JP2020097535A - Composition for lowering blood pressure - Google Patents

Abstract

To provide novel compositions for lowering blood pressure.SOLUTION: According to the present invention, a composition for lowering blood pressure is provided that comprises a milk protein enzymatic degradation product as an active ingredient, the milk protein enzymatic degradation product comprising a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1). The milk protein enzymatic degradation product is preferably a whey protein enzymatic degradation product. The composition of the present invention can be taken by a subject having risk of increased blood pressure.SELECTED DRAWING: None

Description

The present invention relates to compositions for lowering blood pressure.

With the change of lifestyle such as eating habits and exercise habits and the aging of society, so-called lifestyle-related diseases are increasing. Hypertension, which is one of the lifestyle-related diseases, is a state in which the pressure of blood flowing through blood vessels continues to increase. When this state progresses, the elasticity and suppleness of the blood vessel wall is lost, and when the blood vessel wall is further damaged, LDL cholesterol and the like are deposited on the damage, promoting arteriosclerosis that causes cerebral infarction and myocardial infarction. To be done. In addition, the hypertensive state adversely affects various organs such as the heart, kidneys, brain and retina. Therefore, hypertension is called silent killer, and prevention and improvement of hypertension are extremely important as measures against lifestyle-related diseases.

Up to now, food materials having a blood pressure lowering effect have been developed for the purpose of daily intake. For example, a blood pressure-lowering agent containing a tripeptide contained in an enzymatic decomposition product of milk-derived casein as an active ingredient is known (Patent Document 1). However, there are still few food materials that can maintain and improve blood pressure in a healthy state and can be easily taken in daily life.

International Publication No. 2005/012542

The present invention aims to provide a novel material capable of lowering blood pressure and a composition containing the same.

The present inventors have now found that a tetrapeptide having the amino acid sequence of GTWY (SEQ ID NO: 1) and an enzymatic degradation product of whey protein containing it have angiotensin converting enzyme inhibitory activity. The present inventors have also found that an enzymatic degradation product of whey protein containing the tetrapeptide has a hypotensive action in humans. The present invention is based on these findings.

According to the present invention, the following inventions are provided.
[1] A composition for lowering blood pressure, which comprises an enzymatic degradation product of milk protein as an active ingredient, and an antihypertensive agent, wherein the enzymatic degradation product of milk protein has the amino acid sequence of GTWY (SEQ ID NO: 1). Compositions and agents comprising a peptide having.
[2] The composition and agent according to the above [1], wherein the milk protein enzymatic degradation product is a whey protein enzymatic degradation product.
[3] The composition and agent according to the above [1] or [2], wherein the whey protein enzymatic degradation product is ingested in an amount of 0.01 to 100 g (solid content conversion) per day for human.
[4] A composition for lowering blood pressure and an antihypertensive agent, which comprises, as an active ingredient, a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1).
[5] The composition and agent according to the above [1] or [4], wherein the peptide is ingested in an amount of 0.01 to 100 mg (solid content conversion) per day for human.
[6] The composition and agent according to any one of [1] to [5] above, which is in the form of food.

The compositions of [1] and [4] above are referred to herein as the “composition of the present invention”, and the agents of [1] and [4] above are referred to as the “agent of the present invention” herein. There are things to say.

The tetrapeptide and the enzymatically decomposed product of whey protein containing the tetrapeptide, which are used as active ingredients in the compositions and agents of the present invention, are materials that have been eaten for many years. Therefore, the composition and agent of the present invention are advantageous in that they can be used as a functional food having a blood pressure lowering action and can be used as a functional food that is safe for mammals including humans.

It is the figure which showed the effect of the whey decomposition product (test food group) with respect to the systolic blood pressure in a human test. * Indicates P<0.05 (t-test vs. control food group). It is the figure which showed the effect of the whey decomposition product (test food group) with respect to the diastolic blood pressure in a human test.

