JP2010155788A - New tripeptides and method for producing these tripeptides, and method for producing angiotensin converting enzyme-inhibiting substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new peptide having an angiotensin converting enzyme-inhibiting activity. <P>SOLUTION: Five kinds of new tripeptides each having the angiotensin converting enzyme-inhibiting activity are obtained by reacting dried bonito as a raw material with a protease produced by Trametes saguinea. The enzymolysis product can be adsorbed to a hydrophobic adsorptive resin and then eluted with a water-containing organic solvent to produce the more highly active peptide fraction. The new tripeptide is a safe and highly effective raw material as a daily ingestible food, and its utilization for specified health foods, functional foods, and the like can be expected. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to five new and useful tripeptides having an activity to inhibit angiotensin converting enzyme (ACE) and thereby exhibiting blood pressure lowering action. The novel peptide of the present invention is a novel tripeptide obtained by hydrolyzing fish protein, in particular, insoluble protein remaining as a residue when bonito is extracted with hot water using a production enzyme of Trametes saguinea, which is converted to angiotensin. It can be used as an active ingredient of enzyme inhibitors and antihypertensive agents. The novel tripeptide of the present invention is expected to be useful for the treatment or prevention of hypertension.

  Hypertension is a typical lifestyle-related disease, and the number of patients, including the reserve army, is said to be 54.9 million (Ministry of Health, Labor and Welfare: 2006 National Health and Nutrition Survey). Hypertension is known to cause various complications such as cerebral hemorrhage, subarachnoid hemorrhage, cerebral infarction, myocardial infarction, angina pectoris, nephrosclerosis, and various studies on the mechanism of hypertension have been conducted. Yes.

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, 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.

Recently, it has been reported that peptides that are enzymatic degradation products of food material proteins have ACE inhibitory activity. For example, gelatin collagenase degradation product (Japanese Patent Laid-Open No. 52-148431), casein trypsin degradation product (Japanese Patent Laid-Open No. 58-109425, Japanese Patent Laid-Open No. 61-36226, and Japanese Patent Laid-Open No. 61-36227), and pepsin degradation product of sardine muscle. (Japanese Patent Laid-Open No. 3-11097), Thermolysin degradation product of bonito (Japanese Patent Laid-Open No. Hei 4-144696), Thermolysin degradation product of sesame protein (Japanese Patent Laid-Open No. 8-231588), Degradation product of kappa-casein pepsin, etc. Many reports have been made. This is because a natural product-derived angiotensin converting enzyme inhibitor is obtained from foods or food materials, and is expected to be a low-toxic and highly safe antihypertensive agent.
An ACE inhibitor is also found in microorganisms or various foods, and its practical application as an antihypertensive agent is being studied (Kunio Sueami, Fermentation and Industry, Vol . 46 (No. 3), pp. 179-182 (1988)). ).
On the other hand, an enzyme-degraded seasoning has been proposed in which animal protein-containing raw materials, especially bonito soup stock extract (residue), are decomposed with an agaric-producing enzyme and extracted to produce free amino acids and peptides. Abundantly contained (Japanese Patent Laid-Open No. 2006-94756).
Giant bamboo is a kind of wood decay fungus that naturally grows on dead and fallen trees of broad-leaved trees. Hilotake produces cellulase, xylase, pectinase, acidic carboxypeptidase, and acidic protease, and a mixture of these enzymes is Hilotake producing enzyme. The method for producing the oyster mushroom producing enzyme is described in the aforementioned Japanese Patent Application Laid-Open No. 2000-94756, and is specifically described in Reference Example 1 of the publication.
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 2006-94756 A Kunio Sueami, “Fermentation and Industry”, 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 food.
However, many of the peptides reported above have 5 or more constituent amino acids (Japanese Patent Laid-Open Nos. 52-148431, 58-109425, 61-36226, 61-61). 36227, JP-A-3-11097, JP-A-8-269088). 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.

Accordingly, an object of the present invention is to provide a novel tripeptide having an ACE inhibitory activity that is difficult to be digested by digestive enzymes when taken orally, is less likely to lose ACE inhibitory activity in the body, and can be absorbed as it is in the small intestinal mucosa. And to provide a powder product containing such a novel tripeptide.
Furthermore, the present invention provides an angiotensin converting enzyme inhibitor, an antihypertensive agent, or a composition for eating and drinking (food and beverage) comprising at least one of the above novel tripeptides.

  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.

In the product degradation product obtained by hydrolyzing the water-insoluble protein remaining as a residue after extraction of bonito with hot bamboo shoots, there is a peptide that solves the above problem, and the product degradation product An investigation was made as to whether a peptide having an ACE inhibitory activity with 3 or fewer amino acids was contained therein.
As a result, five types of tripeptides having the following amino acid sequences and having ACE inhibitory activity were found in the hydrolysis products of the water-insoluble protein, which is the residue of hot water extraction from bonito, by the oyster mushroom-producing enzyme. The present invention has been completed successfully.

In the first aspect of the present invention, a tripeptide having an amino acid sequence represented by Val-Ile-Pro, Phe-Ile-Tyr, Leu-Pro-Tyr, Phe-Ile-Phe, or Leu-Val-Trp, Furthermore, a tripeptide having an angiotensin converting enzyme inhibitory activity is provided.
Moreover, this invention provides the composition for eating and drinking which comprises 1 or more types of the said tripeptide, the angiotensin converting enzyme inhibitor, and the 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, this inhibitor can be obtained in high yields by decomposing insoluble protein residues obtained from hot bonito using hot bonito as a raw material, degrading it with a bamboo shoot-producing enzyme, adsorbing it on a hydrophobic adsorption resin, and eluting it with a water-containing organic solvent. Obtained results that can be easily concentrated, and for each component in the obtained inhibitory substance, a component having strong ACE inhibitory activity was isolated using chromatography, and inhibition of that component was achieved. When the activity value (IC ₅₀ value) was measured and the structure was analyzed, this component was Val-Ile-Pro, Phe-Ile-Tyr, Leu-Pro-Tyr, Phe-Ile-Phe or Leu-Val-Trp. This is because it has been found to be a tripeptide having the amino acid sequence shown and having angiotensin converting enzyme inhibitory activity.

