JP2014005243A - Dipeptide inhibitor of angiotensin-converting enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide five useful dipeptides having hypotensive effect and to provide peptide compositions containing them.SOLUTION: The composition is obtained as follows: the residual water-insoluble protein after the hot water extraction of fish meat protein is pulverized and dispersed in water, to which protease is applied under the optimum condition of pH5.0-7.0 at a temperature of 40-60°C for enzymatic hydrolysis of the water-insoluble protein; the enzyme reaction is ended, water-insoluble particles are removed from the obtained hydrolysis reaction product to obtain an aqueous solution containing hydrophobic or hydrophilic, high molecular weight or low molecular weight peptides and water soluble amino acids; then the aqueous solution is fractioned by the hydrophobic resin column method, the adsorbed fraction is then subjected to ultrafiltration (M.W. 1000) to obtain the permeate as the final purified product. Dipeptides have sequences of valine - tryptophan, tryptophan - tyrosine, tryptophan - methionine, methionine - tryptophan or isoleucine - tryptophan.

Description

  The present invention relates to five useful dipeptides having an activity to inhibit angiotensin converting enzyme (ACE) and thereby exhibiting a blood pressure lowering action, and peptide compositions containing the same. The production method for obtaining the dipeptide of the present invention and the peptide composition is obtained by hydrolyzing a fish protein, particularly an insoluble protein remaining as a residue when bonito is extracted with hot water with a protin NY100 (Amano Enzyme) enzyme. After adsorbing to a hydrophobic resin, desorption with water-containing alcohol, permeation through an ultrafiltration membrane (molecular weight 1000), and its strong ACE inhibitory activity fraction is an active ingredient for angiotensin converting enzyme inhibitors and blood pressure lowering agents. Available. The dipeptide having high ACE inhibitory activity of the present invention and the peptide composition containing it are expected to be useful for the treatment or prevention of hypertension.

  Hypertension is a typical lifestyle-related disease, and it is said that the number of patients in Japan including the reserve army is 54.9 million (Ministry of Health, Labor and Welfare: 2006 National Health and Nutrition Survey). According to a 2012 WHO survey, a quarter of the world's population is reported to have hypertension or its reserves. Hypertension has few subjective symptoms and is also called a silent killer, and is known to cause various complications such as cerebral hemorrhage, subarachnoid hemorrhage, cerebral infarction, myocardial infarction, angina pectoris, nephrosclerosis, etc. Various studies have been conducted on the mechanism.

The renin / angiotensin system involved in pressurization and the kallikrein / kinin system involved in hypotension play important roles in the regulation of blood pressure. In the renin-angiotensin system, angiosinogen secreted from the liver is converted into angiotensin I by renin produced in the kidney, and further converted into angiotensin II by angiotensin converting enzyme (ACE). This angiotensin II contracts vascular smooth muscles and increases blood pressure. On the other hand, antihypertensive kallikrein acts on kininogen to produce bradykinin. This bradykinin has the effect of dilating blood vessels and lowering blood pressure, while ACE has the action of degrading this bradykinin. Thus, it is known that ACE is involved in the increase of blood pressure by two actions of production of angiotensin II which is a pressor peptide and inactivation of bradykinin which is a hypotensive peptide.
Therefore, it is possible to suppress an increase in blood pressure by suppressing the enzyme activity of ACE. Captopril (D-2-methyl-3-mercaptopropanoyl-L-proline) and enalapril, which are proline derivatives developed as ACE inhibitory active substances, are widely used for the treatment of hypertension. However, in the case of pharmaceuticals, dry cough, which is a side effect, is recognized by taking it, and it is also true that there is a problem in terms of QOL. It is also known to rebound when a drug is taken off.

Therefore, there is a trend of investigating components having antihypertensive action from natural products, or purifying them from natural products and utilizing them. Among them, it has recently been reported that peptides that are enzymatic degradation products of food material proteins have ACE inhibitory activity. For example, a collagenase degradation product of gelatin (patent document 1), a trypsin degradation product of casein (patent document 2, patent document 3 and patent document 4), a pepsin degradation product of sardine muscle (patent document 5), and a thermolysin degradation product of bonito ( A number of reports have been made such as Patent Document 6), thermolysin degradation product of sesame protein (Patent Literature 7), degradation product of κ-casein such as pepsin (Patent Literature 8). This is because these angiotensin converting enzyme inhibitors derived from natural products can be obtained from foods or food raw materials, and therefore are expected to be low-toxic and highly safe antihypertensive agents.
ACE inhibitors are also found in microorganisms and various foods, and their practical application as antihypertensive agents has been studied (Non-patent Document 1).
Several reports have been made on methods for producing peptides having ACE inhibitory activity (Patent Documents 9, 10, 11, 12, 13, 14, 15).

Japanese Patent Laid-Open No. 52-148631 JP 58-109425 A JP-A 61-36226 JP-A-61-36227 Japanese Patent Laid-Open No. 3-11097 JP-A-4-144696 JP-A-8-231588 JP-A-8-269088 JP-A-6-7188 Japanese Patent No. 2794094 Japanese Patent No. 2873318 JP 2006-347937 A JP 2001-240600 A JP-A-6-298794 JP 2010-155788 A

Sueami Kunio, Fermentation and Industry, Volume 46 (No. 3), 179-182 (1988)

Peptides having ACE inhibitory activity derived from food materials have few problems in terms of safety such as side effects and toxicity, and it is a great advantage that they can be taken as normal foods.
However, many of the peptides reported above have 5 or more constituent amino acids (Patent Documents 1, 2, 3, 4, 5, 8). Since these peptides with a large number of amino acid residues are easily degraded by digestive enzymes such as pepsin, trypsin, chymotrypsin after ingestion, their ACE inhibitory activity disappears in the body, and even when they are not degraded, their molecular structure is large. It is said that it is difficult to absorb.
Furthermore, with regard to the ACE-inhibiting peptide composition and its production method, Patent Document 9 discloses ACE inhibition having a blood pressure lowering action of a fraction removed from a permeate by ultrafiltration membrane (molecular weight 3000-10000) with a low-molecular-weight reverse osmosis membrane. Although a peptide mixture has been obtained, the purpose is to increase the inhibitory activity by removing salts and free amino acids, the fraction has not been reached, and the tracking of specific component peptides in the reverse osmosis membrane has not been clarified. In Patent Document 10, it is said that the target oligopeptide can be obtained by separation and purification by column chromatography from a substance having a molecular weight of 10,000 or less derived from figs. In particular, industrial production that is industrially effective from natural products has not been achieved, and only the purification of the ultrafiltration membrane 10000 has been shown. Patent Document 11 is a thermolysin hydrolyzate of zein or gluten meal, and there is a method obtained by gel filtration or ultrafiltration in which the content of a fraction having a molecular weight of 10,000 or less is 30% or more based on the solid content, There is no evidence that the molecular weight of 1000 or less contained in the permeated liquid having a molecular weight of 10,000 is 95%. In Patent Document 12, an antihypertensive peptide derived from livestock meat protein, which is a peptide-containing composition obtained by hydrolyzing myosin and actin with an enzyme such as Amano S, is obtained. However, purification is limited to a lab scale. The synthesis of isolated peptides has been described. Its industrial mass production only describes general enzyme preparation methods. In addition, a resin is used to purify the inhibitory peptide, but its purpose is to determine the structure. Patent Document 13 describes a method of treating a protein ACE-inhibiting enzyme degradation product with activated carbon having an average pore diameter of 3 nm or less, and can remove bitterness and odor without reducing ACE inhibitory activity. I'm trying. It is not aimed at increasing inhibitory activity. Patent Document 14 proposes a method of removing a bitter peptide from a non-adsorbed fraction by contacting with a synthetic resin, and describes that inhibitory activity remains in the non-adsorbed fraction. On the other hand, in the present invention, high ACE inhibitory activity is observed in the adsorbed fraction, ACE non-inhibitory activity is shown in the non-adsorbed fraction, and the low-molecular-weight ACE having superior digestion resistance in the permeate of ultrafiltration. Inhibitory active peptides can be obtained.
Furthermore, the obtained ACE inhibitory activity peptide can be purified, isolated and identified by high-inhibition activity dipeptide by one HPLC operation by loading on HPLC. In general, it has been pointed out that the isolation of a peptide from an enzymatic degradation product is complicated because a plurality of column operations are required about 5 times, and this indicates that the ACE-inhibiting peptide of the present invention has high accuracy. The fact that it is fractionated suggests that there are few unnecessary peptides. Furthermore, the ACE inhibitory peptide of the present invention is a collection of peptides having high ACE inhibitory activity, and therefore exhibits a high titer of the antihypertensive effect.