Detailed explanation of the invention

The milk protein enzymatic hydrolyzate (hereinafter sometimes referred to as “milk protein hydrolyzate of the present invention”), which is an active ingredient of the composition and agent of the present invention, is not particularly limited as long as it is an enzymatic hydrolyzate of milk protein. As the milk protein, whole milk, milk powder, milk protein or whey can be used, and whey is preferable. Here, "whey" is also called whey, whey, or whey, and means an aqueous solution obtained by removing milk fat, casein, and the like from milk. Whey is composed of β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulin and the like. The whey-derived animal or plant used in the present invention is not limited, but milk-derived whey is preferably used. The milk protein hydrolyzate of the present invention is preferably a whey protein enzymatic hydrolyzate (hereinafter sometimes simply referred to as “whey hydrolyzate”).

In the composition and agent of the present invention, the milk protein hydrolyzate of the present invention can be used alone, or can be used as a mixture with other components. The content (in terms of solid content) of the milk protein hydrolyzate of the present invention in the composition and agent of the present invention can be arbitrarily determined according to its purpose, use, form, dosage form, symptom, age, etc., Although the present invention is not limited to this, for example, it can be 0.001 to 99% by mass (preferably 0.01 to 95% by mass) with respect to the total amount. In the present invention, the agent of the present invention may comprise the milk protein hydrolyzate of the present invention, and the composition of the present invention may comprise the milk protein hydrolyzate of the present invention and other components. it can.

The milk protein hydrolyzate (particularly, whey hydrolyzate) of the present invention preferably has a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) (hereinafter, may be referred to as “the peptide of the present invention”) and the amino acid sequence of WY. It is possible to use the one containing the peptide. Here, the "peptide having an amino acid sequence" means a peptide whose sequence is specified by the amino acid sequence.

The content (solid content) of the tetrapeptide GTWY in the milk protein hydrolyzate (particularly, whey hydrolyzate) of the present invention is, for example, 0.01 to 1% by mass (preferably 0.05 to 0.5% by mass). ), and the content of dipeptide WY (in terms of solid content) is, for example, 0.005 to 0.5% by mass (preferably 0.01 to 0.1% by mass).

The content (in terms of solid content) of the peptide of the present invention in the composition and agent of the present invention can be arbitrarily determined according to the purpose, use, form, dosage form, symptom, body weight, etc. Although not limited to this, for example, it can be 0.00001 to 50 mass% (preferably 0.0001 to 10 mass%) with respect to the total amount.

The method for producing the milk protein hydrolyzate of the present invention (in particular, the milk protein hydrolyzate containing the peptide of the present invention) is known, and can be produced, for example, according to the description in International Publication WO2017/086303. Alternatively, a commercially available decomposed product of whey (for example, HW-3 (manufactured by Megmilk Snow Brand)) may be used as the decomposed product of milk protein of the present invention.

The milk protein hydrolyzate containing the peptide of the present invention can be produced, for example, by reacting a raw material protein with an enzyme preparation containing a proteolytic enzyme.

When using whey protein as a raw material, the concentration of whey protein to be subjected to the enzymatic reaction is not limited as long as the protein can be dissolved, but is not limited as long as the protein can be dissolved, but suppresses gelation and aggregation, the concentration of From the viewpoint of saving labor, it is preferably 1 to 30 w/v%, more preferably 1 to 20 w/v%, and further preferably 5 to 15 w/v%.

When the raw material protein is an aqueous solution such as whey or whey, it may be used as it is, or may be concentrated or diluted and then subjected to the enzyme reaction, and pH adjustment or the like can be performed as necessary. When the raw material protein is a solid such as powder, it may be dissolved in any aqueous solvent as long as the enzymatic reaction proceeds, but considering use as a food, it is dissolved in water or a food additive grade buffer solution. It is preferable. It is preferable to use a buffer solution so that the pH of the reaction solution does not change due to the amino acid generated by the enzymatic reaction. The type of the buffer solution is arbitrary, and may be determined in consideration of the subsequent use and the flavor, taste, and mineral amount, but the pH of the reaction solution is 4 to 9, preferably 5 to 8, and more preferably 7 to 8. A composition that can be maintained at Most preferably, it is a potassium phosphate buffer. The concentration of the buffer solution is arbitrary as long as a buffering effect can be obtained, but considering the flavor, taste and mineral amount, it can be 0.01 to 0.5 M, preferably 0.05 to 0. 2M, more preferably about 0.1M.

