro-Asn-Met (LKPNM) , Lys-Pro-Asn- Met (KPNM) (SEQ ID NO: 3) , Lys-Pro-Asn (KPN) , Pro-Asn-Met (PNM) , Leu-Lys (LK) , Lys-Pro (KP) , Pro-Asn (PN) , Asn-Met (NM) , Tyr-Pro (YP) , Glu-Ala-Pro (EAP) , Tyr-Lys-Pro (YKP) , and Leu-Ala-Pro (LAP) and salts thereof.
  • the peptides are produced by forming a homogeneous mixture of a source protein of milk, soybeans, corn, wheat, rice, peanuts, beef muscle, lamb muscle, pork muscle, turkey muscle, chicken muscle, fish muscle other than dried bonito fish, yeast, bacteria, or products thereof and an aqueous liquid.
  • the pH of the mixture is adjusted to a pH that is favorable for a selected cleaving agent.
  • the mixture is boiled, cooled, and a cleaving agent is added to digest the protein present in the mixture.
  • the cleaving agent is inactivated after a desired period of time to stop the digestion reaction and the mixture is separated into solid and supernatant phases.
  • the desired peptides are thereafter isolated from the supernatant .
  • LKP anti-hypertensive peptide Leu- Lys-Pro
  • LKP is produced by forming a homogeneous mixture of a source protein of wheat, rice, peanuts, beef muscle, lamb muscle, pork muscle, turkey muscle, chicken muscle, yeast, bacteria, or products thereof and an aqueous liquid.
  • the pH of the mixture is adjusted to a pH that is favorable for a selected cleaving agent.
  • the mixture is boiled, cooled, and the cleaving agent is added to digest the protein present in the mixture.
  • the cleaving agent is inactivated after a desired period of time to stop the digestion reaction and the mixture is separated into solid and supernatant phases.
  • LKPN Leu-Lys-Pro-Asn
  • LKPN Leu-Lys-Pro-Asn
  • the pH of the mixture is adjusted to a pH that is favorable for a selected cleaving agent .
  • the mixture is boiled, cooled, and the cleaving agent is added to digest the protein present in the mixture.
  • the cleaving agent is inactivated after a desired period of time to stop the digestion reaction and the mixture is separated into solid and supernatant phases.
  • the desired peptides are thereafter isolated from the supernatant.
  • Fig. 1 is a graph of LKP detected in the digestion of bonito fish, soybeans, and Calpis milk.
  • Fig. 2 is a graph of LKPNM detected in the digestion of bonito fish, soybeans, and Calpis milk.
  • Fig. 3 is a graph of VPP detected in the digestion of bonito fish, soybeans, and Calpis milk.
  • Fig. 4 is a compilation of HPLC/mass spectrometry (MS) chromatograms of LKPNM-related peptides.
  • Fig. 5 is a compilation of HPLC/MS chromatograms of LKPNM-related peptides from samples of fish digested in thermolysin.
  • Fig. 6 is a compilation of screening results for peptides released during the enzymatic digestion of pork protein.
  • Fig. 7 is a compilation of screening results for peptides released during the enzymatic digestion of pork and beef protein.
  • Fig. 8 is a compilation of screening results for peptides released during the enzymatic digestion of beef protein.
  • Fig. 9 is a compilation of screening results for peptides released during the enzymatic digestion of chicken and turkey protein.
  • Fig. 10 is a compilation of screening results for peptides released during the enzymatic digestion of turkey and fish protein.
  • Fig. 11 is a compilation of screening results for peptides released during the enzymatic digestion of fish and soy protein.
  • Table 1 is the tabulation of the Quantitative Results for LKP released in the Thermolysin Digestion of Bonito Fish, Soy and Calpis Milk.
  • Table 2 is the tabulation of the Quantitative Results for LKPNM released in the Thermolysin Digestion of Bonito Fish, Soy and Calpis Milk.