  As a means for achieving the above-mentioned object, an angiotensin converting enzyme inhibitory peptide comprising Val-Ile-Pro, Phe-Ile-Tyr, Leu-Pro-Tyr, Phe-Ile-Phe and Leu-Val-Trp is mainly used. Angiotensin-converting enzyme inhibitor that inhibits angiotensin-converting enzyme, which is obtained by decomposing bonito hot water extract residue protein with Hilotake-producing enzyme, immediately adsorbing to hydrophobic adsorption resin and eluting with water-containing organic solvent A method for producing a substance is provided.

  Hydrophobic adsorption resin, that is, aromatic modified resin (Mitsubishi Chemical: Sepabead SP207) is an aromatic (styrene-divinylbenzene) synthetic adsorbent in which bromine is chemically introduced into the aromatic ring. Due to its strong hydrophobic adsorptivity, it is considered to exhibit excellent adsorption performance for highly hydrophilic organic substances (substances with low hydrophobicity), such as amino acid separation purification, protein removal, natural extract purification, fermentation broth pretreatment, etc. May be used.

  The second invention is a tripeptide having an amino acid sequence represented by Val-Ile-Pro, Phe-Ile-Tyr, Leu-Pro-Tyr, Phe-Ile-Phe or Leu-Val-Trp, or a pharmacologically thereof. An angiotensin converting enzyme inhibitor containing as an active ingredient at least one acceptable acid addition salt.

  The third aspect of the present invention is a tripeptide having an amino acid sequence represented by Val-Ile-Pro, Phe-Ile-Tyr, Leu-Pro-Tyr, Phe-Ile-Phe or Leu-Val-Trp, or a pharmacologically thereof. An antihypertensive agent containing at least one acceptable acid addition salt as an active ingredient.

  In the above, acid addition salt of tripeptide means addition salt with pharmaceutically acceptable acid (inorganic acid and organic acid), for example, hydrochloride, hydrogen bromide, acid salt, sulfate, nitrate, acetate, benzoic acid Salt, maleate, fumarate, succinate, tartrate, citrate, oxalate, methanesulfonate, toluenesulfonate, aspartate, glutamate and the like.

  The tripeptide used in the present invention is a method of enzymatic degradation of hot water extract residue protein, a method of stepwise introduction of amino acids by an organic chemical synthesis method, a peptide synthesis method utilizing reverse reaction of hydrolase, genetic engineering It can be manufactured by a method or the like.

  A method for producing the above five novel tripeptides by enzymatic decomposition of water-insoluble protein, which is the 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.

  As the working enzyme, endo-protease and oyster mushroom-producing enzyme including carboxypeptidase can be used. The oyster mushroom-producing enzyme may be of high purity (pharmaceutical grade) or industrial grade (for example, HP enzyme (Kirin Foodtech Co., Ltd.)). The substrate concentration may be any as long as it is within the range in which stirring and mixing can be performed during the reaction, but it is preferably performed in the range of protein concentration of 2 to 30% (w / v) where stirring is easy. The added amount of the oyster mushroom-producing enzyme 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 2.0 to 6.0, preferably 3.0 to 5.0, and the temperature is 30 to 70 ° C, preferably 40 to 60 ° C. It is. The pH during the reaction is adjusted with an aqueous sodium hydroxide solution, hydrochloric acid or the like as necessary.

  The enzyme reaction time varies depending on the amount of enzyme added, the reaction temperature, and the reaction pH, and is not constant, but is usually about 0.5 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. The hydrolyzed liquid is then 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 a fraction having a molecular weight of 10,000 or less. This liquid contains the tripeptide of the present invention, and the liquid or its concentrate (for example, spray dried) can be further fractionated to obtain the target tripeptide.

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

  This tripeptide 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 tripeptide and its acid addition salt are used as such or usually in a pharmaceutical composition with at least one pharmaceutical adjuvant.

  The present tripeptide 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 in the above form 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 tripeptide or its acid addition salt in the present ACE inhibitor or antihypertensive agent can vary, but usually 5 to 100% (w / w), especially 10 to 60% (w / w) Is appropriate. The dose of the present ACE inhibitor or antihypertensive agent is suitably 0.1 to 500 mg / kg / day as an active ingredient when administered to humans.