Accordingly, an object of the present invention is to provide a dipeptide having a high activity of inhibiting ACE, which is difficult to be degraded by digestive enzymes when ingested orally, is less likely to lose ACE inhibitory activity in the body, and can be absorbed as it is in the small intestinal mucosa. Providing a peptide composition.
Furthermore, the present invention provides an angiotensin converting enzyme inhibitor, an antihypertensive agent, or a food or drink composition (food or drink or food for specified health use) containing one or more of the above-mentioned dipeptides.

  As described above, natural organic substances and food-derived angiotensin converting enzyme inhibitors are highly important because of their safety to the human body, and are a major research subject for the prevention of lifestyle-related diseases.

  The present invention has been made to solve this problem, and by effectively inhibiting an angiotensin converting enzyme, a novel and safe substance that suppresses an increase in blood pressure is found from a food material. The purpose is to provide a food material containing an angiotensin converting enzyme inhibitory substance by clarifying the structure and developing an appropriate concentration method in terms of quality and price.

Peptides that solve the above problems are not present in the product degradation product obtained by hydrolyzing water-insoluble protein remaining as a residue after hot water extraction of bonito with protease Protin NY100 (Amano Enzyme). Therefore, a search was performed to determine whether the product degradation product contains a peptide having an amino acid number of 2 or less and having ACE inhibitory activity.
As a result, five dipeptides having the following amino acid sequences and having ACE inhibitory activity were found in the hydrolyzed product of water-insoluble protein protin NY100 (Amano Enzyme), which is a residue extracted from bonito hot water. The present invention was completed successfully.

  A dipeptide comprising the sequence of valine-tryptophan, a dipeptide comprising the sequence of tryptophan-tyrosine, a dipeptide comprising the sequence of tryptophan-methionine, a dipeptide comprising the sequence of methionine-tryptophan or a dipeptide comprising the sequence of isoleucine-tryptophan, and angiotensin Provided are dipeptides having converting enzyme inhibitory activity. Moreover, this invention provides the composition for eating and drinking which comprises 1 or more types of the said dipeptide, an angiotensin converting enzyme inhibitor, and a blood pressure lowering agent.

The present inventors have conducted various studies, and as a result, the presence of an angiotensin converting enzyme inhibitor in the cocoon protein is presumed, and the angiotensin converting enzyme inhibitor in the bonito protein is a reverse phase partition resin. It has been found that it has a property of being adsorbed on the surface. Furthermore, it was also found that a highly active fraction resistant to digestion with an ultrafiltration (molecular weight 1000) membrane permeate was obtained. This inhibitor decomposes the insoluble protein residue obtained by hot water extraction from bonito with a protease, preferably a protease for the food industry, in particular, protin NY100 (Amano Enzyme) into a hydrophobic adsorption resin. Adsorption, elution with a water-containing organic solvent, and the ultrafiltration membrane (molecular weight 1000) treatment give a high yield of the ACE inhibitory permeate, and it can be easily concentrated. for ingredients, using a UPLC chromatography to isolate a component having a strong ACE inhibitory activity, was subjected to measurements and structural analysis of inhibitory activity value (IC ₅₀ value) of its components, the amino acid sequence represented by this component This is because it has been found to be a dipeptide having an angiotensin converting enzyme inhibitory activity.

  In order to achieve the above-mentioned object, a dipeptide consisting of a valine-tryptophan sequence, a dipeptide consisting of a tryptophan-tyrosine sequence, a dipeptide consisting of a tryptophan-methionine sequence, a dipeptide consisting of a methionine-tryptophan sequence, or an isoleucine-tryptophan An angiotensin converting enzyme inhibitory substance mainly comprising an angiotensin converting enzyme inhibitory peptide consisting of a dipeptide consisting of the above-mentioned sequence, and after decomposing bonito hot water extract residue protein with a protease, for example, protin NY-100 (Amano Enzyme) An angiotensin converting enzyme inhibitor characterized by being immediately adsorbed on a hydrophobic adsorption resin, eluted with a water-containing organic solvent, and having high ACE inhibitory activity in an ultrafiltration membrane (molecular weight 1000) permeate. It is intended to provide. In general, since ultrafiltration membrane treatment takes several days, the problem of spoilage has not been solved for its use, and there are no examples of its use in foods. As a problem, there is no clear solution for lowering the temperature and lowering the pH because of problems of equipment costs, running costs, and quality problems due to the use of hydrochloric acid. Further, although there is a combined use of an ultrafiltration membrane (molecular weight 10,000) and a reverse osmosis membrane, an accurate molecular weight fraction is not achieved as in the present invention. By improving this point and performing desorption of hydrophobic resin treatment with a 50% ethanol solution, the fraction having no inhibitory activity and free amino acids are removed to increase the ACE inhibitory activity in the first purification stage, Next, an ACE-inhibitory peptide obtained from a stable production process free from spoilage, in which highly active components are observed in the permeate obtained by the ultrafiltration membrane (molecular weight 1000) treatment process in the presence of alcohol, is provided.

  Hydrophobic adsorption resin, that is, aromatic modified resin (for example, manufactured by Mitsubishi Chemical: Sepabead SP207) is an aromatic (styrene-divinylbenzene) synthetic adsorbent in which bromine is chemically introduced into an aromatic ring, and has a pore size. Since the surface has a strong hydrophobic adsorptivity, it is considered to exhibit excellent adsorption performance even for highly hydrophilic organic substances (substances with low hydrophobicity). Amino acid separation purification, protein removal, natural extract purification, before fermentation broth It may be used for processing or the like.

  The dipeptide used in the present invention is an enzymatic degradation method of bonito hot water extraction residue protein, a method of introducing amino acids stepwise by an organic chemical synthesis method, a peptide synthesis method utilizing a reverse reaction of hydrolase, a genetic engineering method Etc. can be manufactured.

  A method for producing the above-mentioned dipeptide by an enzymatic decomposition method of water-insoluble protein, which is a residue of hot water extraction from bonito, will be described. The case where the hot water extraction insoluble fraction which refine | purified it further as a raw material koji protein is demonstrated.

  Preferred examples of the working enzyme include protease for food industry. An example is protin NY100 (Amano Enzyme). Here, Protin NY100 (Amano Enzyme) is derived from Bacillus amyloliquefaciens, has an optimum pH of 7.0, and an optimum temperature of 55 ° C. On the other hand, the substrate concentration may be any as long as it is within the range where stirring and mixing can be performed during the reaction, but it is preferably performed within the range of protein concentration of 2 to 30% (w / v) where stirring is easy. The addition amount varies depending on the titer, but is usually 0.01% by weight or more, preferably 0.1 to 10% by weight per protein. The pH and temperature of the reaction may be the optimum pH and the vicinity of the optimum temperature. The pH is 5.0 to 9.0, preferably 6.0 to 7.5, and the temperature is 40 to 60 ° C, preferably 45 to 55 ° C. It is. The pH during the reaction is adjusted with an aqueous sodium hydroxide solution, hydrochloric acid, or the like, if necessary.

  The enzyme reaction time varies depending on the amount of enzyme added, the reaction temperature, and the reaction pH, but is not constant, but is usually about 1 to 50 hours.

  The enzymatic decomposition reaction can be stopped according to a known method such as heating of the hydrolyzate or deactivation of the enzyme due to a change in pH. Next, the hydrolyzed liquid is subjected to solid-liquid separation (for example, centrifugation, filtration, etc.), and the separated liquid is fractionated by ultrafiltration, gel filtration, or the like to obtain a liquid containing, for example, a fraction having a molecular weight of 10,000 or less. This liquid contains the dipeptide of the present invention, and the liquid or its concentrate (for example, spray dried) can be further fractionated to obtain the target dipeptide.