  • Table 3 is the tabulation of the Quantitative Results for VPP released in the Thermolysin Digestion of Bonito
  • Table 4 is a tabulation of VPP, IPP, and LKPNM yields from the digestion of soybeans, wheat, milk, bonito fish, turkey muscle, lamb muscle, pork muscle, beef muscle, chicken muscle, yeast, E. coli , and Calpis milk.
  • Table 5 is a tabulation of LK, LKPN, KPNM, PNM, LKP, LKPNM, YP, EAP, YKP, AND LAP yields from the digestion of pork muscle using pepsin, chymotrypsin, trypsin, and thermolysin proteases.
  • Table 6 is a tabulation of LK, LKPN, KPNM, PNM, LKP, LKPNM, YP, EAP, YKP, AND LAP yields from the digestion of beef muscle using pepsin, chymotrypsin, trypsin, and thermolysin proteases.
  • Table 7 is a tabulation of LK, LKPN, KPNM, PNM, LKP, LKPNM, YP, EAP, YKP, AND LAP yields from the digestion of chicken muscle using pepsin, chymotrypsin, trypsin, and thermolysin proteases.
  • Table 8 is a tabulation of LK, LKPN, KPNM, PNM, LKP, LKPNM, YP, EAP, YKP, AND LAP yields from the digestion of turkey muscle using pepsin, chymotrypsin, trypsin, and thermolysin proteases.
  • Table 9 is a tabulation of LK, LKPN, KPNM, PNM, LKP, LKPNM, YP, EAP, YKP, AND LAP yields from the digestion of fish muscle using pepsin, chymotrypsin, trypsin, and thermolysin proteases.
  • Table 10 is a tabulation of LK, LKPN, KPNM, PNM, LKP, LKPNM, YP, EAP, YKP, AND LAP yields from the digestion of soybeans using pepsin, chymotrypsin, trypsin, and thermolysin proteases.
  • Table 11 is the tabulation of LC/MS detection results for peptide standard solutions.
  • a and Ala represent alanine
  • R and Arg represent arginine
  • N and Asn represent asparagine
  • D and Asp represent aspartic acid
  • C and Cys represent cysteine
  • Q and Gin represent glutamine
  • E and Glu represent glutamic acid
  • G and Gly represent glycine
  • H and His represent histidine
  • I and lie represent isoleucine
  • L and Leu represent leucine
  • K and Lys represent lysine
  • M and Met represent methionine
  • F and Phe represent phenylalanine
  • P and Pro represent proline
  • S and Ser represent serine
  • T and Thr represent threonine
  • W and Trp represent tryptophan
  • Y and Tyr represent tyrosine
  • V and Val represent valine.
  • ACE angiotensin converting enzyme
  • the present invention relates to the process of producing and isolating from a variety of protein materials short peptides which exhibit anti-hypertensive activity which effectively depress elevated blood pressure, but which, unlike current anti-hypertensive pharmaceutical compounds, do not have adverse side effects such as altering normal blood pressure, over depressing blood pressure, producing a dry cough or other side effects.
  • the novel processes of the present invention can be beneficially used to produce the anti- hypertensive peptides including LKPNM, LKPN, KPNM, LKP, KPN, PNM, LK, KP, PN, NM, VPP, YP, EAP, YKP, LAP and the acid addition salts thereof.
  • These peptides have significant anti-hypertensive activities and therefore are useful in the diagnosis, treatment and prophylaxis of hypertension and related conditions such as left ventricular systolic dysfunction, myocardial infarction, diabetes mellitus and progressive renal impairment/failure.
  • the peptides are not expected to have the negative side effects associated with the anti-hypertensive pharmaceutical products.
  • the processes of the present invention may derive antihypertensive peptides from a variety of food protein digest products.
  • the present invention described herein as applied to LKPNM and its fragment oligopeptides is a process for producing the oligopeptides from alternative sources of protein other than dried-bonito.