In addition, since this tripeptide has the advantage of not adversely affecting the living body even if it is ingested in large amounts, it can be used as it is, or with various nutrients added or contained in food or drink, to lower blood pressure and prevent hypertension. It may be eaten as a functional food or health food with a function. 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 present 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 types of organic chemical synthesis methods for the above novel tripeptides, liquid phase method and solid phase method, both of which are conventional methods such as Nobuo Izumiya, Tetsuo Kato, Toshihiko Aoyagi and Noriaki Wakimichi, Basic and Experiment ", Maruzen Co., Ltd., 1985. In the liquid phase method, for example, an amino acid that should be located at the C-terminal of the present tripeptide and whose carboxyl group is protected with a benzyl group (Bzl), t-butyl group (t-Bu), etc., and the C-terminal An amino acid that should be located next to an amino acid and whose α-amino group is protected with a t-butyloxycarbonyl group (Boc), a benzyloxycarbonyl group (Z), etc. is converted into dimethylformamide (DMF), dimethylacetamide, etc. Dissolve and react them 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 present tripeptide 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 groups for alcoholic hydroxyl groups include Bzl, t-Bu, etc., protecting groups for phenolic hydroxyl groups include Bzl, protective groups for guanidino groups include tosyl groups (Tos), and protecting groups for imidazolyl groups include Tos, etc. . After completion of the final reaction, all protecting groups are removed to give the tripeptide. 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. Basically, phenylacetamidomethyl (PAM) resin L-Xaa-O-CH2-PAM (where Xaa is an amino acid residue) to which an amino acid located at the C-terminus of this tripeptide is bound (obtained from Applied Biosystems) The α-amino acid (Boc-amino acid) whose amino group is protected with Boc is extended stepwise by repeating peptide bonds and removal of Boc. Boc-amino acids are subjected to an extension reaction via their symmetrical anhydrides as intermediates through the 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 solvents are used: N, N-diisopropylethylamine (TFA neutralizing agent), 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), Neutralizer (70% ethanolamine, 29.5% methanol) (in waste liquid 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 room temperature. The above procedure yields tripeptide-O-CH2-PAM in which the reactive group in the tripeptide is protected, and the actual operation of the solid phase peptide synthesis is described by Applied Biosystems 43. Performed according to the 0A type peptide synthesizer user's manual.

  The obtained reactive functional group-protected tripeptide-O-CH2-PAM was prepared by a conventional method, for example, the method described in the above-mentioned “Basics and Experiments of Peptide Synthesis” or 430A Type Peptide Synthesizer User's Manual, for example, protection. Resin and protecting groups treated with trifluoromethanesulfonic acid (TFMSA) (TFA is a diluent of TFMSA) in the presence of thioanisole and / or ethanedithiol as a scavenger to capture cations generated by group cleavage To obtain the desired tripeptide.

  The tripeptide of the present invention may be produced by organic synthesis as described above. However, for the purpose of exerting ACE inhibitory activity by adding it to foods and beverages or pharmaceuticals to be taken orally, it is obtained by degrading proteins derived from bonito and the like with a bamboo shoot-producing enzyme and further by isolation and purification. Preferably, it is produced as an orally ingestible composition containing at least one of the above five tripeptides.

  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 tripeptide of the present invention is obtained by decomposition with the oyster mushroom-producing enzyme, it is preferable to first remove the amino acid and water-soluble protein by heat treatment as a pretreatment for the koji protein as a raw material. Further, in order to efficiently decompose the bamboo shoot-producing enzyme, it is preferable that the raw material is finely pulverized and then stirred and suspended in water. Moreover, although the obtained protein is sparingly soluble, hydrochloric acid is added to achieve an optimum pH for the enzyme reaction, and the resulting protein is uniformly dispersed, suspended and dissolved. To this, 0.1 to 10% by weight of a bamboo shoot-producing enzyme is added per 100 g of protein, and the protein is degraded while stirring at pH 3.5 to 4.5 and temperature 40 to 55 ° C. for 0.5 to 30 hours. After that, the pH is adjusted to 6.0 to 7.5 with caustic soda. And the activity of the oyster mushroom producing enzyme is inactivated by heat treatment (98 ° C., 15 minutes). After removing undegraded protein with a vibe screen, remove the undegraded material and precipitates with a decanter, DeLaval, ultra-high speed centrifuge (15000 rpm) or filtration (Celite filtration: Hyflo Super Cell, etc.) The filtrate obtained is neutralized with caustic soda or hydrochloric acid and then concentrated. Furthermore, it can also be improved by performing a problem on flavor, for example, activated carbon treatment (purified white rice cake: product of Kirin Foodtech Co., Ltd., etc.) such as bitterness, pungent taste, and offensive odor. The bonito peptide thus obtained contains 0.0005% by weight to 0.005% by weight of Val-Ile-Pro, Phe-Ile-Tyr, Leu-Pro-Tyr, Phe-Ile-Phe and Leu-Val-Trp, respectively. 5% by weight is included.

  As the tripeptides of the present invention, the above-obtained oyster mushroom-producing enzyme degradation product and this are further treated with a high porous polymer resin (hydrophobic adsorption resin), an ion exchange resin or the like, so that a high molecular protein, a monomeric amino acid, Further, the salt can be removed to obtain a crude product containing abundant tripeptides of the present invention, which can be used as it is. Hereinafter, such decomposed products and crudely purified products are collectively referred to as compositions containing abundant tripeptides.

  When the peptide of the present invention is obtained by purification, the concentrate is subjected to gel filtration column chromatography, chromatography using an ion exchange resin or high porous polymer resin, affinity chromatography, etc. Peptide fractions are collected, and the active fraction 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 tripeptide of the present invention is not limited to bonito and bonito hot water extraction residue, cocoon, bonito hot water extraction residue, Soda bonbushi, Soda bonbushi hot water extraction residue, Soda tsumugi, soda bonito hot water extraction residue In addition, it can be obtained from fish meat protein by the method described above. The ACE inhibitory activity of a tripeptide or a composition rich in it can be measured, for example, by the method described in the Examples.

  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.

  Since the specific activity of the ACE inhibitory action of the thus obtained tripeptide or a composition rich in it is strong, it can be used as an extremely 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 of the said tripeptides is provided. The present invention also provides an angiotensin converting enzyme inhibitor and an antihypertensive agent comprising one or more of the above-mentioned tripeptides.