  The acid addition salt of this dipeptide can be manufactured by a conventional method. For example, it can be obtained by reacting the present dipeptide (containing a basic amino acid residue) with 1 equivalent of an appropriate acid in water and freeze-drying.

  This dipeptide and its acid addition salt have an ACE inhibitory action and thus a blood pressure lowering action, and are expected to be effective in the treatment and prevention of hypertension in mammals including humans.

  The present dipeptides and their acid addition salts are used as such or usually in at least one pharmaceutical adjuvant and pharmaceutical composition.

  The present dipeptide and acid addition salts thereof can be administered parenterally (that is, intravenous injection, rectal administration, etc.) or orally, and can be formulated into a form suitable for each administration method.

  Formulation forms as injections usually include sterile aqueous solutions. Formulations of the above forms are also buffer pH adjusters (sodium hydrogen phosphate, citric acid, etc.), isotonic agents (sodium chloride, glucose, etc.), preservatives (methyl paraoxybenzoate, propyl p-hydroxybenzoate, etc.) And other pharmaceutical adjuvants other than water. The preparation can be sterilized by filtration through a bacteria-retaining filter, mixing of a bactericide into the composition, irradiation of the composition or heating. The preparation can also be produced as a sterilized solid composition and dissolved in sterilized water before use.

  Orally administered drugs are formulated in a form suitable for absorption by the gastrointestinal tract. Tablets, capsules, granules, fine granules, powders are conventional pharmaceutical adjuvants such as binders (syrup, gum arabic, gelatin, sorbit, tragacanth, polyvinylpyrrolidone, hydroxypropylcellulose, etc.), excipients (lactose Sugar, corn starch, calcium phosphate, sorbit, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silica, etc.), disintegrants (potato starch, carboxymethyl cellulose, etc.), wetting agents (sodium lauryl sulfate, etc.) Can be included. Tablets can be coated by conventional methods. The oral solution can be made into a dry product such as an aqueous solution. Such oral solutions may contain conventional additives such as preservatives (methyl or propyl p-hydroxybenzoate, sorbic acid, etc.).

  The amount of the present dipeptide or its acid addition salt in the present ACE inhibitor or antihypertensive agent can vary, but usually 5 to 10% (w / w), especially 10 to 60% (w / w) is appropriate. It is. The dosage of the present ACE inhibitor or antihypertensive agent is suitably 0.01 to 50 mg / kg / day as an active ingredient when administered to humans.

In addition, since this dipeptide has the advantage that it does not adversely affect the living body even if it is ingested in large amounts, it has the function of lowering blood pressure and preventing hypertension as it is, or with various nutrients added or contained in foods and drinks. It may be eaten as a functional food or health food. That is, for example, by adding nutrients such as various vitamins and minerals, for example, liquid foods such as energy drinks, soy milk and soup, solid foods of various shapes, and powders as they are or added to various foods. You can also. The content and intake of the active ingredient in the ACE inhibitor or antihypertensive agent as a functional food and a health food may be the same as the content and dose in the above-mentioned pharmaceutical product, respectively.
There are two kinds of organic chemical synthesis methods of the above-mentioned dipeptides, a liquid phase method and a solid phase method, both of which are conventional methods, for example, Nobuo Izumiya, Tetsuo Kato, Tohiko Aoyagi and Noriaki Wakimichi, Experiment ", Maruzen Co., Ltd., 1985. In the liquid phase method, for example, an amino acid that should be located at the C-terminus of the present dipeptide and whose carboxyl group is protected with a benzyl group (Bzl), t-butyl group (t-Bu) or the like, and the C-terminal amino acid An amino acid that should be located next to the amino acid and whose α-amino group is protected with t-butyloxycarbonyl group (Boc), benzyloxycarbonyl group (Z), etc. is dissolved in dimethylformamide (DMF), dimethylacetamide, etc. They are reacted in the presence of dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBT), usually at room temperature overnight. Next, the dipeptide derivative after removal of the amino protecting group of the product by a conventional method is reacted in the same manner as the third amino acid with the amino group protected if necessary, the amino protecting group is removed, and the same procedure is performed as necessary. Repeat to obtain the dipeptide derivative. If the amino acid to be reacted has a hydroxyl group, a guanidino group or an imidazolyl group, these groups should generally be protected prior to the reaction. Protecting group for alcoholic hydroxyl group includes Bzl, t-Bu, etc., protecting group for phenolic hydroxyl group includes Bzl, etc., protecting group for guanidino group includes tosyl group (Tos), and protecting group for imidazolyl group includes Tos etc. . After completion of the final reaction, all protecting groups are removed to obtain the dipeptide. Introduction and removal of these protecting groups can be carried out by conventional methods.

  On the other hand, regarding the solid phase method, a method using a peptide synthesizer has been widely used in recent years. For example, the present tripeptide can be produced using a 430A type peptide synthesizer manufactured by Applied Biosystems. That is, basically, phenylacetamidomethyl (PAM) resin L-Xaa-O-CH2-PAM (Xaa) to which an amino acid located at the C-terminus of this dipeptide is bonded is an amino acid residue) (obtained from Applied Biosystems) The α-amino acid (Boc-amino acid) whose amino group is protected with Boc is extended stepwise by repeated peptide bonds and removal of Boc. A Boc-amino acid is subjected to an extension reaction via its symmetrical anhydride as an intermediate by use of DCC. In the above Boc-amino acid or L-Xaa-O-CH2-PAM, if there is a reactive functional group that should not participate in the reaction, it should generally be protected by a suitable protecting group. In the synthesis system using the 430A type peptide synthesizer, in addition to the amino acid raw material, the following reagents and solvent are used: N, N-diisopropylethylamine (TFA neutralizer), TFA (Boc cleavage), MeOH (dissolution of the generated urea compound and Removal), HOBT (0.5M HOBT / DMF), DCC (0.5M DCC / dichloromethane (DCM), DCM and DMF (solvent), neutralizing agent (70% ethanolamine, 29.5% methanol) Neutralization) The amino acid raw material and these reagents and solvent are charged in place, and their use is automatically performed by the peptide synthesizer, and the reaction temperature and time are adjusted automatically, but the reaction temperature is usually The above procedure yields dipeptide-O-CH2-PAM in which the reactive group in the dipeptide is protected. The actual operation of the solid-phase peptide synthesis is carried out by Applied Biosystems according 430A peptide synthesizer User's Manual.

  The obtained reactive functional group-protected dipeptide-O—CH 2 -PAM was prepared by a conventional method, for example, the method described in “Peptide Synthesis Basics and Experiments” or the 430A type peptide synthesizer user's manual, for example, a protecting group. Treatment with trifluoromethanesulfonic acid (TFMSA) (TFASA is a diluent of TFMSA) in the presence of thioanisole and / or ethanedithiol as a scavenger to capture cations generated by cleavage of Cleavage to obtain the desired dipeptide.

  The dipeptide of the present invention may be produced by organic synthesis as described above. However, for the purpose of exerting ACE inhibitory activity by adding to foods and beverages or drugs to be taken orally, protein derived from bonito and the like is decomposed with protin NY100 (Amano Enzyme), and further isolated and purified. The obtained composition is preferably prepared as an orally ingestible composition containing at least one of the above five dipeptides.

  As raw materials, fish meat such as salmon, salmon roar, salmon koji, soda salmon, soda salmon, salmon, salmon, salmon, salmon, salmon, salmon, salmon, other dried knots, etc., and hot water extract residues thereof Can be used.

