WO2001068115A1

WO2001068115A1 - Process for producing peptide sequences possessing anti-hypertension activity

Info

Publication number: WO2001068115A1
Application number: PCT/US2001/007531
Authority: WO
Inventors: Zhonghong Guan; Michael Kellogg; Foe Siong Tjoeng; Qi Wang; Jian-Min Zhao; Kathy Mandrell; Wei Li
Original assignee: Monsanto Technology LLC; Monsanto Co
Current assignee: Monsanto Technology LLC; Monsanto Co
Priority date: 2000-03-13
Filing date: 2001-03-09
Publication date: 2001-09-20
Anticipated expiration: 2002-09-13
Also published as: AU2001245547A1

Abstract

A process for producing anti-hypertensive peptides that are isolated from a variety of protein raw materials. The process includes identifying a protein raw material that contains that contains Val-Pro-Pro, Ile-Pro-Pro, or Leu-Lys-Pro-Asn-Met sequences within the protein. The protein is digested with a cleaving agent that produces anti-hypertensive peptide fragments of 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) or a mixture thereof. The fragments are then separated and isolated.

Description

PROCESS FOR PRODUCING PEPTIDE SEQUENCES POSSESSING ANTI-HYPERTENSION ACTIVITY

This application claims priority to copending United States provisional patent application Ser. No. 60/188,794, filed March 10, 2000, the entire contents of which are incorporated herein by reference.

BACKGROUND OF INVENTION

Field of the Invention

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)

(SEQ ID NO: 1), Val-Pro-Pro (VPP), Ile-Pro-Pro (IPP) , Tyr- Pro (YP) , Glu-Ala-Pro (EAP) , Tyr-Lys-Pro (YKP) , and Leu-Ala- Pro (LAP) within the protein.

Description of Related Art 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. In this mechanism, 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.

Several drugs and dietary supplements have been studied for their ability to reduce blood pressure. Much focus has been on inhibiting ACE in the renin-angiotensin mechanism and preventing the formation of angiotensin II. While the inhibition of ACE affects the blood pressure, the reduction in blood pressure often can not be explained by the inhibition of ACE alone. Thus anti -hypertensive compounds may reduce blood pressure through other mechanisms or through a combination of physiologic mechanisms.

Currently, hypertension is often treated with the use of anti -hypertension pharmaceutical products such as ACE inhibitors such as Captopril and Enalapril, calcium channel blockers, beta blockers, diuretics, alpha blockers, central alpha agonists, and angiotensin II antagonists. Dietary supplements have also been studied as an alternative means to reduce blood pressure. While 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. While it is desirable to ultimately lower blood pressure, lowering elevated blood pressure in a short time decreases blood flow and may lead to insufficient blood circulation to the organs. Thus the use of anti- hypertensive pharmaceutical products can result in functional disorders from organs receiving inadequate blood flow. Furthermore, a sudden elevation of blood pressure may occur once an individual ceases to take anti -hypertensive pharmaceutical products. Nippon Synthetic Chemical, Antihypertensive Material Found in Japanese Traditional Food, page 8, 15. Thus it would be desirable to obtain a nutritional or dietary supplement that possesses anti- hypertensive properties without having the negative side effects associated with the pharmaceutical products.

Alternatively, compounds produced from food stuffs have also been studied for their ability to reduce blood pressure. These 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.

Few non-pharmaceutical products currently exist for treating hypertension in the nutritional or dietary supplement markets. Several studies, however, have found a variety of peptide sequences that have anti-hypertensive properties. The peptides or oligopeptides exhibiting anti- hypertensive activity have been produced in varying quantities from protease digestion of a variety of protein sources. Anti-hypertensive oligopeptides have been isolated from sardine and tuna muscle, dairy products, corn protein, soybean products, yeast, and other plant and animal proteins. Astawan, E. et al . , Effects of Angiotensin I- Converting Enzyme Inhibitory Substances Derived from Indonesian Dried-Salted Fish on blood Pressure of Rats, Biosci. Biotech. Biochem. , 59 (3), 425-429, 1995; Ariyoshi , A. , Angiotensin-Converting Enzyme Inhibitors Derived from Food Proteins, Trends in Food, Science, & Technology, 4, 139-144, May 1993; and Yokoyama, K. et al . , Peptide Inhibitors for Angiotensin I-Converting Enzyme from Thermolysin Digest of Dried Bonito, Biosci. Biotech. Biochem., 56(10), 1541-1545, 1992. Recently, VPP and IPP, tripeptides having strong ACE inhibiting activity, have been derived from lactic acid bacteria-fermented milk. See Nakamura et al . , J. Dairy Sci . 78:777-783, 1995. These tripeptides are reported to exhibit strong anti-hypertensive effects in spontaneously hypertensive rats (SHR) . See Nakamura et al . , J. Dairy Sci. 78:1253-1257, 1995. However, since 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 .