Any enzyme can be used as long as it is an enzyme agent containing a proteolytic enzyme, but an enzyme preparation containing a neutral protease is preferable, and one or more kinds can be used in combination. As the enzyme preparation, for example, those derived from microorganisms such as Bacillus subtilis, Aspergillus oryzae, and Alpergillus mereus can be used. Among them, enzyme preparations derived from Aspergillus oryzae and enzyme preparations derived from Alpergillus meleus are The enzyme preparation derived from Alpergillus meleus is more preferable, and more preferable.

In the present invention, a commercially available enzyme preparation can be used. For example, the enzyme preparation can be obtained from Amano Enzyme, Shin Nippon Chemical Co., DSM, Danisco, Novozyme, HBI and the like. The amount of the enzyme preparation added is arbitrary, but considering an appropriate hydrolysis reaction rate and cost, for example, 0.01 to 5 w/v%, preferably 0.05 to 4 w/v%, more preferably 0. It can be set to 0.1 to 0.5 w/v %.

The enzymatic reaction temperature and the enzymatic reaction time can be set so that the raw material protein is sufficiently hydrolyzed and the quality of the enzymatic degradation product is maintained. That is, the enzyme reaction temperature can be, for example, 30 to 70°C, preferably 40 to 70°C, and more preferably 45 to 65°C. The enzyme reaction time can be set to 1 to 12 hours, preferably 2 to 10 hours, and more preferably 4 to 5 hours. The reaction temperature and reaction time can be appropriately adjusted while confirming the production amount of the peptide of the present invention.

The enzymatic reaction can also be carried out while raising the temperature. For example, the reaction can be performed while raising the temperature from 30°C to 75°C over 4 to 10 hours. Preferably, the reaction can be performed while raising the temperature from 35°C to 75°C over 5 to 8 hours, and more preferably the reaction can be performed from 35°C to 75°C over 6 to 8 hours. The temperature rising speed is arbitrary, but the holding time between 45°C and 55°C is lengthened (5 to 7 hours), then gradually raised to 60°C, and then prolonged between 60°C and 75°C. It is preferable to hold (for example, 1 to 3 hours). Most preferred is a method in which the enzyme is added at 50° C., the temperature is maintained for 5 to 7 hours, the temperature is raised at an arbitrary rate, and the target temperature of 60 to 65° C. or 65 to 75° C. is maintained for 1 to 3 hours.

During the reaction, it is preferable to stir the reaction solution from the viewpoint of reaction efficiency. The liquid stirring rate is preferably high so that the substrate is in good contact with the enzyme, but since the reaction solution may scatter if it is too fast, for example, it can be set to 100 to 500 rpm, preferably 200 to 400 rpm, More preferably, it is about 250 rpm.

When the desired peptide is obtained, the reaction solution is preferably subjected to a step of stopping the enzymatic reaction. In the enzyme reaction stopping step, a method of raising the temperature of the reaction solution, a method of adding a chelating agent to change the chemical structure of the enzyme, or a method of removing the enzyme by membrane treatment can be adopted. A preferred technique is high temperature deactivation. The method can be carried out by holding at 80 to 90°C for 5 to 30 minutes, preferably at 80 to 90°C for 20 to 30 minutes. Further, when the temperature becomes high in the concentration step described later, it can be performed also as the concentration step.

The reaction liquid (milk protein degradation product) that has undergone the enzymatic reaction step and the enzymatic reaction termination step may be further subjected to a sterilization step. Examples of the sterilization process include a film treatment process and a high temperature sterilization process described later. The heat sterilization step can also serve as the enzymatic reaction stopping step, which is advantageous in simplifying the manufacturing process.