  When the tripeptide of the present invention is used and blended in foods and drinks, pharmaceuticals, etc., it is possible to use a product obtained by sufficiently purifying the tripeptide from the degradation product of the bamboo shoot hot water extract residue protein by the bamboo shoot producing enzyme, or by chemical synthesis. The obtained synthetic product may be used. However, since the peptide of the present invention is stable and has strong ACE inhibitory activity, it is possible to obtain a sufficient ACE inhibitory activity by using the crude product or the enzyme-degraded enzyme product as described above as a composition rich in tripeptides as described above. I can do it.

  The composition for eating and drinking (food and beverage) of the present invention is produced by adding one or more of the above-described tripeptides as 0.001 mg to 100 mg, preferably 0.01 mg to 20 mg as a single intake. The tripeptide-containing 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 and beverage composition containing the tripeptide 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 tripeptide 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 tripeptide 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 an active ingredient tripeptide in physiological saline and subjecting it to normal aseptic operation. The ACE inhibitory activity value (IC ₅₀ ) of Leu-Val-Trp, one of the tripeptides of the present invention, is 0.14 μM (0.06 μg / ml), and the ACE inhibitory antihypertensive drug captopril is 5 × 10 ⁻³ μM (1. 1 × 10 ⁻³ μg / ml), the drug titer (titer) is about 1/30, and the titer of ACE-inhibiting peptides generally reported so far is one-hundredth of captopril. 1 to several hundred thousand or more (activity value of ACE inhibitory peptide 1 μg / ml to 1000 μg / ml), an effect of ACE inhibitory action and antihypertensive action can be expected.

  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 decomposition treatment with agaric producing 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 preferably about 3 to 10 times the volume of the column. After loading the column, water is then passed through about 3 to 10 times the amount of the column. All non-adsorbed fractions are eluted. Further, a stepwise gradient of ethanol with a concentration of 10%, 25%, 50%, 99.5% is performed to obtain a target adsorption fraction.

  The fraction containing an angiotensin converting enzyme inhibitor substance eluted with an ethanol solution is concentrated under reduced pressure and spray-dried. The product is obtained in powder form.

  This food material mainly composed of angiotensin-converting enzyme inhibitory peptide inhibits angiotensin-converting enzyme by isolating the aforementioned eluate using high performance liquid chromatography and elution with acetonitrile / trifluoroacetic acid. A purified and isolated form of a highly active ingredient is obtained.

Test example 1
The ACE inhibitory activity was measured as follows. That is, the ACE inhibitory activity of the present tripeptide obtained as described above was obtained by changing the buffer solution of Cheung and Cushman's method (Biochemical Pharamacology, 20, 1637 (1971)) from phosphate buffer to borate buffer. It measured according to the method.
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, and the supernatant was further purified with hydroxyapatite. 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 the solution of each concentration of the present tripeptide is put into a test tube, and then 0.1 ml of the angiotensin converting enzyme solution is added and reacted at 37 ° C. for 5 minutes. Next, 0.25 ml of hippuryl histidyl leucine (containing Peptide Institute, Bz-Gly-His-Leu.H ₂ O, final concentration 5 mM, NaC 1300 mM) as a substrate is added and reacted at 37 ° C. for 30 minutes. I let you. Thereafter, 0.25 ml of IN 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), dissolved in 3 ml of distilled water, the absorption value at 228 nm of hippuric acid extracted into ethyl acetate was measured, and this was defined as enzyme activity.
The inhibition rate was calculated from the following formula. A: 228 nm absorption value when no inhibitor is contained B: 228 nm absorption value when an inhibitor is added Further, the concentration of the present tripeptide 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

  Table 1 below shows the ACE inhibitory activity values of the 5 tripeptides isolated in the enzyme-degraded enzyme product of the bamboo shoot hot water extracted from the dried bonito hot water.

Example 1
(A) Preparation and purification of peptides:
After adding 2000 mL of water to 160 g of bonito protein, heat treatment (95 ° C., 35 minutes), removing amino acids and water-soluble protein, adding 1200 mL of water to 120 g of the obtained hot water extraction residue, adjusting to pH 4 with 6N HCl, Agaricum producing enzyme 1.0 wt% (enzyme amount: per protein, product of Kirin Foodtech) was added and reacted at 50 ° C. for 17 hours while stirring. After the reaction, caustic soda was added to adjust the pH to 6.8 and heated at 98 ° C. for 15 minutes to inactivate the bamboo shoot-producing enzyme. Thereafter, undegraded protein was removed by a vibe screen, DeLaval, and a centrifuge, and the supernatant was filtered through Celite. The filtrate was spray-dried (with a spray dryer) to obtain 73 g of peptide powder.

25 g of this peptide powder was dissolved in 2500 ml of water and loaded onto hydrophobic chromatography.
The conditions for hydrophobic chromatography are described below.
Column: SP-207 (50mm ID x 255mm L, manufactured by Nippon Nensui)
Eluent: Stepwise gradient of ethanol solution of 0, 10%, 25%, 50%, 99.5% concentration Flow rate: 16.6 ml / min

The eluate from the column packed with the hydrophobic adsorption resin was fractionated into 5 fractions with a stepwise gradient of alcohol concentration, and 5 fractions of 2500 ml were fractionated. Each fraction was measured for ACE inhibitory activity by the method of Test Example 1. As a result, the ACE inhibitory activity fraction obtained under the above conditions was an elution fraction with 10%, 25% or 50% ethanol. The values IC ₅₀ were 0.030 mg / ml, 0.038 mg / ml and 0.017 mg / ml, respectively. These fractions were spray-dried to obtain white powders in amounts of 5.3 g, 3.4 g and 1.8 g, respectively. No ACE inhibitory activity was observed in the fraction eluted with water.