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. In the manufacturing process of dried bonito, 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 . Thus while LKPNM oligopeptides have been isolated from thermolysin digests of dried bonito fish, 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. Although 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.

Another peptide subunit that is a fragment of LKPNM also producing anti-hypertensive properties is Leu-Lys-Pro- Asn (LKPN) (SEQ ID NO: 2) . LKPN has been isolated from the enzymatic digestion of fish muscle and soybeans. See Suetsuna et al . , Kiso to Rinsho (Clinical Report) 25: 477304784 (1991); Japanese Application No. 7138287. Other peptides which have also been found possess antihypertensive properties include YP, EAP, YKP, LAP. In a study by the inventors, blood pressure of spontaneously hypertensive rats decreased after the administration of synthetic EAP and YKP. While the peptides are promising anti -hypertensive agents, synthetically peptides are costly to produce .

Accordingly, it would be beneficial to produce nutritional or dietary supplements from natural sources that possess anti-hypertensive properties such as the peptides VPP, YP, EAP, YKP, LAP, and LKPNM and its fragments without the unpredictable production quantities associated with current VPP production methods or the high costs of producing LKPNM and its fragments from dried bonito. Thus a process to produce anti -hypertensive oligopeptides from alternative protein sources that have predictable production quantities, lower raw material costs, higher quantities of anti-hypertensive peptides produced, or a combination thereof than can be currently attained is desired.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a novel process for economically preparing and isolating naturally occurring peptides from a protein material that demonstrate anti-hypertensive activity. It is another object of the present invention to provide a novel process for preparing and isolating naturally occurring peptides from a protein material that demonstrate anti -hypertensive activity without the negative physiological side effects associated with commercial anti- hypertensive pharmaceutical products.

It is a further object of the present invention to provide a novel process for preparing and isolating naturally occurring peptides from a protein material that demonstrate a milder anti -hypertensive effect than are available with commercial anti-hypertensive pharmaceutical products.

Briefly, therefore, 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 .

Another aspect of the present invention is directed to a process for producing the anti-hypertensive peptide Leu- Lys-Pro (LKP) and its acid addition salts. 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. The desired peptides are thereafter isolated from the supernatant. Still another aspect of the present invention is directed to a process for producing the anti-hypertensive peptide LKP and its acid addition salts. Leu-Lys-Pro-Asn (LKPN) is produced by forming a homogeneous mixture of a source protein of milk, wheat, rice, corn, 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. The desired peptides are thereafter isolated from the supernatant.

Other features of the present invention will be in part apparent to those skilled in the art and in part pointed out in the detailed description provided below.

BRIEF DESCRIPTION OF DRAWINGS 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

Fish, Soy and Calpis Milk.

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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All publications, patents, patent applications or other references cited in this application are herein incorporated by reference in their entirety as if each individual publication, patent, patent application or reference are specifically and individually indicated to be incorporated by reference.

Abbreviations and Definitions To facilitate understanding of the invention, a number of terms as used herein are defined below:

The amino acid residues are abbreviated herein according to convention to their single letters symbols and/or their three letter symbols: 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; and V and Val represent valine.

As used herein, "ACE" means angiotensin converting enzyme .

Preparation of Peptides 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.

Specifically, 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. Furthermore, 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. As applied to VPP, 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 .

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

(for example, a Tris-HCl buffer or a phosphate 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.

Once the mixture is thoroughly homogenized, it is adjusted to a pH that is favorable for the activity of a cleaving agent. For 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. For a acidic cleaving agent such as pepsin, the most favorable pH range is about 0.1 to 4, most preferably about 2. Likewise, 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. After the mixture is boiled, it is allowed to cool to approximately 50°C or below, preferably about 37°C. 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. Commercially available 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. Although the addition amount of the enzyme thermolysin is varied depending on its titer, the final concentration within the reaction mixture is preferably about 100 μg/ml to 1000 μg/ml , and most preferably a final thermolysin concentration of approximately 800 to 880 μg/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. To obtain LKPNM, and/or and its fragment oligopeptides, 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. As the time permitted for digestion increases, 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. Thus, in an example of a combination of methods to halt the reaction, the pH of a digestion mixture can be changed, then the digestion mixture may be heated followed by ultrafiltration.