The reaction liquid (milk protein degradation product) that has undergone the enzymatic reaction step and the enzymatic reaction termination step may be further subjected to a purification step. Examples of the purification step include a membrane treatment step, and a preferable membrane treatment is ultrafiltration. The molecular weight cut-off for ultrafiltration is preferably from 3 to 100 kDa, more preferably from 5 to 50 kDa. Performing the purification step is advantageous in that it can improve the flavor of the peptide composition as compared to the case where it is not performed. In addition, the purification step can serve as both the enzymatic reaction stopping step and the sterilization step, which is advantageous in terms of simplification of the manufacturing step.

The reaction liquid (milk protein degradation product) that has undergone the enzymatic reaction step and the enzymatic reaction termination step may be further subjected to a concentration step from the viewpoint of storage and transportation. Although any method can be selected for the concentration step, concentration by reduced pressure, freeze drying and spray drying (spray drying), concentration by membrane treatment (for example, a method using a reverse osmosis membrane) are preferable, and freezing is more preferable. Drying and spray drying. Spray drying is particularly preferred from the viewpoint of efficiently performing a large amount of concentration.

The content of the peptide of the present invention can be measured by liquid chromatography tandem mass spectrometry (LC/MS/MS). Those skilled in the art can easily set the conditions, and needless to say, a standard peptide having a purity for LC/MS/MS measurement is used in the measurement, for example, SIGMA ALDRICH AQUA peptides produced by the company can be used.

The compositions and agents of the present invention are for lowering blood pressure. Here, "to lower the blood pressure" is used in the sense of literally lowering the blood pressure and suppressing the increase in blood pressure. Here, “suppressing an increase in blood pressure” includes reducing the risk in a subject at risk of increasing blood pressure (for example, risk of increasing blood pressure due to excessive intake of salt, lack of exercise, smoking, drinking, etc.). Further, "blood pressure" includes systolic blood pressure and diastolic blood pressure. Blood pressure can be measured according to a known method, for example, using a non-invasive blood pressure monitor.

As shown in Examples below, the blood pressure of a subject can be lowered by ingesting an enzymatic degradation product of whey protein. Therefore, the compositions and agents of the present invention can be taken by subjects with hypertension and those at risk of hypertension. Here, "hypertension" refers to a state in which the systolic blood pressure is 140 mmHg or higher and the diastolic blood pressure is 90 mmHg or higher. In addition, “a subject at risk of hypertension” is used to include a subject who is not hypertension but may develop hypertension in the future, and, for example, has a systolic blood pressure of 130 to 139 mmHg and a diastolic blood pressure. Includes a subject with normal high blood pressure that satisfies at least one of 85 to 89 mmHg.

The composition and agent of the present invention can be provided in the form of a drug (eg, pharmaceutical composition), quasi drug, food (eg, food composition), feed (including pet food), etc. Can be carried out as described in.

The compositions and agents of the present invention can be orally administered to humans and non-human animals. Examples of oral agents include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. These preparations can be prepared using a pharmaceutically acceptable carrier according to a method commonly used in the art. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc. Are listed.

When the milk protein hydrolyzate of the present invention and the peptide of the present invention are provided as a food, it can be provided as a food as it is, or can be provided by including it in a food. For example, when the milk protein hydrolyzate of the present invention or the peptide of the present invention is provided as a food, an enzymatic hydrolyzate of a milk protein such as a whey hydrolyzate or the peptide of the present invention can be directly provided as a food, Alternatively, it can be provided by being contained in food. The food thus provided is a food containing the milk protein hydrolyzate of the present invention or the peptide of the present invention in an effective amount. In the present specification, "containing an effective amount of the milk protein hydrolyzate of the present invention or the peptide of the present invention" means that the amount of the present invention is within the range as described below when ingested in an amount that is usually consumed in individual foods. The content is such that the digestion product of milk protein or the peptide of the present invention is ingested. Further, "food" means health food, functional food, dietary supplement, health functional food (for example, food for specified health use, food with nutritional function, food with functional claims), special-purpose food (for example, food for infants, pregnant women). Food, food for the sick) and supplements. Needless to say, when the non-human animal is ingested with the milk protein hydrolyzate or the peptide of the present invention, the food of the present invention is used as a feed.