  When the dipeptide of the present invention is obtained by degradation with protin NY100 (Amano Enzyme), it is preferable to first remove the amino acid and water-soluble protein by heat treatment as a pretreatment of the koji protein as a raw material. In addition, in order to efficiently decompose protin NY100 (Amano Enzyme), it is preferable that the raw material is finely ground and then stirred and suspended in water. Moreover, although the obtained protein is sparingly soluble, temporary soda is added so as to obtain an optimum pH for the enzyme reaction, and uniformly dispersed, suspended and dissolved. To this, 0.1 to 10% by weight of protin NY100 (Amano Enzyme) is added per 100 g of protein, and the protein is added while stirring at pH 5.0 to 9.0 and temperature 40 to 55 ° C. for 0.5 to 30 hours. After the decomposition, the enzyme activity is deactivated by heat treatment (98 ° C., 15 minutes). After removing undegraded protein with a vibe screen, remove the undegraded product and precipitate by decanter, DeLaval, ultra-high speed centrifuge (15000 rpm), filtration (Celite filtration: Hyflo Super Celite, etc.) The filtrate obtained is neutralized with caustic soda or hydrochloric acid and then concentrated. The bonito peptide thus obtained contains 0.0005% to 0.5% by weight of valine-tryptophan, tryptophan-tyrosine, tryptophan-methionine, methionine-tryptophan, and isoleucine-tryptophan, respectively.

  As the dipeptide of the present invention, the above-obtained protin NY100 (Amano Enzyme) enzyme degradation product, and this is further treated with a high porous polymer resin (hydrophobic adsorption resin), an ion exchange resin or the like to obtain a high molecular protein or monomer The amino acid and further salts can be removed, and the polymer peptide can be removed by ultrafiltration, so that a crude product containing abundant dipeptide of the present invention can be obtained and used as it is. Hereinafter, such decomposed products and crude purified products are collectively referred to as a composition containing abundant dipeptides.

  When the peptide of the present invention is obtained by purification, the above-mentioned concentrate is subjected to gel filtration column chromatography, chromatography using an ion exchange resin or high porous polymer resin, affinity chromatography, etc. The peptide fractions are collected, and the active fractions can be further purified to almost pure peptides by a normal peptide purification method using high performance liquid chromatography using a reverse phase column such as an ODS column. In addition, the dipeptide of the present invention is not limited to bonito and bonito hot water extraction residue, but also from fish meat protein, potato, bonito hot water extraction residue, Soda cocoon, Soda bonito, Soda, Soda hot water extraction residue, etc. It can be obtained by the method shown. The ACE inhibitory activity of a dipeptide or a composition rich in it can be measured, for example, by the method described in Test Example 1.

  When the peptide of the present invention is obtained by chemical synthesis, the peptide can be synthesized by either a solid phase method or a liquid phase method used for usual peptide synthesis. The peptide of the present invention obtained by synthesis can be purified by a conventional purification method using reverse phase high performance liquid chromatography, chromatography using ion exchange resin or high porous polymer resin, affinity chromatography or the like. Further, the polymer peptide can be removed by ultrafiltration to obtain a peptide having increased ACE inhibitory activity and resistance to digestion.

  The specific activity of the ACE inhibitory action of the thus obtained dipeptide or a composition containing it abundantly can be used as a very useful ACE inhibitor. Furthermore, since it is well absorbed from the gastrointestinal tract and is relatively stable against heat, it can be applied to any form of food and drink and pharmaceutical preparation.

  Therefore, in this invention, the composition (food / beverage) which can anticipate the exhibit of the angiotensin converting enzyme inhibitory effect formed by adding one or more said dipeptides is provided. The present invention also provides an angiotensin converting enzyme inhibitor and an antihypertensive agent comprising one or more of the above-mentioned dipeptides.

  When the dipeptide of the present invention is used and blended in foods and drinks, pharmaceuticals, etc., a product obtained by sufficiently purifying the dipeptide from a decomposition product of prosthesis NY100 (Amano Enzyme) enzyme of bonito hot water extraction residue protein may be used. A synthetic product obtained by synthesis may be used. However, since the peptide of the present invention is stable and has a strong ACE inhibitory activity, as described above, a crude product or a protin NY100 (Amano Enzyme) enzymatic degradation product can be used as it is as a composition rich in dipeptides to obtain a sufficient ACE inhibitory activity. Can be obtained.

  The composition for eating and drinking of the present invention (food and beverage) is produced by adding 0.001 mg to 100 mg, preferably 0.01 mg to 20 mg of one or more of the above dipeptides as a single intake. The peptide composition of the present invention is a solid or powder that is easy to handle and stable, and has good solubility in water. Absorption from the gastrointestinal tract is also good. Therefore, there are no particular restrictions on the timing and method of addition to food, and it can be used as a powder, solution, suspension, etc., in the raw material stage, intermediate process, and final process of food production by a method commonly used in the food field. It is possible to add. By ingesting the food / drink composition containing the dipeptide of the present invention temporarily, intermittently, continuously or daily, an angiotensin converting enzyme is inhibited, and for example, a blood pressure lowering action is possible. Examples of the form of food and drink include solid, semi-fluid, and fluid. Examples of solid foods include sheet foods, tablets such as tablets and capsules, and general foods and health foods such as granular powders. Examples of the semi-fluid food include paste, jelly, and gel. Examples of the fluid food include general food and health food in the form of juice, soft drink, tea drink, and drink. It is also possible to suppress an increase in blood pressure by continuously ingesting the dipeptide of the present invention using food and drink as an energy drink or seasoning.

  The pharmaceutical composition in the form of an ACE inhibitor or an antihypertensive agent according to the present invention contains the dipeptide of the present invention in the same amount as the above-mentioned composition for eating and drinking. The pharmaceutical composition of the present invention may be temporarily administered to a patient with hypertension in order to inhibit the angiotensin converting enzyme of the patient and exert a hypotensive effect, for example, or the effectiveness of the pharmaceutical composition of the present invention Since the ingredients are derived from natural products, they can be used safely continuously. Hypertension can be treated or prevented by the pharmaceutical composition of the present invention. The form of the pharmaceutical composition is preferably an oral administration agent such as a tablet, capsule, granule or syrup. Formulations for parenteral administration include sterile solutions for administration through veins, arteries, subcutaneous, muscle, or for inhalation through the nasal passages. The liquid agent may be a dry solid that can be dissolved at the time of use. An injectable preparation can be produced as an injectable preparation by dissolving a dipeptide as an active ingredient in physiological saline and carrying out normal aseptic operation.

  In the means of the present invention, bonito is used as a raw material, and amino acid and water-soluble protein are removed by hot water extraction to obtain an insoluble protein residue. This bonito hot water extraction residue protein is subjected to an enzymatic degradation treatment with a protin NY100 (Amano Enzyme) enzyme.

  Next, this liquid after enzymatic decomposition is subjected to a vibe screen, DeLaval, sharp press, and celite filtration treatment, which is effective in increasing the adsorption capacity, and then loaded onto a column filled with a hydrophobic adsorption resin. Adsorption is carried out by passing through the column.

  The angiotensin converting enzyme inhibitor adsorbed on the hydrophobic adsorption resin is eluted using a water-containing organic solvent such as water-containing ethanol.

  Even when elution is performed with this solvent, the substance that dissolves in water is eluted by supplying water before supplying the solvent in order to effectively perform the elution by the solvent of the adsorbed inhibitory substance. It is effective to perform the processing. That is, the enzyme decomposition product aqueous solution is desirably about 2 to 10 times the volume of the column. After loading the column, water is then passed through about 2 to 10 times the amount of the column. All non-adsorbed fractions are eluted. Further, desorption is carried out at a 50% ethanol concentration to obtain the target adsorption fraction.

  For fractions containing angiotensin converting enzyme inhibitor eluted with ethanol solution, load the eluate to ultrafiltration (molecular weight 1000), concentrate the highly inhibitory permeate fraction under reduced pressure, and spray dry (spray dry) By doing so, a food material product mainly comprising an angiotensin converting enzyme-inhibiting peptide as a powder is obtained in the form of a powder.

  The food material mainly composed of this angiotensin converting enzyme inhibitory peptide can be converted to angiotensin by isolating the above eluate using high performance liquid chromatography and isocratic elution with acetonitrile / trifluoroacetic acid. A purified and isolated form of a component having strong enzyme inhibitory activity is obtained.