  • the present invention discloses a process for producing VPP from alternative sources of protein other than lactic acid bacteria-fermented milk.
  • the first step in the process of producing VPP, YP, EAP, YKP, LAP, LKPNM, and/or and its fragment oligopeptides from a food protein is the selection of the protein raw material.
  • the preferred protein raw material is a protein that contains VPP, YP, EAP, YKP, LAP, or LKPNM sequences within the protein.
  • Non-exhaustive examples of proteins having such sequences include milk, soybeans, corn, wheat, rice, peanuts, beef muscle, lamb muscle, pork muscle, turkey muscle, chicken muscle, fish muscle, yeast, bacteria, acid addition salts (inorganic acid or organic acid addition salts, for example, hydrochloride, hydrobromide, sulfate, nitrate, acetate, benzoate, maleate, fumarate, succinate, tartrate, citrate, oxalate, methanesulfonate, toluenesulfonate, aspartate, glutamate, etc.) and base addition salts thereof .
  • acid addition salts inorganic acid or organic acid addition salts, for example, hydrochloride, hydrobromide, sulfate, nitrate, acetate, benzoate, maleate, fumarate, succinate, tartrate, citrate, oxalate, methanesulfonate, toluenesulfonate, aspartate, glut
  • the protein raw material is selected, measured, and mixed with an aqueous liquid such as water, preferably distilled water, or other aqueous liquid such as a buffer
  • the protein and liquid mixture is preferably homogenized.
  • the activity of a protein cleaving agent is improved as the mixture is more thoroughly homogenized. Homogenization of the mixture promotes the direct contact of the cleaving agent to the protein by increasing protein surface area exposed to the cleaving agent, thereby facilitating the protein digestion process.
  • the concentration of protein raw material is preferably in the range of about 1 to 50% (w/v) , preferably about 10%, which promotes the ease of homogenization.
  • the mixture is thoroughly homogenized, it is adjusted to a pH that is favorable for the activity of a cleaving agent.
  • a cleaving agent such as thermolysin which has a cleaving activity that is most favorable at neutral pH
  • the preferred pH range is 6 to 8 and most preferably about 7.
  • a acidic cleaving agent such as pepsin
  • the most favorable pH range is about 0.1 to 4, most preferably about 2.
  • an alkaline cleaving agent would work most effectively in alkaline conditions such as a pH of 8 to 10.
  • the pH adjusted mixture is raised to a boiling temperature for a period of approximately 5 to 60 minutes, preferably for about 10 to 15 minutes. The boiling step inactivates all endogenous enzymes present in the source protein.
  • the pH in course of reaction can, if necessary, be adjusted with a base such as aqueous sodium hydroxide solution, an acid, such as hydrochloric acid, or the like.
  • a base such as aqueous sodium hydroxide solution, an acid, such as hydrochloric acid, or the like.
  • Oligopeptides contained in the protein raw material used in the invention can be prepared by a process to hydrolyze or digest the selected protein with a cleaving agent.
  • a cleaving agent such as a protease is added to the mixture to hydrolyze the protein material within the mixture.
  • proteases such as thermolysin, pepsin, trypsin, chymotrypsin, papain, Pronase E, Proteinase K, or Actinase E may be used to digest the protein.
  • Thermolysin is particularly preferred as the digesting enzyme .
  • the optimal concentration of the cleaving agent within the digestion mixture is dependent upon the cleaving agent selected.
  • the addition amount of the enzyme thermolysin is varied depending on its titer, the final concentration within the reaction mixture is preferably about 100 ⁇ g/ml to 1000 ⁇ g/ml , and most preferably a final thermolysin concentration of approximately 800 to 880 ⁇ g/ml based on the protein. It is also possible to add part of the thermolysin in the course of the reaction. Treatment of the various protein raw materials with thermolysin is described further in the examples below. To promote protein digestion, the temperature of the reaction mixture during the reaction may be maintained within approximately 30°C to 50°C, preferably about 37°C.