Since the milk protein hydrolyzate of the present invention or the peptide of the present invention has the above-described blood pressure lowering action, it can be contained in foods that are ingested daily, or can be provided as a supplement. That is, the composition and agent of the present invention can be provided in the form of food. In this case, the composition and agent of the present invention can be provided in a unit package form in which the amount to be taken per meal is predetermined. Examples of the unit packaging form per meal include a form in which a fixed amount is specified in a pack, a package, a can, a bottle, or the like. In order to better exert various effects of the composition and agent of the present invention, the intake amount per meal can be determined according to the daily intake amount of the blood pressure lowering function of the present invention described below. The food of the present invention may be provided with a description of the intake amount on the packaging, or may be provided together with a document or the like in which the description is described.

The predetermined intake amount per meal in the unit packaging form is divided into two or more (preferably 2 or 3 times) the effective intake amount per day even if it is the effective intake amount per day. It may be an intake amount. Therefore, the composition and the agent of the present invention may contain the milk protein hydrolyzate of the present invention or the peptide of the present invention in a unit daily intake amount of the human, which will be described later. The milk protein hydrolyzate of the present invention or the peptide of the present invention can be contained in an amount of ½ to ⅓ of the daily intake. The composition and agent of the present invention are preferably provided in a “unit package form per meal”, where the intake per meal is an effective intake per day for convenience of intake.

The form of the “food” is not particularly limited and may be, for example, a drink, a semi-liquid or gel form, a solid body or a powder form. In addition, as the “supplement”, a tablet, a capsule or the like produced by kneading the milk protein hydrolyzate of the present invention or the peptide of the present invention with an excipient, a binder and the like and kneading the mixture and then encapsulating the same Examples include capsules.

The food provided in the present invention is not particularly limited as long as it contains the milk protein hydrolyzate of the present invention or the peptide of the present invention, but includes, for example, soft drinks, carbonated drinks, beverages containing fruit juice, and vegetable juice. Beverage, fruit juice and vegetable juice beverage, milk such as milk, soy milk, milk beverage, lactic acid bacteria beverage, drink type yogurt, drink type or stick type jelly, coffee, cocoa, tea beverage, energy drink, energy drink, Non-alcoholic drinks such as sports drinks, mineral water, near water, and non-alcoholic beer-taste drinks; carbohydrate-containing foods and drinks such as rice, noodles, breads and pasta; cheeses, hard or soft yogurt, livestock Milk and other dairy products such as fresh creams and ice creams based on oils and fats; Western confectionery such as cookies, cakes and chocolates; Japanese confectionery such as buns and yokan; Tablet confectionery (cool confectionery) such as ramune; candies and gums; Various confectioneries such as frozen desserts such as jelly and pudding, frozen desserts and snacks; whiskey, bourbon, spirits, liqueur, wine, fruit liquor, sake, Chinese liquor, shochu, beer, non-alcoholic beer with an alcohol content of 1% or less, sparkling liquor , Other liquor, alcoholic beverages such as shochu; processed products using eggs, processed products of seafood and meat (including liver and other offal) (including delicacy), processed foods such as miso soup and soups, Examples thereof include miso, soy sauce, sprinkles, seasonings such as seasoning, liquid foods such as concentrated liquid foods, and the like. The mineral water includes both effervescent and non-effervescent mineral water. Further, the food products provided by the present invention include both food production raw materials and food additives.

The tea beverage includes any of fermented tea, semi-fermented tea and non-fermented tea, for example, black tea, green tea, barley tea, brown rice tea, sencha, gyokuro tea, houjicha, oolong tea, turmeric tea, puer tea, rooibos tea. , Rose tea, chrysanthemum tea, ginkgo leaf tea, and herbal tea (for example, mint tea and jasmine tea).