Next, 50 mg of the 50% ethanol-eluted fraction obtained by hydrophobic chromatography was dissolved in 3 ml of purified water and further fractionated using an ODS cartridge column.
The chromatographic conditions are described below.
Column: Sep-Pak Plus C-18 column (Waters)
Moving layer: 0, 10, 25, 30, 50, 99.5% ethanol solution

Under the above conditions, 3 ml of each ethanol concentration solution was eluted to obtain an active fraction.
The fraction was evaporated to dryness under reduced pressure and then used as a sample for measuring ACE inhibitory activity. As a result, a strong ACE inhibitory activity of 8.9 μg / ml was observed in the 25% ethanol elution fraction. Freeze drying was performed to obtain 18.4 mg of powder. Moreover, the activity values (IC ₅₀ ) of the water-eluted fraction and the eluted fraction with 10%, 30%, and 50% ethanol are 64.9, 13.3, 50.7, and 86.9 μg / ml, respectively. From each of its fractions, powders were obtained in amounts of 5.79, 6.20, 15.80 and 3.77 mg, respectively. The results are shown in Table 1 below.

8000 μg of the ACE-inhibiting peptide in 25% ethanol eluate was dissolved in 100 μl of purified water, loaded onto a high performance liquid chromatograph using a C-18 column at 2000 μg / 25 μl, and the peptide was fractionated. The chromatographic conditions are described below.
Column: Cosmosil 5C-18 ARII (4.6 mm ID × 250 mm L, Nacalai Chemical)
Moving layer: 5-35% CH ₃ CN in 0.1% TFA (10-40 minutes)
Flow rate: 0.4ml / min
Temperature: 35 ° C
Detection: UV 210nm

  Under the above conditions, one fraction was collected every minute. From each fraction, after evaporating to dryness under reduced pressure, the sample was used as an ACE inhibitory activity measurement sample, and the ACE inhibitory activity was measured according to the method described above. As a result, strong ACE inhibitory activity was observed in fractions 43, 44, 46, 47, 48, 49, 52, 53, 55, and 56. Each of these 10 fractions was freeze-dried to obtain a trace amount of peptide.

(A) Purification of ACE inhibitory peptide in fractions 43 and 44 obtained above Lyophilized peptide in fractions 43 and 44 was dissolved in 30 μl of purified water and loaded onto an ultrahigh performance liquid chromatograph using an ODS column. The peptide was fractionated. The implementation conditions are described below.
Column: Acquity UPLC BEH C18 (2.1 mm ID × 100 mm L, 1.7 μm)
Moving layer: 5-35% CH ₃ CN in 0.1% TFA (10-40 minutes)
Flow rate: 0.2ml / min
Temperature: 40 ° C
Detection: UV 210nm

  Under the above conditions, one fraction was collected every 30 seconds. From each fraction, after evaporating to dryness under reduced pressure, the sample was used as an ACE inhibitory activity measurement sample, and the ACE inhibitory activity was measured according to the method described above. As a result, strong ACE inhibitory activity was observed in peptide fractions 39 and 40. Each fraction was freeze-dried to obtain a trace amount of peptide. The fractions 39 and 40 were subjected to amino acid analysis and TOF MS analysis, and the peptide of each fraction was found to be a Leu-Pro-Tyr tripeptide.

(B) Purification of ACE-inhibiting peptide in fractions 48 and 49 obtained above The lyophilized peptide in fractions 48 and 49 was dissolved in 30 μl of purified water and loaded onto an ultrahigh performance liquid chromatograph using an ODS column. The peptide was fractionated. The conditions are described below.
Column: Acquity UPLC BEH C18 (2.1 mm ID × 100 mm L, 1.7 μm)
Moving layer: 5-35% CH ₃ CN in 0.1% TFA (10-40 minutes)
Flow rate: 0.2ml / min
Temperature: 40 ° C
Detection: UV 210nm

  Under the above conditions, one fraction was collected every 30 seconds. From each fraction, after evaporating to dryness under reduced pressure, the sample was used as an ACE inhibitory activity measurement sample, and the ACE inhibitory activity was measured according to the method described above. As a result, strong ACE inhibitory activity was observed in peptide fractions 57 and 58. Each fraction was freeze-dried, and a trace amount of peptide was obtained. Fractions 57 and 58 were subjected to amino acid analysis and TOF MS analysis, and the peptides in each fraction were found to be a Val-Ile-Pro tripeptide and a Phe-Ile-Tyr tripeptide.

(C) Purification of ACE-inhibiting peptide in fractions 52 and 53 obtained above Lyophilized peptide in fractions 52 and 53 was dissolved in 30 μl of purified water and loaded onto an ultrahigh performance liquid chromatograph using an ODS column. The peptide was fractionated. The implementation conditions are described below.
Column: Acquity UPLC BEH C18 (2.1 mm ID × 100 mm L, 1.7 μm)
Moving layer: 5-35% CH ₃ CN in 0.1% TFA (10-40 minutes)
Flow rate: 0.2ml / min
Temperature: 40 ° C
Detection: UV 210nm

  Under the above conditions, one fraction was collected every 30 seconds. From each fraction, after evaporating to dryness under reduced pressure, the sample was used as an ACE inhibitory activity measurement sample, and the ACE inhibitory activity was measured according to the method described above. As a result, strong ACE inhibitory activity was observed in peptide fractions 70 and 71. Each of these two fractions was freeze-dried, and a trace amount of peptide was obtained. Fractions 70 and 71 were subjected to amino acid analysis and TOF MS analysis, and it was found that the peptides in each fraction were Leu-Val-Trp tripeptides.