  • the reaction time varies depending on the amount of the enzyme, reaction temperature and reaction pH, but is usually on the order of 1 to 24 hours, preferably 1 to 6 hours.
  • a reaction time of 3 to 6 hours is preferred.
  • the period of time the protein digestion should be permitted to react is dependent upon the cleaving agent used and the desired peptide to be isolated. For example, a mixture digested for one hour with thermolysin may produce a more optimal quantity of the LKPNM peptide and very little of the shorter peptides such as LKP, LK, and so forth.
  • the quantity of LKPNM which may be isolated may decrease while the quantities of the shorter peptide sequences will increase.
  • the period of time identified for the digestion reaction may therefore be identified by quantifying the isolated peptides over time such as is illustrated in Figs. 1 - 3.
  • the digestion reaction can halted according to a known method, for example, according to inactivation of the cleaving agent either by pH change with addition of an organic acid such as citric acid or malic acid, an inorganic acid such as hydrochloric acid or phosphoric acid or an alkali such as sodium hydroxide or potassium hydroxide, by heating of the reaction mixture, by separation of the enzyme by filtration using an ultrafiltration membrane, by a combination of the aforementioned steps, or the like.
  • an organic acid such as citric acid or malic acid
  • an inorganic acid such as hydrochloric acid or phosphoric acid or an alkali such as sodium hydroxide or potassium hydroxide
  • the pH of a digestion mixture can be changed, then the digestion mixture may be heated followed by ultrafiltration.
  • the digestion mixture is cooled to approximately 0°C to 30°C, preferably about 4°C.
  • the oligopeptides can be isolated from the resulting digestion solution through solid-liquid separation, for example, centrifugation or filtration.
  • the resulting liquid is fractionated by ultrafiltration, gel filtration or the like to obtain liquid containing the desired target oligopeptides of the invention.
  • the fractionated liquid is further fractionated to obtain each objective oligopeptide according to its size and retention time.
  • the cooled mixture is transferred into centrifuge tubes and separated into solid and liquid phases by centrifugation at approximately 1500 to 3000 rpm. Centrifugation is conducted for a period of about 10 to 20 minutes, preferably about 15 minutes. The supernatant is removed and transferred to a clean centrifuge tube and centrifuged a second time at approximately 8000 rpm for a period of about 10 to 20 minutes, preferably about 10 minutes, at a temperature below about 30°C.
  • the contents of the centrifuge tubes are then dried under nitrogen gas at a temperature of approximately 30°C to 80°C, preferably about 37°C.
  • a mobile phase solvent is added to the centrifuge tubes and the dried contents are resuspended to form a mobile phase mixture.
  • the mobile phase mixture in then centrifuged at about 1000 to 10,000 rpm, preferably approximately 8000 rpm at a temperature below about 30°C for a period of about 10 to 20 minutes.
  • the resulting supernatant is then fractionated by chromatographic methods such as liquid chrom tography, HPLC, or the like.
  • the desired peptides are then isolated according to their respective retention times.
  • VPP VPP
  • YP EAP
  • YKP EAP
  • LAP lactic acid bacteria-fermented milk
  • Acid addition salts of the present peptides can be prepared according to a conventional method.
  • an acid addition salt can be obtained by reacting one of the isolated peptides containing a basic amino acid residue with a suitable acid in one equivalent amount thereto in water and then freeze-drying the product.
  • alkali or alkaline earth metal salts, ammonium salts or organic base salts can also be prepared according to a conventional method.
  • a base salt can be obtained by reacting one of the present peptides containing an acidic amino acid residue with a suitable base in one equivalent amount thereto in water and then freeze-drying the product.