Examples of fruits used in the beverage containing fruit juice and the beverage containing fruit juice and vegetable juice include apple, mandarin orange, grape, banana, pear, peach, mango, acai, blueberry and plum. Examples of vegetables used in the beverage containing vegetable juice, the fruit juice, and the beverage containing vegetable juice include tomato, carrot, celery, pumpkin, cucumber, and watermelon.

Since the milk protein hydrolyzate of the present invention and the peptide of the present invention are components contained in milk protein hydrolyzate and the like that humans have ingested as food for many years, toxicity is low, and mammals in need thereof (for example, (Human, mouse, rat, rabbit, dog, cat, cow, horse, pig, monkey, dolphin, sea lion, etc.). The intake amount or dose of the milk protein hydrolyzate of the present invention and the peptide of the present invention can be determined depending on the sex, age and weight of the recipient, symptoms, administration time, dosage form, administration route, combined drug and the like. The adult daily intake and dose (in terms of solid content) of the milk protein hydrolyzate for the purpose of lowering blood pressure are, for example, 0.01 to 100 g (preferably 0.1 to 10 g). The adult daily intake and dose (in terms of solid content) of the peptide of the present invention for the purpose of lowering blood pressure are, for example, 0.01 to 100 mg (preferably 0.1 to 10 mg).

It is desirable that the milk protein hydrolyzate of the present invention and the peptide of the present invention start ingestion before, for example, the date and time at which the antihypertensive effect is expected, and the start time is 3 days before the expected date and time (preferably 5 days before). , And more preferably 10 days before). It is also preferable that the milk protein hydrolyzate of the present invention and the peptide of the present invention continue to be ingested during a period in which a hypotensive action is expected.

The milk protein hydrolyzate of the present invention and the peptide of the present invention can be taken for at least 3 days (preferably 1 week) in the above-mentioned daily intake. In addition, the intake interval of the milk protein hydrolyzate of the present invention and the peptide of the present invention can be such that the above-mentioned daily intake is once every 3 days, once every 2 days, or once daily.

Regarding the above intake amount, intake timing, intake period and intake interval for the milk protein hydrolyzate of the present invention and the peptide of the present invention, the milk protein hydrolyzate of the present invention and the peptide of the present invention are used for both non-therapeutic purposes and therapeutic purposes. In case of treatment, ingestion can be read as administration for therapeutic purposes.

The composition, agent and food of the present invention may be labeled with a hypotensive effect. In this case, in order to make it easy for consumers to understand, the composition, the agent, and the food of the present invention may be provided with some or all of the following labels. It goes without saying that in the present invention, "lowering blood pressure" is used in the meaning including the following indications.
・For those who are concerned about blood pressure ・For those who have high blood pressure (high normal values) ・Keep blood pressure healthy ・Maintain blood pressure normal ・Improve high blood pressure

According to another aspect of the present invention, an effective amount of the enzymatic degradation product of the milk protein of the present invention or the peptide of the present invention or a composition containing the same is ingested or administered to a subject in need thereof. A method of lowering blood pressure is provided comprising. The method of the present invention can be carried out as described for the compositions and agents of the present invention.

According to still another aspect of the present invention, a milk protein enzymatic hydrolyzate of the present invention or a peptide of the present invention for producing a blood pressure lowering agent, as a blood pressure lowering agent, or in the blood pressure lowering method of the present invention, Use provided. The use according to the invention can be carried out as described for the compositions and agents according to the invention.

According to still another aspect of the present invention, a peptide having an amino acid sequence of a milk protein enzymatic degradation product or GTWY (SEQ ID NO: 1) for use in lowering blood pressure or for use in the method for lowering blood pressure of the present invention. Will be provided. The milk protein enzymatic degradation product and the tetrapeptide described above can be carried out according to the description regarding the composition and agent of the present invention.