(D) Purification of ACE inhibitory peptide in fractions 55 and 56 obtained above Lyophilized peptide in fractions 55 and 56 was dissolved in 30 μl of purified water and loaded onto an ultrahigh performance liquid chromatograph using an ODS column. The peptide was fractionated. The implementation conditions are described below.
Column: Acquity UPLC BEH C18 (2.1 mm ID × 100 mm L, 1.7 μm)
Moving layer: 5-35% CH ₃ CN in 0.1% TFA (10-40 minutes)
Flow rate: 0.2ml / min
Temperature: 40 ° C
Detection: UV 210nm

  Under the above conditions, one fraction was collected every 30 seconds. From each fraction, after evaporating to dryness under reduced pressure, the sample was used as an ACE inhibitory activity measurement sample, and the ACE inhibitory activity was measured according to the method described above. As a result, strong ACE inhibitory activity was observed in peptide fractions 86 and 87. Each of these two fractions was freeze-dried, and a trace amount of peptide was obtained. Fractions 86 and 87 were subjected to amino acid analysis and TOF MS analysis, and it was found that the peptides in each fraction were Phe-Ile-Phe tripeptides.

Example 2
Synthesis of peptides by synthetic methods:
Using an automated peptide synthesizer (Applied Biosystems) (ABI 430 model), the target protected peptide resin was synthesized by sequentially extending the peptide chain from the C end by the BOC method according to the program.

After the construction of the peptide on the resin was completed, the protected peptide resin was dried. The resulting protected peptide was separated from the deprotecting group and the resin carrier by anhydrous hydrogen fluoride treatment (HF / p-Creso 18: 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 desired peptide.
Column: YMC-Pack ODS-A (30mm ID x 250mm L, YMC)
Moving 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 10-90 min: 0-100% Buffer B
Flow rate: 20ml / min
Detection: UV 220nm

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 x 150 mm L, Agilent Technologies)
Moving 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: UV 220nm

(A) Synthesis of Val-Ile-Pro tripeptide:
Boc-Pro (BrZ) resin (0.5 mmol) was used as the starting amino acid resin carrier, and the peptide chain was elongated using 2 mM each of the amino acid derivatives Boc-Ile and Boc-Val. Purification was performed by the above method to obtain a purified Val-Ile-Pro product. As a result of measuring the purity of the purified product by the above method, it was 97.69%.

(B) Synthesis of Phe-Ile-Tyr tripeptide:
Boc-Tyr (BrZ) resin (0.5 mmol) was used as the starting amino acid resin carrier, and the peptide chain was elongated using 2 mM each of the amino acid derivatives Boc-Ile and Boc-Phe. Purification was performed by the method described above to obtain a purified product of Phel-Ile-Tyr. As a result of measuring the purity of the purified product by the above method, it was 98.54%.

(C) Synthesis of Leu-Pro-Tyr tripeptide:
Boc-Tyr (BrZ) resin (0.5 mmol) was used as the starting amino acid resin carrier, and the peptide chain was elongated using 2 mM each of amino acid derivatives Boc-Pro and Boc-Leu. Purification was performed by the above method to obtain a purified product of Leu-Pro-Tyr. As a result of measuring the purity of the purified product by the above method, it was 97.95%.

(D) Synthesis of Phe-Ile-Phe tripeptide:
Boc-Phe (BrZ) resin (0.5 mmol) was used as the starting amino acid resin carrier, and the peptide chain was elongated using 2 mM each of the amino acid derivatives Boc-Ile and Boc-Phe. Purification was performed by the above method to obtain a purified product of Phe-Ile-Phe. As a result of measuring the purity of the purified product by the above method, it was 96.73%.

(E) Synthesis of Leu-Val-Trp tripeptide:
Boc-Trp (BrZ) resin (0.5 mmol) was used as the starting amino acid resin carrier, and the peptide chain was elongated using 2 mM each of the amino acid derivatives Boc-Val and Boc-Leu. Purification was performed by the above method to obtain a purified product. As a result of measuring the purity of the purified product by the above method, it was 96.30%.

For the obtained five types of tripeptides, ACE inhibitory activity was measured according to the method of Test Example 1 above, and IC ₅₀ was determined. In addition, the ACE inhibitory activity of the bamboo shoot hot water extract residue-derived oyster mushroom-producing enzyme degradation product obtained as a target was measured. The results are shown in Table 2 below.

Example 3
Using the tripeptide synthetic product obtained in Example 2, a stock beverage having the following composition was produced.
(A) Material and blending amount:
A mixture of 500 ml of bonito hot water extract (mentsuyu) and 5 kinds of synthetic tripeptides synthesized and isolated in Example 2 (230 mg of Val-Ile-Pro tripeptide, Phe-Ile-Tyr tripeptide 215 mg, Leu-Pro-Tyr tripeptide 115 mg, Phe-Ile-Phe tripeptide 55 mg, Leu-Val-Trp tripeptide 75 mg).

(B) Manufacturing method:
After hot water extraction of bonito (95 ° C., opened for 35 minutes), Celite filtration was performed, and the filtrate was cooled to room temperature. The mixture of the above five types of tripeptides was added to the extract after cooling and stirred and dissolved. This produced a dashi drink.

Example 4
(A) Isolation and quantification of Leu-Val-Trp tripeptide from the reaction mixture obtained by decomposing the residue of hot water extract of bonito protein with agaricum-producing enzyme Add 2000 ml of water to 160 g of bonito protein and extract with hot water (95 ° C., 35 Minutes). After adding 10-fold amount of water to the obtained residue (insoluble protein), adjusting to pH4, decomposing agaric syrup, adjusting to pH 6.8, heating (98 ° C, 15 minutes), vibe screen, decanter, DeLaval, Sharp Less processing and Celite filtration are performed, and 73 g of powder is obtained by vacuum concentration and spray drying.