  • the mixture was transferred to centrifuge tubes and centrifuged at 3000 rpm at 4°C for a period of 15 minutes. 200 ⁇ l of the supernatant was collected and placed into a clean microfuge tube and centrifuged at 8000 rpm at a temperature about 30°C for a period of 10 minutes. The contents of the microfuge tube were dried down under nitrogen gas (N 2 ) at a temperature of 37°C. The dried contents of the microfuge tube were then resuspended in 100 ⁇ l of the mobile phase solvent (water) and centrifuged at 8000 rpm at about 30°C for a period of 10 minutes.
  • N 2 nitrogen gas
  • the resulting supernatant was transferred to an HPLC vial and 25 ⁇ l of the supernatant was injected into the HPLC.
  • the LKPNM, LKP, and VPP peptide fragments were isolated and quantified.
  • the quantities of the peptides resulting from various digest times are compared in Figs. 1, 2, and 3.
  • the concentrations of the peptides corresponding to Figs. 1-3 are quantified in Tables 1, 2, and 3.
  • LKPNM protein from dried bonito, soybean, wheat, milk, turkey muscle, lamb muscle, pork muscle, beef muscle, chicken muscle, yeast, E. coli, and Calpis milk were digested according to the method described in Example 1.
  • the quantities of the peptides resulting from various digest times are identified in Table 4.
  • Table 4 after enzymatic digestion with thermolysin, significant amounts of LKPNM were measured from most of the food proteins tested using LC/MS isolation and detection methods.
  • the yield rate of LKPNM from digested chicken, turkey, and pork muscle was close to or even higher than the amount of LKPNM produced from dried bonito hydrolysate.
  • Protein from pork muscle, beef muscle, chicken muscle, turkey muscle, fish muscle and soybeans were each digested in pepsin, chymotrypsin, trypsin, and thermolysin. From each digest, LK, LKPN, KPNM, PNM, LKP, LKPNM, YP, EAP, YKP, and LAP peptides were quantified and identified in Tables 5 - 11.
  • the mixture was held at a temperature of 37°C and enzymatically digested for a period of 0 , 1, 3, and 5 hours.
  • the enzymatic reaction was stopped at the end of each digestion period by boiling the mixture for 10 minutes to inactivate the cleaving agent.
  • the mixture was transferred to centrifuge tubes and centrifuged at 1500 rpm at a temperature of about 30°C for a period of 10 minutes.
  • the supernatant was removed and analyzed using liquid chromatography/mass spectroscopy (LC/MS) to quantify the presence of oligopeptide fragments.
  • LC/MS liquid chromatography/mass spectroscopy
  • the supernatant was diluted 1:10 in deionized water with 0.1% trifluoroacetic acid (TFA) to form samples for
  • LC/MS injections 10 ⁇ g samples were injected into an LC/MS operated in the positive ion electrospray ionization (ESI) mode. Components of the samples were separated using a reverse phase HPLC method and a Waters C 18 Symmetry column operated at 70°C. Positive ESI mass spectra of standard reference materials showed that the major ions formed were MH + . This mass was monitored to quantify peptides present in the samples.
  • Water and acetonitrile contained 0.1% TFA. The water/acetonitrile flow rate was 1.0 mL/min with a gradient that began at 0% acetonitrile which increased linearly to 100% acetonitrile between 4 and 10 minutes. A 2 minute post time was used to allow the column to equilibrate between injections.
  • Fig. 4 shows LC/MS chromatograms from the analysis of a standard solution containing oligopeptides related to LKPNM. Each chromatogram is the mass specific response of the protonated oligopeptide. The chromatographs are thus very specific for the peptides. Since the protonated molecular ions are the most abundant ions in the mass spectra of this class of compounds, good sensitivities were obtained from this analysis.
  • Fig. 5 shows the oligopeptide analysis for fish which was processed for three hours with thermolysin. Since the oligopeptide responses in the fish samples were not completely resolved from other matrix components, the values that were obtained were therefore typically larger than the true values for the oligopeptides.
  • the oligopeptide concentrations were calculated using the following formula:
  • Tables 5 to 11 summarize the quantities of the peptides identified from the pepsin, chymotrypsin, trypsin, and thermolysin digestions of the protein.