The methods of the invention and uses of the invention may be for use in mammals, including humans, and is intended for both therapeutic and non-therapeutic uses. In the present specification, “non-therapeutic” means not including an act of operating, treating or diagnosing a human (that is, a medical act for a human), specifically, a doctor or a doctor's instruction. It means that the person does not include a method of performing surgery, treatment or diagnosis on a human.

The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples.

Example 1: Preparation of tablet containing whey degradation product and measurement of content of tetrapeptide GTWY and dipeptide WY (1) Preparation of tablet Whey degradation product (HW-3, manufactured by Megmilk Snow Brand Co., Ltd.), excipient and binder were used. After mixing and kneading, a tablet containing whey degradation product (300 mg/grain) was produced by tableting. The content of decomposed whey products in one tablet was 168 mg. The above-mentioned whey degradation product is a product obtained by allowing an enzyme preparation containing a proteolytic enzyme to act on whey protein, then performing a membrane treatment to remove undegraded products, and drying the product. The tetrapeptide GTWY is described below. And dipeptide WY.

(2) Preparation of Analytical Sample 100 tablets (about 30 g) prepared in (1) above were well ground in a mortar, 20 mg of the tablet was weighed, and 1 mL of sterilized water was added and well suspended. The suspension was centrifuged (15000 rpm, room temperature, 3 minutes), and 500 μL of the obtained supernatant was filtered with an ultrafiltration filter (10 kDa), and the filtrate was diluted 1000 times to obtain a measurement sample.

(3) Analytical Method The concentrations of the tetrapeptide GTWY and dipeptide WY in the measurement sample obtained in (2) above were quantified by the LC/MS/MS method under the following analytical conditions. The GTWY concentration of the measurement sample was calculated by the calibration curve method using AQUA Peptide (manufactured by Sigma Aldrich) as a standard sample.

<Analysis conditions>
Mass spectrometer: 4000Q TRAP (manufactured by AB SIEX)
HPLC device: Agilent 1200 Series (manufactured by Agilent Technologies)
Column: TSK gel ODS-100V 3 μm 2.0 mm I.V. D. X150 mm (made by Tosoh Corporation)
Column temperature: 70℃
Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid acetonitrile solution Gradient conditions: The gradient conditions shown in Table 1 were applied.

Flow rate: 0.2 mL/min Sample injection rate: 2 μL
Ionization method: ESI (positive ion detection mode)
Curtain gas: 40 psi
Nebulizer gas: 50 psi
Dry gas: 80 psi
Dry gas temperature: 600°C
Collision gas: Nitrogen ionization voltage: 5000V

<Analysis conditions for tetrapeptide GTWY>
Set mass number (m/z)/collision energy (eV): 526.4→159.2/47, 526.4→368.3/23
DP voltage (V): 36

<Analysis conditions for dipeptide WY>
Set mass number (m/z)/collision energy (eV): 368.2→351.1/19, 368.2→159.2/33
DP voltage (V): 51

(4) Analysis Results One tablet (300 mg) containing a whey degradation product contained 0.27 mg (0.097% by mass) of tetrapeptide GTWY and 0.10 mg (0.033% by mass) of dipeptide WY. It was confirmed.

Example 2: Effect of whey degradation product and two kinds of peptides on angiotensin converting enzyme activity (1) Outline of test Angiotensin, which is a system involved in blood pressure regulation for whey degradation products and peptides contained in whey degradation products (GTWY, WY) The converting enzyme inhibitory activity was evaluated.

(2) Test procedure The measurement of angiotensin converting enzyme inhibitory activity was performed by Le Hoang Lam et al. (Biochem., 2007, 364, 104.) and Le Hoang Lam et al. (Anal. Sci., 2008, 24, 1057.). ) Based measurement kit (ACE Kit-WST, Dojindo Co., Ltd.) was used. The kit utilized coloring to determine the amount of 3-hydroxybutyric acid (3HB) produced when angiotensin converting enzyme (ACE) was allowed to act on 3-hydroxybutyryl-Gly-Gly-Gly (3HB-GGG). It is obtained as an absorbance at 450 nm by an enzymatic method. The 50% inhibitory concentration (IC ₅₀ ) of the test sample was calculated from the color development intensity obtained when the test sample coexisted with the seven measurement samples with different dilution ratios and the color development intensity when the test sample was not present.