  A 25 g of the above powder is loaded onto a hydrophobic chromatograph, and a fraction with a high activity for 50% ethanol elution is obtained by a stepwise gradient with an alcohol concentration of 0, 10, 25, 50, 99.5% / 250 ml. The eluate is concentrated under reduced pressure (40 ° C.) to 40% solids and then spray-dried (inlet temperature 150 to 200 ° C., outlet temperature 50 to 90 ° C.) to obtain 500 mg of a highly active powder product.

  Using the powder product as a raw material, a functional food containing 500 mg of this is obtained. The processed food is also used for beverages, tablets, soups and the like.

Example 5
Leu-Val-Trp was isolated and quantified as follows. That is, Leu-Val-Trp, which is one of the tripeptides of the present invention, was isolated and quantified from the powder product or the processed product as follows.

Sep-PakC18 pretreatment:
Weighed 25 mg and 5 g of processed food, respectively, of the dried bonito extract residue and its processed food, loaded onto a Sep-Pak C18 cartridge, removed the water-soluble fraction, and eluted the adsorbed fraction with a 50% ethanol solution. Use the solution as a sample.

8000 μg of ACE-inhibited purified peptide recovered from the sample obtained by treatment with Sep-PakC18 as described above was dissolved in 100 μl of purified water, and loaded on a high-performance liquid chromatograph using a C-18 column at 2000 μg / 25 μl. Peptides were fractionated. The conditions are described below.
Column: Cosmosil 5C-18 ARII (4.6 mm ID × 250 mm L, manufactured by Nacalai Chemical)
Moving layer: 5-35% CH ₃ CN in 0.1% TFA (10-40 minutes)
Flow rate: 0.4ml / min
Temperature: 35 ° C
Detection: UV 210nm
A synthetic product, Leu-Val-Trp, was loaded as a standard at 1 μg / μl. The elution time of Leu-Val-Trp was 51.50 minutes.

  Fractions (1 minute intervals) fractions 52 and 53 obtained under the above conditions were collected, dried under reduced pressure, dissolved in 100 μl of distilled water, and subjected to the following UPLC.

Fractions 52 and 53 were dried under reduced pressure, dissolved in 100 μl of purified water, and 20 μl was loaded onto an ultrahigh performance liquid chromatograph using an ODS column to separate peptides. The conditions for performing chromatography are described below.
Column: Acquity UPLC BEH C18 (2.1 mm ID × 100 mm L, 1.7 μm)
Moving layer: 5-35% CH ₃ CN in 0.1% TFA (10-40 minutes)
Flow rate: 0.2ml / min
Temperature: 40 ° C
Detection: UV 210nm
Fractions 70 and 71 were fractionated, and the separated peptide was found to be Leu-Val-Trp by amino acid analysis, TOF MS analysis (Waters), and amino acid sequencer (Applied Biosystems Procise 492cLC). .

  A standard curve was prepared by loading the standard Leu-Val-Trp onto the Acquity UPLC BEH C18 under the same conditions as described above and plotting the peak area against the load.

  The peak area of the Leu-Val-Trp fraction from Acquity UPLC BEH C18 column chromatography was applied to this calibration curve. As a result, the amount of the Leu-Val-Trp tripeptide in 100 g of the hydrolyzed bamboo shoot-producing enzyme degradation product derived from bonito hot water extraction residue protein was 15 mg.

Example 6
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 koji-bush hot-water extraction residue obtained as a by-product as a raw material, this was decomposed by a oyster mushroom-producing protease. The enzymatic decomposition reaction mixture was screened (100 mesh), DeLaval (three-layer continuous discharge centrifuge), shear press (ultracentrifugation 15000 rpm), celite filtration (Hyflo Super Celite: Hyflo Super Cell 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). Further, this powder product had an IC ₅₀ of angiotensin converting enzyme inhibitory activity of 65.0 μg / ml.

The above filtrate (10 kg protein) was dissolved in 600 liters of water, loaded onto a column (φ45 cm × 150 cm) that had been 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 stepwise gradient elution with 600 L of 10%, 25%, 50%, 99.5% 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.
Furthermore, each elution fraction obtained by the above gradient elution was concentrated under reduced pressure (solid content 40%) and then spray-dried (spray dryer), and the yield and angiotensin converting enzyme inhibitory activity were measured. The results are shown in Table 3 below. As shown in Table 3, no angiotensin converting enzyme inhibitory activity was observed in the fraction eluted with water (0% ethanol), that is, the non-adsorbed component.

Further, high activity was observed in the fractions eluted with 10%, 25% and 50% ethanol. This suggests that the angiotensin converting enzyme-inhibiting peptide has a property of adsorbing to the hydrophobic adsorption resin. Further, as is clear from this result, a substance having a high angiotensin converting enzyme inhibitory activity can be obtained in a high yield by a single operation. The active body was isolated and purified. Further, the ACE inhibitory activity IC ₅₀ and the structural analysis results of each tripeptide of the isolated component were the same as those shown in Table 1 above.

  As described above, according to the present invention, an angiotensin converting enzyme inhibitory activity is obtained by reaction with proteolytic enzymes produced by agaric bamboo shoots using bonito, which is a food material that has been proven safe from a long dietary experience. Five new tripeptides were obtained. It was also found that a highly active peptide fraction can be produced by adsorbing the enzymatic degradation product on a hydrophobic adsorption resin and eluting it with a water-containing organic solvent. It is clear that the novel tripeptide of the present invention is a safe and highly effective material for daily ingested food, and is a food material that is very meaningful for the future aging society. Expected to be used for food.