  • Figs. 6 to 10 summarize the screening results for PN, KPN, KP, NM, LK, LKPN, KPNM, VPP, YP, PNM, LKP and LKPNM peptides resulting from the enzymatic digestion of pork, beef, chicken, turkey, fish, and soy protein.
  • standards were analyzed for quality control purposes. These quality control samples were labeled SCRN01, SCRN01(1:2), SCRN02, and SCRN01(1:2).
  • the SCRNOl and SCRN02 samples are standards described in Table 8. The total height of the stacked bars for each of these samples, shown in Figs. 6 to 10, should be consistent.
  • the SCRNOl bars should be twice as tall as the SCRNOl (1:2) bars and the SCRN02 bars should be twice as tall as the SCRN02(1:2) bars.
  • the SCRMOl bars should also be constant at approximately

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

L'invention se rapporte à un procédé de production de peptides à action hypotensive qui sont isolés à partir d'une variété de matières premières protéiques. Ledit procédé consiste à identifier une matière première protéique qui contient, dans la protéine, les séquences Val-Pro-Pro, Ile-Pro-Pro, ou Leu-Lys-Pro-Asn-Met. La protéine est digérée par un agent de clivage qui produit des fragments peptiques à action hypotensive de Leu-Lys-Pro-Met-Asn (LKPNM), Leu-Lys-Pro-Asn (LKPN), Lys-Pro-Asn-Met (KPNM), Leu-Lys-Pro (LKP), Lys-Pro-Asn (KPN), Pro-Asn-Met (PNM), Leu-Lys (LK), Lys-Pro (KP), Pro-Asn (PN), Asn-Met (NM),Val-Pro-Pro (VPP), Tyr-Pro (YP), Glu-Ala-Pro (EAP), Tyr-Lys-Pro (YKP), Leu-Ala-Pro (LAP) ou d'un mélange de ces séquences. Les fragments sont alors séparés et isolés.
PCT/US2001/007531 2000-03-13 2001-03-09 Procede de production de sequences peptidiques a activite hypotensive WO2001068115A1 (fr)

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Cited By (10)

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WO2003070267A1 (fr) * 2002-02-25 2003-08-28 Valio Ltd Amelioration de la disponibilite de mineraux au moyen de peptides biologiquement actifs
WO2004082709A1 (fr) 2003-03-18 2004-09-30 Suntory Limited Peptides inhibiteurs de l'enzyme de conversion de l'angiotensine (ace)
WO2006067163A2 (fr) * 2004-12-22 2006-06-29 Dsm Ip Assets B.V. Peptides abaissant la pression sanguine obtenus en une seule operation enzymatique
WO2006084560A1 (fr) * 2005-02-09 2006-08-17 Unilever N.V. Composition comprenant un peptide
WO2006084383A1 (fr) * 2005-02-14 2006-08-17 Ocean Nutrition Canada Limited Supplement dietetique antidiabetique et antihypertensif
US7179793B2 (en) 2005-02-14 2007-02-20 Ocean Nutrition Canada Limited Anti-hypertensive dietary supplement
KR20200017223A (ko) * 2018-08-08 2020-02-18 중앙대학교 산학협력단 한우육 유래 펩타이드의 제조 방법 및 이로부터 제조된 한우육 유래 펩타이드를 유효성분으로 포함하는 고혈압 또는 퇴행성 신경질환의 예방, 치료, 또는 개선용 조성물