  According to the present invention, five kinds of dipeptides having angiotensin converting enzyme inhibitory activity are obtained by reaction with protin NY100 (Amano Enzyme) using bonito, which is a food that has been proven to be safe from a long dietary experience. Was made. It was also found that a highly active peptide fraction can be produced by adsorbing an enzymatic degradation product on a hydrophobic adsorption resin and eluting with a hydrous organic solvent. Furthermore, it was also found that a high ACE inhibitory peptide resistant to digestion was obtained by circulating through an ultrafiltration (molecular weight 1000 membrane). Furthermore, it was also found that an antihypertensive effect was expressed in a small amount in animal experiments. It is clear that bonito peptide containing dipeptide obtained by this production method is a safe and highly effective material for daily consumption, and is a food material that is very meaningful for the aging society in the future. Expected to be used for foods and functional foods. This production method can be widely used for the production of functional materials on an industrial scale.

It is a graph which shows the blood pressure lowering effect with respect to SHR of the koji peptide of Example 7. It is a graph which shows the blood pressure lowering effect with respect to SHR of the koji peptide of Example 7 by heart rate.

Test example 1
The ACE inhibitory activity was measured as follows. That is, the ACE inhibitory activity of the present dipeptide obtained as described above is obtained by changing the buffer solution of Cheung and Cushman's method (Biochemical Pharmacology, 20, 1637 (1971)) from a phosphate buffer solution to a borate buffer solution. It measured according to.
That is, 5 g of rabbit Lang acetone powder was dissolved in 50 ml of 0.1M sodium borate buffer (pH 8.3), centrifuged at 40,000 G for 40 minutes, the supernatant was further purified with hydroxyapatite, and 1 unit. An angiotensin converting enzyme solution of / mg protein was obtained. Alternatively, rabbit lang-derived purified ACE (Sigma, 0.25 unit) was used.

0.030 ml of each concentration of the dipeptide solution is placed in a test tube, and then 0.1 ml of the angiotensin converting enzyme solution is added, followed by reaction at 37 ° C. for 5 minutes. Next, 0.25 ml of hippuryl histidyl leucine (Peptide Institute, Bz-Gly-His-Leu.H ₂ O, final concentration 5 mM, NaCl 300 mM included) is added as a substrate and reacted at 37 ° C. for 30 minutes. I let you. Thereafter, 0.25 ml of 1N hydrochloric acid was added to stop the reaction, 1.5 ml of ethyl acetate was added, and the mixture was stirred with a vortex mixer for 20 seconds, followed by centrifugation (3000 rpm, 5 minutes). 1 ml of the ethyl layer was collected. After heating at 105 ° C. for 30 minutes (aluminum block), the absorption value at 228 nm of hippuric acid dissolved in 3 ml of distilled water and extracted into ethyl acetate was measured, and this was defined as enzyme activity.
The inhibition rate was calculated from the following formula. A: Absorption value at 228 nm when no inhibitor is included B: Absorption value at 228 nm when inhibitor is added Further, the concentration of the present otripeptide at an inhibition rate of 50% was defined as an IC ₅₀ value. Inhibition rate = [1- (A−a) / (B−b)] × 100
A: Sample added a: Sample added, buffer added instead of enzyme B: Distilled water added instead of sample b: Distilled water added instead of sample, buffer added instead of enzyme

Example 1
(A) Industrial production of peptide composition having ACE inhibitory activity Add 10000 L of water to 1000 kg of bonito protein, heat treatment (95 ° C., 35 minutes), remove amino acids and water-soluble protein, and obtain 1153 kg of hot water extraction residue 2884 L of water was added to (576 kg of protein), adjusted to pH 7 with 6N caustic soda, 1.0 wt% of protin NY100 (Amano Enzyme) enzyme (enzyme amount: per protein) was added, and the mixture was stirred at 50 ° C., 17 Reacted for hours. After the reaction, caustic soda was added to adjust the pH to 6.8, and the enzyme was inactivated by heating at 98 ° C. for 15 minutes. Thereafter, undegraded protein was removed by a vibe screen, DeLaval, and a centrifuge, and the supernatant was filtered through Celite to obtain a degradation solution (protein 200 kg). The ACE inhibitory activity IC50 value was 0.136 mg / ml (protein).

200 kg of this peptide was loaded onto hydrophobic chromatography.
The conditions for hydrophobic chromatography are described below.
Column load: 50 kgb
Column: SP-207 (1000 L capacity column; Nippon Netsusui)
Eluent: 0.5% ethanol solution flow rate: 2000 L / hour cycle: 4 cycles

Elution from the column packed with the hydrophobic adsorption resin was divided into two fractions according to the alcohol concentration, and two fractions of 8000 L each were collected. As a result of measuring the ACE inhibitory activity of each fraction by the method of Test Example 1, the ACE inhibitory activity value IC ₅₀ of the fraction eluted with 0% or 50% ethanol was not detected and was 0.047 mg / ml. . 4 cycles were repeatedly collected to obtain 74.9 kg of protein.

  Next, the ACE inhibitory activity fraction (50% ethanol-eluted fraction) obtained by hydrophobic chromatography was subjected to ultrafiltration membrane (GE 7.9 inch × 40 inch × 2, filter area 48.4 m 2; Molecular weight 1000, pressure 1.8 MPa; Model No. GE8040F1002) was passed through to obtain a 20-fold concentrated solution as the non-permeate. The ACE inhibitory activity of the non-permeate and the permeate was measured, and values of 0.034 mg / ml for the permeate and 0.110 mg / ml for the non-permeate were obtained.

The permeate was concentrated under reduced pressure and spray-dried to mass-produce a powder with a protein amount of 47.6 kg. The balance is shown in Table 1.

透過液のＡＣＥ阻害活性に高い値が示されたことより、消化酵素耐性の可能性が推定された。非透過液のＡＣＥ阻害活性（ＩＣ50）に１．６倍の強い値が示されたが、透過液の値には及ばなかった。 In addition, in order to confirm the degree of degradation by digestive enzymes in vivo when ingested orally, artificial digestion was performed on permeate and non-permeate with pepsin, trypsin, and chymotrypsin (pepsin, trypsin, chymotrypsin 4). Time resolution). The results are shown in Table 2.

The high value of the ACE inhibitory activity of the permeate showed the possibility of digestive enzyme resistance. The ACE inhibitory activity (IC50) of the non-permeate was 1.6 times stronger, but not as high as that of the permeate.

その結果、分解液と樹脂脱着非透過液の最大分子量はともに５０００であった。
目的とする樹脂脱着透過液の最大分子量は１５００で、うち分子量１０００以下の割合は７９．７５％であった。 The molecular weight analysis by the HPLC method was performed under the following conditions (Table 3). The results are shown in Table 4.

As a result, the maximum molecular weights of the decomposition solution and the resin desorption non-permeate were both 5000.
The maximum molecular weight of the target resin desorption permeate was 1500, and the ratio of the molecular weight of 1000 or less was 79.75%.

酵素ろ過液の樹脂処理により、タンパク質３９．９８％の収率で、バリン−トリプトファン１００％の回収率であった。
さらに、限外ろ過膜処理により、タンパク質６７．６６％の収率、濃縮液側１/２０倍の液量で、バリン−トリプトファンのロス率は１０％であった。よって、精製タンパク質収率は２７．０５％、バリン−トリプトファンの回収率は９０％であった。 The recovery rates in dipeptide purification are shown in Table 5 below.

By the resin treatment of the enzyme filtrate, the yield of protein was 39.98% and the recovery rate of valine-tryptophan was 100%.
Furthermore, by the ultrafiltration membrane treatment, the loss rate of valine-tryptophan was 10% with a protein yield of 67.66% and a liquid volume 1/20 times the concentrated liquid side. Therefore, the purified protein yield was 27.05% and the recovery rate of valine-tryptophan was 90%.

その結果、スケール１、２、３のいずれにおいても工業生産できることが分かった。 The production scale for mass production of bonito peptide is shown in Table 6.

As a result, it was found that industrial production was possible on any of scales 1, 2, and 3.