Once the reaction is halted, 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. As demonstrated in the examples described below, applicants successfully produced VPP, YP, EAP, YKP, LAP, and LKPNM and its fragment oligopeptides from a variety of alternative sources of protein other than dried-bonito and lactic acid bacteria-fermented milk in appreciable quantities.

Addition Salts

Acid addition salts of the present peptides can be prepared according to a conventional method. For example, 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.

Further, alkali or alkaline earth metal salts, ammonium salts or organic base salts (hereinafter these are referred to as base salts) can also be prepared according to a conventional method. For example, 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.

EXAMPLES

The following examples illustrate the invention.

Example 1 - Method of Producing LKPNM, LKP and VPP From Thermolysin Digested Dried Bonito, Calpis, and Soybean Protein

To evaluate whether LKPNM can be released from other food proteins by enzymatic digestion, dried bonito (positive control) , Calpis milk, and soybeans were digested and the peptide sequences of LKPNM and its fragments were isolated and the peptide quantities compared according to the procedure as outlined below:

Five grams of protein (dried bonito muscle, Calpis milk, or soybeans) were added to 45 ml of distilled water and homogenized for a period of 1 to 2 minutes. The homogenized mixture was adjusted to pH 7.0 using sodium hydroxide. The neutralized homogenized mixture was boiled for a period of 10 minutes. Thermolysin was then added to the mixture in a quantity resulting in a final concentration of 880 μg/ml. 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 thermolysin. The mixture was transferred to centrifuge tubes and centrifuged at 3000 rpm at 4°C for a period of 15 minutes. 200 μl 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 (N₂) at a temperature of 37°C. The dried contents of the microfuge tube were then resuspended in 100 μl of the mobile phase solvent (water) and centrifuged at 8000 rpm at about 30°C for a period of 10 minutes. The resulting supernatant was transferred to an HPLC vial and 25 μl 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.

Table 1 LKP Quantitative Results for Thermolysin Digestion of Bonito Fish, Soy and Calpis Milk

SAMI ³LE CONCENTRATION

BLMJK 0

BLANK 2 0

BONITO 0 0

BONITO 0' 0

BONITO 1 0

BONITO 1' 0

BONITO 3 0

BONITO 3 ' 0

BONITO 5 0

BONITO 5' 0

CALPIS 0

CALPIS' 0

SOY 0 0

SOY 0' 0

SOY 1 0

SOY 3 0

SOY 3' 0

SOY 5 0

SOY 5' 0

STD 1 LKP 0, .418

STD 10 LKP 10. .733

STD 2.5 LKP 1. .985

STD 25 LKP 26, .292

STD 5 LKP 4, .809

STD 50 LKP 49, .264 Table 2

LKPNM Quantitative Results for Thermolysin Digestion of Bonito Fish, Soy and Calpis Milk

SAMI ?LE CONCENTRATION

BLAI•JK 0.032

BLANK 2 0.146

BONITO 0 0.898

BONITO 0' 1.138

BONITO 1 82.582

BONITO 1' 81.88

BONITO 3 80.5

BONITO 3 ' 81.072

BONITO 5 2.572

BONITO 5' 114.218

CALPIS 28.784

CALPIS' 29.266

SOY 0 4.696

SOY 0' 4.756

SOY 1 1.634

SOY 1' 0.598

SOY 3 29.238

SOY 3 ' 30.406

SOY 5 28.338

SOY 5' 29.128

STD 1 LKP -0.024

STD 10 LKP 0.996

STD 2.5 LKP -0.029

STD 25 LKP 10.394

STD 50 LKP -0.019

STD 50 LKP 2.481

Table 3 VPP Quantitative Results for Thermolysin Digestion of Bonito Fish, Soy and Calpis Milk

SAMI ?LE CONCENTRATION

BON:[TO 0 24.768

BONITO 0' 74.548

BONITO 1 65.164

BONITO 1' 69.36

BONITO 3 54.184

BONITO 3 ' 68.128

BONITO 5 66.092

BONITO 5' 47.802

CALPIS 0

CALPIS' 0

SOY 0 74.772

SOY 0' 21.348

SOY 1 16.55

SOY 3 0

SOY 3' 0

SOY 5 0

SOY 5' 0

STD 1 LKP 0.378

STD 10 LKP 10.276

STD 2.5 LKP 2.02

STD 25 LKP 26.224

STD 5 LKP 5.258

STD 50 LKP 49.343

Example 2 - Method of Producing LKPNM and VPP From Thermolysin Digested Protein

To evaluate whether LKPNM can be released from other food proteins by enzymatic digestion, 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 LKPNM and VPP peptide sequences were quantified. The quantities of the peptides resulting from various digest times are identified in Table 4. As shown in 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. Significantly, 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.