CN111073944A (zh) * 2019-12-31 2020-04-28 江南大学 一种基于Caco-2细胞模型的活性肽定向制备方法
CN111484545A (zh) * 2020-03-31 2020-08-04 华南农业大学 一种米酒糟来源的降血压寡肽及其制备方法与应用
CN111548387A (zh) * 2020-03-31 2020-08-18 华南农业大学 一种具有降血压功效的寡肽及其制备方法与应用

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WO2003070267A1 (fr) * 2002-02-25 2003-08-28 Valio Ltd Amelioration de la disponibilite de mineraux au moyen de peptides biologiquement actifs
JP2009084294A (ja) * 2003-03-18 2009-04-23 Suntory Ltd アンジオテンシン変換酵素阻害ペプチド
CN100402089C (zh) * 2003-03-18 2008-07-16 三得利株式会社 血管紧张素转化酶抑制肽
AU2004222435B2 (en) * 2003-03-18 2009-12-17 Suntory Holdings Limited Angiotensin-converting enzyme inhibitory peptides
JP2009084295A (ja) * 2003-03-18 2009-04-23 Suntory Ltd アンジオテンシン変換酵素阻害ペプチド
JP2006520809A (ja) * 2003-03-18 2006-09-14 サントリー株式会社 アンジオテンシン変換酵素阻害ペプチド
TWI411441B (zh) * 2003-03-18 2013-10-11 Suntory Holdings Ltd 血管收縮素轉化酶抑制性肽類
US7943578B2 (en) 2003-03-18 2011-05-17 Suntory Holdings Limited Angiotensin-converting enzyme inhibitory peptides
EP1938832A1 (fr) 2003-03-18 2008-07-02 Suntory Limited Peptides inhibitoires d'enzyme à conversion d'angiotensine
EP2145630A3 (fr) * 2003-03-18 2010-02-17 Suntory Holdings Limited Peptides inhibitoires d'enzyme à conversion d'angiotensine
WO2004082709A1 (fr) 2003-03-18 2004-09-30 Suntory Limited Peptides inhibiteurs de l'enzyme de conversion de l'angiotensine (ace)
US7833985B2 (en) 2003-03-18 2010-11-16 Suntory Holdings Limited Angiotensin-converting enzyme inhibitory peptides
WO2006067163A3 (fr) * 2004-12-22 2007-05-10 Dsm Ip Assets Bv Peptides abaissant la pression sanguine obtenus en une seule operation enzymatique
US7879804B2 (en) 2004-12-22 2011-02-01 Dsm Ip Assets B.V. Blood pressure lowering peptides in a single enzymatic step
WO2006067163A2 (fr) * 2004-12-22 2006-06-29 Dsm Ip Assets B.V. Peptides abaissant la pression sanguine obtenus en une seule operation enzymatique
WO2006084560A1 (fr) * 2005-02-09 2006-08-17 Unilever N.V. Composition comprenant un peptide
US7179793B2 (en) 2005-02-14 2007-02-20 Ocean Nutrition Canada Limited Anti-hypertensive dietary supplement
WO2006084383A1 (fr) * 2005-02-14 2006-08-17 Ocean Nutrition Canada Limited Supplement dietetique antidiabetique et antihypertensif
KR20200017223A (ko) * 2018-08-08 2020-02-18 중앙대학교 산학협력단 한우육 유래 펩타이드의 제조 방법 및 이로부터 제조된 한우육 유래 펩타이드를 유효성분으로 포함하는 고혈압 또는 퇴행성 신경질환의 예방, 치료, 또는 개선용 조성물
KR102081852B1 (ko) 2018-08-08 2020-02-26 중앙대학교 산학협력단 한우육 유래 펩타이드의 제조 방법 및 이로부터 제조된 한우육 유래 펩타이드를 유효성분으로 포함하는 고혈압 또는 퇴행성 신경질환의 예방, 치료, 또는 개선용 조성물
CN111073944A (zh) * 2019-12-31 2020-04-28 江南大学 一种基于Caco-2细胞模型的活性肽定向制备方法
CN111548387A (zh) * 2020-03-31 2020-08-18 华南农业大学 一种具有降血压功效的寡肽及其制备方法与应用
CN111484545A (zh) * 2020-03-31 2020-08-04 华南农业大学 一种米酒糟来源的降血压寡肽及其制备方法与应用

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