(B) Isolation of 5 Dipeptides Dipeptides in the ultrafiltration membrane permeate (bonito peptide) of the alcohol desorbed fraction after column adsorption of the koji protin NY100 enzymatic degradation solution were isolated.
Column: Acquity UPLC BEH C18
(2.1mmID x 100mmL, 1.7μm)
Mobile layer: 15% CH ₃ CN in 0.1% TFA
Flow rate: 0.2 ml / min
Temperature: 40 ° C
Detection: UV 200-300nm

  Under the above conditions, one fraction was collected every 30 seconds. From each fraction, after evaporating to dryness under reduced pressure, ACE inhibitory activity was measured according to the above method using a sample for ACE inhibitory activity measurement. As a result, a strong ACE inhibitory activity was observed in the peptide fraction. Each fraction was freeze-dried to obtain a trace amount of peptide. The fractions were subjected to amino acid analysis and TOF MS analysis, and the peptides of each fraction were found to be valine-tryptophan, tryptophan-tyrosine, tryptophan-methionine, methionine-tryptophan, and isoleucine-tryptophan.

Confirmation of further isolation of the fractions obtained above was performed by changing the concentration of the mobile phase and fractionating the peaks.
Column: Acquity UPLC BEH C18
(2.1mmID x 100mmL, 1.7μm)
Mobile layer: 10% CH ₃ CN in 0.1% TFA
Flow rate: 0.2 ml / min
Temperature: 40 ° C
Detection: UV 200-300nm
As a result of HPLC analysis, it was found that the purity was 95% or more.

Table 7 shows the ACE inhibitory activity values of 5 dipeptides isolated in the protin NY100 (Amano Enzyme) enzymatic degradation product of bonito hot water extraction residue.

Example 2
(A) Industrial production of peptide composition having ACE inhibitory activity 10000 L of water is added to 1000 kg of bonito protein, and after heat treatment (95 ° C., 35 minutes), amino acids and water-soluble proteins are removed, and 1774 kg of the obtained hot water extraction residue is obtained. After adding 4436 L of water to (886 kg of protein) and adjusting to pH 7 with 6N caustic soda, 1.0 wt% of Samoase PC10F (Daiwa Kasei) enzyme (enzyme amount: per protein) was added and stirred at 50 ° C. The reaction was carried out for 17 hours. After the reaction, caustic soda was added to adjust the pH to 6.8, and the enzyme was inactivated by heating at 98 ° C. for 15 minutes. Thereafter, undegraded protein was removed with a vibratory screen, DeLaval, and a centrifuge, and the supernatant was filtered through Celite to obtain an enzymatic degradation solution (200 kg of protein). The ACE inhibitory activity IC50 value was 0.204 mg / ml (protein).

200 kg of this peptide was loaded onto hydrophobic chromatography.
The conditions for hydrophobic chromatography are described below.
Column load: 50kg
Column: SP-207 (1000 L capacity column; Nippon Netsusui)
Eluent: 0.5% ethanol solution flow rate: 2000 L / hour cycle: 4 cycles

Elution from the column packed with the hydrophobic adsorption resin was divided into two fractions according to the alcohol concentration, and two fractions of 8000 L each were collected. As a result of measuring the ACE inhibitory activity of each fraction by the method of Test Example 1, the ACE inhibitory activity value IC ₅₀ of the fraction eluted with 0% or 50% ethanol was not detected, and was 0.071 mg / ml. The fractionation was repeated 4 cycles to obtain a protein amount of 74.0 kg.

  Next, the ACE inhibitory activity fraction (50% ethanol-eluted fraction) obtained by hydrophobic chromatography was subjected to ultrafiltration membrane (GE 7.9 inch × 40 inch × 2, filtration area 48.4 m 2; Molecular weight 1000, pressure 1.8 MPa; Model No. GE8040F1002) was passed through to obtain a 20-fold concentrated solution as the non-permeate. The ACE inhibitory activity of the non-permeate and the permeate was measured, and a value of 0.050 mg / ml was obtained for the permeate and 0.165 mg / ml for the non-permeate.

The permeate was concentrated under reduced pressure and spray-dried to mass-produce a powder having a protein amount of 71.4 kg. The balance is shown in Table 8.

透過液のＡＣＥ阻害活性に高い値が示されたことより、消化酵素耐性の可能性が推定された。非透過液のＡＣＥ阻害活性（ＩＣ50）に１．５倍の強い値が示されたが、透過液の値には及ばなかった。 In addition, in order to confirm the degree of degradation by digestive enzymes in vivo when ingested orally, artificial digestion was performed on permeate and non-permeate with pepsin, trypsin, and chymotrypsin (pepsin, trypsin, chymotrypsin 4). Time resolution). The results are shown in Table 9.

The high value of the ACE inhibitory activity of the permeate showed the possibility of digestive enzyme resistance. The ACE inhibitory activity (IC50) of the non-permeate was 1.5 times stronger, but not as high as that of the permeate.

その結果、酵素分解液と樹脂脱着非透過液の最大分子量はともに５０００であった。
目的とする樹脂脱着透過液の最大分子量は１５００で、うち分子量１０００以下の割合は７９．８％であった。 Molecular weight analysis by the HPLC method was performed under the following conditions (Table 10), and the results are shown in Table 11.

As a result, the maximum molecular weight of both the enzyme decomposition solution and the resin desorption non-permeate was 5000.
The maximum molecular weight of the target resin desorption permeate was 1500, and the ratio of the molecular weight of 1000 or less was 79.8%.

その結果、スケール１，２，３のいずれにおいても工業生産できることが分かった。 The production scale for mass production of bonito peptide is shown in Table 12.

As a result, it was found that industrial production was possible on any of scales 1, 2, and 3.

(B) Isolation of 5 Dipeptides The dipeptides in the permeate of the ultrafiltration membrane (bonito peptide) of the alcohol desorption fraction after column adsorption of the bonito samoyase PC10F (Daiwa Kasei) enzyme degradation solution were isolated.
Column: Acquity UPLC BEH C18
(2.1mmID x 100mmL, 1.7μm)
Mobile layer: 15% CH ₃ CN in 0.1% TFA
Flow rate: 0.2 ml / min
Temperature: 40 ° C
Detection: UV 200-300nm

  Under the above conditions, one fraction was collected every 30 seconds. From each fraction, after evaporating to dryness under reduced pressure, ACE inhibitory activity was measured according to the above method using a sample for ACE inhibitory activity measurement. As a result, a strong ACE inhibitory activity was observed in the peptide fraction. Each fraction was freeze-dried to obtain a trace amount of peptide. The fractions were subjected to amino acid analysis and TOF MS analysis, and the peptides of each fraction were found to be dipeptides consisting of the sequences of valine-tryptophan, tryptophan-tyrosine, tryptophan-methionine, methionine-tryptophan, and isoleucine-tryptophan.

Example 3
Synthesis of peptides by synthetic methods:
Using an automatic peptide synthesizer (Applied Biosystems) (ABI 430 model), the peptide chain was sequentially extended from the C end by the BOC method according to the program to synthesize the target protected peptide resin.
After the construction of the peptide on the resin was completed, the protected peptide resin was dried. Cleavage of the obtained protected peptide from the deprotecting group and the peptide from the resin carrier was performed by anhydrous hydrogen fluoride treatment (HF / p-Creso18: 2 v / v, 60 minutes). The resulting crude peptide was extracted with 90% acetic acid and obtained as a powdered solid by lyophilization. Further, the obtained crude peptide was loaded onto a high performance liquid chromatograph using an ODS column and purified to obtain the target peptide.
Column: YMC-Pack ODS-A (30 mm ID × 250 mm L, YMC)
Mobile layer: Buffer A: 5% CH ₃ CN, 0.1% TFA
Buffer B: 40% CH ₃ CN, 0.1% TFA
Gradient: 0-10 min: 0% Buffer B
10 to 90 min: 0 to 100% Buffer B
Flow rate: 20 ml / min
Detection: UV220nm

The purity of the purified peptide was tested by high performance liquid chromatography using an ODS column.
Column: Zorbax 300SB-C18 (4.6 mm ID × 150 mm L,
(Agilent Technologies)
Mobile layer: Buffer A: 1% CH ₃ CN, 0.1% TFA
Buffer B: 60% CH ₃ CN, 0.1% TFA
Gradient: 0-25 min: 0-100% Buffer B
Flow rate: 1 ml / min
Detection: UV220nm

(A) Synthesis of valine-tryptophan dipeptide:
Boc-tryptophan (BrZ) resin (0.5 mmol) was used as the starting amino acid resin carrier, and the peptide chain was elongated using the amino acid derivative Boc-valine 2 mM. Purification was performed by the above method to obtain a purified product of valine-tryptophan.