TABLE 4 Peptide Yield For Source Proteins Digested in Thermolysin

TABLE 4 Peptide Yield For Source Proteins Digested in Thermolysin

TABLE 4 Peptide Yield For Source Proteins Digested in Thermolysin

TABLE 4 Peptide Yield For Source Proteins Digested in Thermolysin

TABLE 4 Peptide Yield For Source Proteins Digested in Thermolysin

Example 3 - Method of Producing Peptides From Thermolysin Digested Protein

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.

Five grams of protein (pork muscle, beef muscle, chicken muscle, turkey muscle, fish muscle and soybeans) were added to 45 ml of distilled water and homogenized for a period of 1 to 2 minutes. The homogenized mixture was adjusted to pH 2.0 with hydrochloric acid for pepsin digestion. For chymotrypsin, trypsin, and thermolysin digestion, the homogenized mixture was adjusted to pH 7.0 using sodium hydroxide. The homogenized mixtures were boiled for a period of 10 minutes. A cleaving agent, pepsin, chymotrypsin, trypsin, and thermolysin, was then added to the mixtures in a quantity resulting in a final concentration of 800 μg/ml. 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.

The supernatant was diluted 1:10 in deionized water with 0.1% trifluoroacetic acid (TFA) to form samples for

LC/MS injections. 10 μg 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 C₁₈ 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:

ExtractConcentration(μg I mL) x ExtractVolume(mL)

Concentration{μg I g) =

SampleSιze(g)

Tables 5 to 11 summarize the quantities of the peptides identified from the pepsin, chymotrypsin, trypsin, and thermolysin digestions of the protein.

Table 5 Peptides Released from Enzyme Digested Pork

Yield rate > 0.1 mg/g fres food Concentration of peptides released mg peptides/g fresh food

Table 6 Peptides Released from Enzyme Digested Beef

Yield rate > 0.1 mg g fresh food Concentration of peptides released = mg peptides/g fresh food

Yield rate > 0.1 mg g fresh food Concentration of peptides released mg peptides/g fresh food

Table 10 Peptides Released from Enzyme Digested Soy

Yield rate > 0.1 mg g fresh food Concentration of peptides released = mg peptides/g fresh food Example 4 - Peptide Screen Results from Various Proteins Digested in various enzymes

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. In all of the figures, 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

1,200 μg/mL while the SCRN02 bars should also be constant at approximately 1,500 μg/mL. A quality assurance feature of Figs. 6 to 10 is that only compounds listed in Table 11 for SCRNOl should be detected in SCRNOl, while only compounds listed in Table 11 for SCRN02 should be detected in SCRN02. All data reported in this Example meet this criteria.

Table 11 LC/MS Detection Results for Peptide Standard Solutions

Claims

We Claim :

1. A process of preparing an anti-hypertension peptide from a protein material which comprises: a. mixing the source protein material with an aqueous liquid to form an initial mixture wherein the source protein material is 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; b. adjusting the initial mixture to a pH favorable for a selected protein cleaving agent; c. adding the cleaving agent to the initial mixture forming a digest mixture wherein a reaction between the cleaving agent and the protein material cleaves the protein material at desired locations; d. halting the reaction; and e. isolating an anti-hypertension peptide from the digest mixture wherein the anti-hypertension peptide is a peptide sequence selected from the group of Val-Pro-Pro, Ile-Pro-Pro, Leu-Lys-Pro- Asn-Met, Lys-Pro-Asn-Met, Lys-Pro-Asn, Pro-Asn- Met, Leu-Lys, Pro-Asn, Asn-Met Tyr-Pro, Glu-Ala- Pro, Tyr-Lys-Pro, and Leu-Ala-Pro and salts thereof .

2. The process of claim 1 wherein the digest mixture is separated into solid and supernatant layers after the reaction is halted.

3. The process of claim 1 wherein the anti -hypertension peptide is isolated from the supernatant layer.

4. The process of claim 1 wherein the pH of the initial mixture is adjusted by addition of an acid or base wherein the acid or base is selected from the group consisting of NaOH or HCl .