Claims (13)

  Tripeptide having the amino acid sequence of Val-Ile-Pro, Tripeptide having the amino acid sequence of Phe-Ile-Tyr, Tripeptide having the amino acid sequence of Leu-Pro-Tyr, or Phe-Ile-Phe Or a tripeptide having an amino acid sequence of Leu-Val-Trp and having an angiotensin converting enzyme inhibitory activity, or an acid addition salt thereof.   Extract hot-water extract of fish protein from bonito, bonito, bonito, soda, suda, sushi, bonito, sardine, bonito, salmon, bonito, boiled or other miscellaneous knots The water-insoluble protein remaining later is pulverized, and the obtained pulverized product is dispersed in water to form an aqueous dispersion of the water-insoluble protein. In the aqueous dispersion, The produced enzyme is reacted under acidic conditions of pH 2.0 to 6.0, preferably pH 3.0 to 5.0 at a temperature of 30 to 70 ° C., preferably 40 to 60 ° C. Hydrolytic hydrolysis, after which the enzymatic reaction is stopped and the water-insoluble particles are removed from the resulting hydrous hydrolysis reaction mixture, thereby obtaining an aqueous solution containing a water-soluble polypeptide and a water-soluble amino acid. From the aqueous solution A bird having an amino acid sequence represented by Val-Ile-Pro or Phe-Ile-Tyr or Leu-Pro-Tyr or Phe-Ile-Phe or Leu-Val-Trp according to claim 1 by a matography method. 2. A process for producing a tripeptide according to claim 1, comprising separating at least one of the peptides.   A tripeptide having an amino acid sequence represented by Val-Ile-Pro, Phe-Ile-Tyr, Leu-Pro-Tyr, Phe-Ile-Phe or Leu-Val-Trp, respectively, according to claim 1, An angiotensin converting enzyme inhibitor comprising at least one peptide acid addition salt as an active ingredient.   4. An angiotensin converting enzyme inhibitor according to claim 3, comprising the tripeptide according to claim 1 in an amount of 0.001 mg to 100 mg in a single dosage unit for oral administration.   A tripeptide having an amino acid sequence represented by Val-Ile-Pro, Phe-Ile-Tyr, Leu-Pro-Tyr, Phe-Ile-Phe or Leu-Val-Trp, respectively, according to claim 1, An antihypertensive agent containing at least one peptide acid addition salt as an active ingredient.   6. The blood pressure lowering agent according to claim 5, comprising the tripeptide according to claim 1 in an amount of 0.001 mg to 100 mg in a single dosage unit for oral administration.   Extract hot-water extract of fish protein from bonito, bonito, bonito, soda, suda, sushi, bonito, sardine, bonito, salmon, bonito, boiled or other miscellaneous knots 2. Angiotensin conversion containing at least one of the five tripeptides or acid addition salts thereof according to claim 1, obtained by hydrolyzing an insoluble protein that remains as a hot water extraction residue later with an agaric enzyme. Enzyme inhibitor.   Extract fish protein of bonito, bonito bonito, bonito bonito, soda bonito, mulberry bonito, bonito, bonito, bonito, bonito, bonito, bonito, boiled or other miscellaneous fish with hot water, then hot water An antihypertensive agent comprising at least one of the five tripeptides or acid addition salts thereof according to claim 1, which is obtained by degrading an insoluble protein remaining as an extraction residue with an agaricum-producing enzyme.   Extraction of bonito protein with hot water, insoluble protein remaining as a residue after the hot water extraction is decomposed with agaricum-producing enzyme, and then insoluble particles are removed from the resulting enzyme hydrolysis reaction mixture. An aqueous solution containing an amino acid is obtained, this aqueous solution is immediately adsorbed on a hydrophobic adsorption resin, and the adsorbed component is eluted from the resin with a water-containing organic solvent, Val-Ile-Pro, Phe-Ile- A method for producing an angiotensin converting enzyme inhibitor comprising five types of tripeptides each having an amino acid sequence represented by Tyr, Leu-Pro-Tyr, Phe-Ile-Phe and Leu-Val-Trp.   The insoluble protein obtained as a residue after extraction with bonito protein is hydrolyzed by the oyster mushroom-producing enzyme, and the resulting enzyme hydrolysis reaction mixture is immediately loaded onto the hydrophobic adsorption resin. An angiotensin-converting enzyme inhibitor, characterized by eluting the component adsorbed on the resin with a water-containing organic solvent and producing an angiotensin-converting enzyme inhibitor containing the tripeptide that is the eluted adsorbent as the main component Manufacturing method.   The insoluble protein obtained as a residue after extraction with bonito protein is hydrolyzed by the oyster mushroom-producing enzyme, and the resulting enzyme degradation reaction mixture is loaded onto the hydrophobic adsorption resin, and then the hydrophobic adsorption resin is used. An angiotensin converting enzyme inhibitor containing an adsorbed component eluted with a water-containing organic solvent as a main component of the inhibitor.   The insoluble protein obtained as a residue after extraction with bonito protein is hydrolyzed with the oyster mushroom-producing enzyme, and the enzyme-decomposition reaction mixture is loaded on a hydrophobic adsorption resin and eluted with a water-containing organic solvent. A method for producing an angiotensin converting enzyme inhibitory substance, comprising isolating and purifying a tripeptide having angiotensin converting enzyme inhibitory activity from an eluate containing an eluted adsorbing component.   It has an amino acid sequence represented by Val-Ile-Pro or Phe-Ile-Tyr or Leu-Pro-Tyr or Phe-Ile-Phe or Leu-Val-Trp according to claim 1 and converts it into an angiotensin converting enzyme. A food or beverage comprising a tripeptide having inhibitory activity or an acid addition salt of the tripeptide.