(B) Synthesis of tryptophan-tyrosine dipeptide:
Boc-tyrosine (BrZ) resin (0.5 mmol) was used as the starting amino acid resin carrier, and the peptide chain was elongated using the amino acid derivative Boc-tryptophan 2 mM. Purification was performed by the above method to obtain a purified product of tryptophan-tyrosine.

(C) Synthesis of tryptophan-methionine dipeptide:
Boc-tryptophan (BrZ) resin (0.5 mmol) was used as the starting amino acid resin carrier, and the peptide chain was elongated using the amino acid derivative Boc-methionine 2 mM. Purification was performed by the above method to obtain a purified product of methionine-tryptophan. As a result of measuring the purity of the purified product by the above method, it was 95.00%.

(D) Synthesis of methionine-tryptophan dipeptide:
Boc-tryptophan (BrZ) resin (0.5 mmol) was used as the starting amino acid resin carrier, and the peptide chain was elongated using the amino acid derivative Boc-methionine 2 mM. Purification was performed by the above method to obtain a purified product of tryptophan-methionine.

(E) Synthesis of isoleucine-tryptophan dipeptide:
Boc-tryptophan (BrZ) resin (0.5 mmol) was used as the starting amino acid resin carrier, and the peptide chain was elongated using 2 mM of the amino acid derivative Boc-isoleucine. Purification was performed by the above method to obtain a purified product of isoleucine-tryptophan.

Example 4
Using the dipeptide isolate obtained in Example 1, a stock beverage having the following composition was produced.
(A) Material and blending amount:
500 ml of bonito hot water extract (mentsuyu) and a mixture of 5 types of dipeptides isolated in Example 1 (valine-tryptophan dipeptide 58.89 mg, tryptophan-tyrosine dipeptide 13.40 mg, tryptophan-methionine 20 0.000 mg, 13.16 mg methionine-tryptophan dipeptide, 39.20 mg isoleucine-tryptophan dipeptide).

(B) Manufacturing method:
After hot water extraction of bonito (95 ° C., opened for 35 minutes), Celite filtration was performed, and the decomposition solution was cooled to room temperature. To the obtained cooled extract, a mixture of the above five dipeptides was added and stirred and dissolved. This produced a dashi drink.

Example 5
(A) Determination of dipeptide from reaction mixture obtained by decomposing bonito protein hot water extraction residue with protin NY100 enzyme 2000 ml of water was added to 160 g of bonito protein and subjected to hot water extraction (95 ° C., 35 minutes). The resulting residue (insoluble protein) was hydrated 10 times, adjusted to pH 7, protein NY100 (Amano Enzyme) enzymatic degradation, adjusted to pH 6.8, heated (98 ° C., 15 minutes), vibe screen, decanter , DeLaval, sharp pressing, celite filtration, vacuum concentration, and spray drying.

  The above powder is loaded on a hydrophobic chromatograph, and after elution with 250 ml of water, a fraction highly active in elution with 250 ml of 50% ethanol is obtained. Furthermore, after the solution of ultrafiltration (molecular weight 1000 membrane) is concentrated under reduced pressure (solid 40%), it is spray-dried (inlet temperature 150 to 200 ° C, outlet temperature 50 to 90 ° C), and a highly active powder product. 500 mg is obtained.

鰹節ペプチドの錠果・錠剤・カプセル製造については、鰹節ペプチド、食品素材計量後、混合・造粒・打錠・コート工程後、ＡＣＥ阻害活性、ジペプチド量を測定して規格品を製品化する。 Using the above powder product (bonito peptide) as a raw material, a functional food containing 500 mg thereof is obtained. Used as processed foods in beverages, tablets, soups, etc. Table 13 shows the blending ratio of the food material blended with bonito peptide.

For the production of tablets, tablets, and capsules of bonito peptide, we measure the ACE inhibitory activity and the amount of dipeptide after the mixing, granulation, tableting and coating steps after weighing the bonito peptide and food material, and commercialize the standard product.

Example 6
The quantification of valine-tryptophan, tryptophan-tyrosine, tryptophan-methionine, methionine-tryptophan, and isoleucine-tryptophan was performed as follows. That is, the quantification of the present dipeptide was performed from the powder product or the processed product as follows.

Sep-Pak C18 pretreatment:
Enzyme digestion residue from bonito extract and its processed food were weighed 25 mg and 5 g of processed food, respectively, loaded onto a Sep-Pak C18 cartridge, and after removing the water-soluble fraction, the adsorbed fraction was eluted with 50% ethanol solution. Use the solution as a sample.

8000 μg of the ACE-inhibited purified peptide recovered from the sample obtained as described above after being treated with Sep-Pak C18 was dissolved in 100 μL of purified water, and a high-performance liquid chromatograph using a C-18 column was loaded with 2000 μg / 25 μL. The peptide was fractionated. The conditions are described below.
Column: Acquity UPLC BEH C18
(2.1mmID × 100mmL, 1.7μm)
Mobile layer: 15% CH ₃ CN in 0.1% TFA
Flow rate: 0.2ml / min
Temperature: 40 ° C
Detection: UV 200-300nm
Synthetic products valine-tryptophan, tryptophan-tyrosine, tryptophan-methionine, methionine-tryptophan, and isoleucine-tryptophan were loaded as standard samples at 1 μg / μL. The elution times of valine-tryptophan, tryptophan-tyrosine, tryptophan-methionine, methionine-tryptophan, and isoleucine-tryptophan were 10.52, 11.21, 13.84, 15.57, and 16.82 minutes, respectively.

The quantified results are as shown in Table 14 below.

Example 7
Plant production of ACE inhibitory peptides:
21.9 kg of bonito protein is extracted with hot water at 95 ° C. for 35 minutes, and 5.5 kg of soluble protein is used for the soup stock. Using 16.4 kg of water-insoluble protein as a residue of bonito hot water obtained as a by-product as a raw material, this was decomposed with a protin NY100 (Amano Enzyme) enzyme. The enzymatic decomposition reaction mixture was screened (100 mesh), DeLaval (three-layer continuous discharge centrifuge), shear press (ultra-centrifugation 15000 rpm), celite filtration (Hyflo Super Celite: Hyflo Super Celite 0.4%) ) After that, a filtrate was obtained. The filtrate was spray-dried (spray dryer, inlet temperature 150 to 200 ° C., outlet temperature 90 ° C. or less) to obtain a powder product (10 kg). Moreover, this powdery product, _{IC 50} of the angiotensin converting enzyme inhibitory activity was 136.25μg / ml.

The above filtrate (10 kg protein) was dissolved in 600 liters of water, loaded onto a column (φ45 cm × 150 cm) that was packed with a hydrophobic adsorption resin (Separbeads SP-207, Mitsubishi Chemical) and previously equilibrated with water, Adsorption was carried out, followed by elution with 600 L of water, followed by elution with 600 L of 50% ethanol solution.
The hydrophobic adsorption resin used here was a styrene-divinylbenzene resin, but the reverse phase distribution resin was octadecyl silica (YMC Co., Ltd.) or any other reverse phase distribution resin, hydrophobic adsorption resin. Can also be used. Moreover, although ethanol was used for elution, it is not limited to this.
Further, the 50% ethanol elution fraction obtained above was passed through an ultrafiltration membrane (GE module 2.4 inch × 40 inch × 2, filtration area 5 m 2; molecular weight 1000 membrane; model number 2540F1072), The permeate was concentrated under reduced pressure (solid content 40%) and spray dried (spray dryer) to obtain bonito peptide.

  From the fact that high activity was observed in the 50% ethanol-eluted fraction, it seems that the angiotensin converting enzyme-inhibiting peptide has the property of adsorbing to the hydrophobic adsorption resin. In addition, a high activity was observed in the ultrafiltration molecular weight 1000 membrane permeate, and an increase in activity was shown in the artificial permeate of the non-permeate fraction. Was estimated. Based on the above, by carrying out purification, in the present invention, a substance having high angiotensin converting enzyme inhibitory activity could be obtained in a high yield on an industrial production scale by column chromatography and ultrafiltration. .