5. The process of claim 1 wherein the protein cleaving agent is an enzyme selected from the group consisting of thermolysin, pepsin, trypsin, chymotrypsin, papain, Pronase E, Proteinase K, or Actinase E.

6. The process of claim 1 wherein the protein cleaving agent is thermolysin.

7. The process of claim 1 wherein the reaction is halted by a reaction halting method selected from the group consisting of boiling the digest mixture, altering the pH of the digest mixture, filtering the cleaving agent from the digest mixture, and a combination thereof.

8. The process of claim 1 wherein the milk or milk product is not fermented by a lactic acid bacteria and the peptide sequence is selected from the group of Val-Pro- Pro or Ile-Pro-Pro.

9. The process of claim 1 wherein the initial mixture is boiled before adding the cleaving agent .

10. The process of claim 1 wherein the aqueous liquid is selected from group consisting of water, distilled water, and a buffer.

11. A process of preparing an anti-hypertension peptide from a protein material which comprises: a. mixing the protein material with an aqueous liquid to form an initial mixture wherein protein material is milk, soybeans, wheat, rice, peanuts, beef muscle, lamb muscle, pork muscle, turkey muscle, chicken muscle, yeast, bacteria, or products thereof; b. adjusting the initial mixture to a pH favorable for a selected protein cleaving agent; c. boiling the initial mixture; d. adding the cleaving agent to the initial mixture forming a digest mixture wherein a reaction between the cleaving agent and the protein material cleaves the protein material at desired locations; e. halting the reaction in the digest mixture; f . isolating anti-hypertension peptide from the digestion mixture wherein the anti -hypertension peptide is Leu-Lys-Pro and its salts.

12. The process of claim 11 wherein the digest mixture is separated into solid and supernatant layers after the cleaving agent is inactivated.

13. The process of claim 12 wherein the anti -hypertension peptide is isolated from the supernatant layer.

14. The process of claim 11 wherein the pH of the initial mixture is adjusted by addition of an acid or base wherein the acid or base is selected from the group consisting of NaOH or HCl.

15. The process of claim 11 wherein the protein cleaving agent is an enzyme selected from the group of thermolysin, pepsin, trypsin, chymotrypsin, papain, Pronase E, Proteinase K, and Actinase E.

16. The process of claim 11 wherein the protein cleaving agent is thermolysin.

17. The process of claim 11 wherein the reaction is halted by a reaction halting method selected from the group consisting of boiling the digest mixture, altering the pH of the digest mixture, filtering the cleaving agent from the digest mixture, and a combination thereof.

18. The process of claim 11 wherein the aqueous liquid is selected from group consisting of water, distilled water, and a buffer.

19. A process of preparing an anti-hypertension peptide from a protein material which comprises: a. mixing the protein material with an aqueous liquid to form an initial mixture wherein protein material is milk, corn, wheat, rice, peanuts, beef muscle, lamb muscle, pork muscle, turkey muscle, chicken muscle, yeast, bacteria, or products thereof; b. adjusting the initial mixture to a pH favorable for a selected protein cleaving agent; c. boiling the initial mixture; d. adding the cleaving agent to the initial mixture forming a digest mixture wherein a reaction between the cleaving agent and the protein material cleaves the protein material at desired locations; e. halting the reaction in the digest mixture; f . isolating anti-hypertension peptide from the digestion mixture wherein the anti-hypertension peptide is Leu-Lys-Pro-Asn and its salts.

20. The process of claim 19 wherein the digest mixture is separated into solid and supernatant layers after the cleaving agent is inactivated.

21. The process of claim 20 wherein the an i -hypertension peptide is isolated from the supernatant layer.

22. The process of claim 19 wherein the pH of the initial mixture is adjusted by addition of an acid or base wherein the acid or base is selected from the group consisting of NaOH or HCl.

23. The process of claim 19 wherein the protein cleaving agent is an enzyme selected from the group of thermolysin, pepsin, trypsin, chymotrypsin, papain, Pronase E, Proteinase K, and Actinase E.

24. The process of claim 19 wherein the protein cleaving agent is thermolysin.

25. The process of claim 19 wherein the reaction is halted by a reaction halting method selected from the group consisting of boiling the digest mixture, altering the pH of the digest mixture, filtering the cleaving agent from the digest mixture, and a combination thereof.

26. The process of claim 19 wherein the aqueous liquid is selected from group consisting of water, distilled water, and a buffer.