Example 8
Animal experiment oral administration test of bonito peptide Animal: SHR / Izm
Number of examples: 24 animals Measurement item: Blood pressure (systolic blood pressure) and heart rate Measurement time: Measured before administration and at 2, 4, 6, 8, and 24 hours after administration.
Measuring method: Measured non-invasively by tail cuff method (blood pressure monitor for rats and mice, MK-2000, Muromachi Kikai Co., Ltd.). The average of 3 times is adopted. For the heart rate, the average value of the heart rate at the time of blood pressure measurement is adopted. Table 15 shows the composition and dosage of each group.

As a result, an antihypertensive effect was observed after administration of 0.3 or 1 mg / kg bonito peptide of Example 7 (Tables 16, 17 and 18 below, and corresponding FIGS. 1 and 2).

Comparative Example An enzymatic degradation product was obtained in the same manner as in Example 1 except that ultrafiltration was not performed, and this was used as a comparative example.

Test Example 2 Superiority of Composition of the Present Invention in Comparison with Comparative Example ACE inhibition between the permeate obtained by ultrafiltration in Example 1 which is the composition according to the present invention and the comparative example A comparison test was performed on the activity and the blood pressure decrease by a single administration test for SHR. The results are shown in Table 19 below.

本発明の鰹節ペプチドのＡＣＥ阻害活性のうち、バリン-トリプトファン、イソロイシン-トリプトファン、メチオニン-トリプトファンは、他ペプチドと比較して高い値を示した。 Table 20 below shows a comparative example of the inhibitory activity value of dipeptides present in the bonito peptide of the present invention and the inhibitory activity values of other dipeptides.

Among the ACE inhibitory activities of the bonito peptide of the present invention, valine-tryptophan, isoleucine-tryptophan, and methionine-tryptophan showed higher values than other peptides.

鰹節ペプチド中の寄与率は、バリン-トリプトファン、イソロイシン-トリプトファンで高い値を示した。よって、ＡＣＥ阻害活性に関して、鰹節ペプチド中の主要なジペプチド（バリン-トリプトファン、イソロイシン-トリプトファン）であることが示され、かつ鰹節ペプチドは精製されていることも裏付けられた。 The contribution ratio of the peptide of the present invention in the bonito peptide was analyzed, and the results shown in Table 21 below were shown.

The contribution rate in the bonito peptide was high for valine-tryptophan and isoleucine-tryptophan. Therefore, regarding ACE inhibitory activity, it was shown that it is a main dipeptide (valine-tryptophan, isoleucine-tryptophan) in the bonito peptide, and it was confirmed that the bonito peptide was purified.

0.030 ml of each concentration of the dipeptide solution is placed in a test tube, and then 0.1 ml of the angiotensin converting enzyme solution is added, followed by reaction at 37 ° C. for 5 minutes. Next, 0.25 ml of hippuryl histidyl leucine (Peptide Institute, Bz-Gly-His-Leu.H ₂ O, final concentration 5 mM, NaCl 300 mM included) is added as a substrate and reacted at 37 ° C. for 30 minutes. I let you. Thereafter, 0.25 ml of 1N hydrochloric acid was added to stop the reaction, 1.5 ml of ethyl acetate was added, and the mixture was stirred with a vortex mixer for 20 seconds, followed by centrifugation (3000 rpm, 5 minutes). 1 ml of the ethyl layer was collected. After heating at 105 ° C. for 30 minutes (aluminum block), the absorption value at 228 nm of hippuric acid dissolved in 3 ml of distilled water and extracted into ethyl acetate was measured, and this was defined as enzyme activity.
The inhibition rate was calculated from the following formula. A: Absorption value at 228 nm when no inhibitor is contained B: Absorption value at 228 nm when an inhibitor is added Further, the concentration of the present dipeptide when the inhibition rate is 50% was defined as an IC ₅₀ value. Inhibition rate = [1− (A−a) / (B−b)] × 100
A: Sample added a: Sample added, buffer added instead of enzyme B: Distilled water added instead of sample b: Distilled water added instead of sample, buffer added instead of enzyme

透過液のＡＣＥ阻害活性に高い値が示されたことより、消化酵素耐性の可能性が推定された。非透過液のＡＣＥ阻害活性（ＩＣ50）に１．６倍の強い値が示されたが、透過液の値には及ばなかった。 In addition, in order to confirm the degree of degradation by digestive enzymes in vivo when ingested orally, artificial digestion was performed on permeate and non-permeate with pepsin, trypsin, and chymotrypsin (pepsin, trypsin, chymotrypsin 4). Time resolution). The results are shown in Table 2.

The high value of the ACE inhibitory activity of the permeate showed the possibility of digestive enzyme resistance. The ACE inhibitory activity (IC50) of the non-permeate was 1.6 times stronger, but not as high as that of the permeate.

Example (a) Industrial production of peptide composition having ACE inhibitory activity Add 10000 L of water to 1000 kg of bonito protein, heat treatment (95 ° C., 35 minutes), remove amino acid and water-soluble protein, and obtain hot water extraction residue After adding 4436 L of water to 1774 kg (886 kg of protein) and adjusting to pH 7 with 6N caustic soda, 1.0 wt% of Samoase PC10F (Daiwa Kasei) enzyme (enzyme amount: per protein) was added and stirred at 50 ° C. And reacted for 17 hours. After the reaction, caustic soda was added to adjust the pH to 6.8, and the enzyme was inactivated by heating at 98 ° C. for 15 minutes. Thereafter, undegraded protein was removed with a vibratory screen, DeLaval, and a centrifuge, and the supernatant was filtered through Celite to obtain an enzymatic degradation solution (200 kg of protein). The ACE inhibitory activity IC ₅₀ value was 0.204 mg / ml (protein).

Claims (9)

  A dipeptide having an angiotensin converting enzyme inhibitory activity, which is a dipeptide consisting of a valine-tryptophan sequence, a dipeptide consisting of a tryptophan-tyrosine sequence, a dipeptide consisting of a tryptophan-methionine sequence, a dipeptide consisting of a methionine-tryptophan sequence, and isoleucine- At least one dipeptide selected from the group of dipeptides consisting of tryptophan sequences or an acid addition salt thereof.   A pharmaceutical composition comprising the dipeptide or an acid addition salt thereof.   Processed food or food for specified health use containing the dipeptide or acid addition salt thereof.   The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is an antihypertensive composition. Dipeptide consisting of valine-tryptophan sequence, dipeptide consisting of tryptophan-tyrosine sequence, dipeptide consisting of tryptophan-methionine sequence, dipeptide consisting of methionine-tryptophan sequence or dipeptide consisting of isoleucine-tryptophan sequence, or acid addition thereof A method for producing a salt-containing composition comprising:
1) Extract the fish-like protein of salmon, salmon koji, salmon koji, soda salmon, soda salmon, salmon, salmon knot, salmon, salmon knot, salmon, salmon knot, niboshi, or other miscellaneous knots with hot water. ,
2) The water-insoluble protein remaining in the hot water extraction section is pulverized, and the obtained pulverized product is dispersed in water, and the protease is added to the particles of the water-insoluble protein obtained at pH 5.0 to 7.0. The reaction is carried out at a temperature of 40-60 ° C. under suitable conditions, whereby enzymatic hydrolysis of the water-insoluble protein is carried out, after which the enzymatic reaction is stopped and the water-containing hydrolysis reaction mixture obtained is water-insoluble. Particles are removed, thereby obtaining an aqueous solution containing a hydrophobic / hydrophilic polymer / low molecular peptide and a water-soluble amino acid,
3) consisting of finally purifying the adsorbed fraction obtained from the aqueous solution by a hydrophobic resin column method by loading on ultrafiltration (molecular weight 1000) and permeating,
The manufacturing method.   A composition obtained by the production method according to claim 5.   A pharmaceutical composition comprising the composition according to claim 6.   Processed food or food for specified health use containing the composition according to claim 6.   The composition according to claim 7, wherein the pharmaceutical composition is an antihypertensive composition.

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