Specifically, a whey decomposition product (HW-3, manufactured by Megmilk Snow Brand) 0.1% aqueous solution, a peptide (synthetic product) 0.1% aqueous solution having an amino acid sequence of GTWY, or a peptide having an amino acid sequence of WY ( Synthetic product) A 0.1% aqueous solution was used as a test sample, and the test sample was diluted with pure water in 6 steps to obtain a measurement sample. A measurement sample or pure water was put into a 96-well plate, and a substrate solution containing (3HB-GGG) attached to the kit and an Enzyme working solution containing an ACE solution were added thereto and incubated at 37° C. for 60 minutes. Further, an Indicator working solution for color development or pure water was added, and the mixture was incubated at room temperature for 10 minutes. The absorbance at 450 nm was measured using a plate reader, and the ACE inhibitory activity value (inhibition rate %) was determined from the obtained absorbance by the following formula.

In addition, as shown in Table 2, a reaction product obtained by adding pure water instead of the measurement sample was used as blank 1, and was reacted with pure water, and pure water was added instead of the enzyme working solution for reaction. The blank was designated as blank 2.

(3) Results As a result of the measurement of the angiotensin converting enzyme inhibitory activity, the IC ₅₀ of the whey degradation product, GTWY and WY were 32.0 μg/mL, 12.0 μg/mL and 5.0 μg/mL, respectively.

(4) Discussion It was found that whey degradation products, GTWY, and WY each have ACE inhibitory activity. Further, it was considered that the contribution of GTWY to the ACE inhibitory effect of the whey degradation product was not small.

Angiotensin II, a potent vasoconstrictor that causes hypertension, is derived from an inactive peptide, angiotensin I, by converting His-Leu on the C-terminal side into angiotensin I-converting enzyme ( It is known to be produced by being cleaved by ACE). It is known that the renal renin-angiotensin system (RAS) involving the conversion of angiotensin I to angiotensin II is closely related to the development of essential hypertension, which accounts for about 90% of hypertension. On the other hand, angiotensin I converting enzyme (ACE) also catalyzes a reaction that inactivates the degradation of bradykinin, which is a hypotensive peptide in the kinin-kallikrein system (Nikkei Univ., 73(1):6-7(2014)). .. From these facts, it is considered that inhibition of ACE activity suppresses an increase in blood pressure and leads to suppression of hypertension. Therefore, the peptide GTWY and the whey degradation product containing the peptide were considered to be effective in lowering blood pressure.

Example 3: Effect of whey degradation product on human blood pressure (1) Outline of test A human test was conducted to verify the effect of whey degradation product on blood pressure reduction. The study was a placebo-controlled, randomized, double-blind, parallel-group comparison study. The test period was 12 weeks, and the test food or the control food was consumed during the test period. Specifically, let healthy men and women aged 50 to 75 years old ingest the "whey hydrolyzate-containing tablet" as a test food and the "whey hydrolyzate-free tablet" as a control food, respectively. The effect on the decrease was confirmed.

(2) Subjects Subjects who were judged to be healthy by the doctor in the preliminary examination were randomly assigned to the test food group (51 persons) and the control food group (53 persons). During the test period, the subjects continued to live as they did before the test period.

(3) Test food During the test period (12 weeks), the test food group contains 6 test foods, and the control food group contains 6 control foods (hereinafter sometimes referred to as “placebo”). It was taken once daily with water or lukewarm water. As the test food, "whey decomposed product-containing tablet" (GTWY contained in 174 mg in dry weight conversion in 6 tablets and WY contained in 0.66 mg in dry weight conversion) was used. As a control food, "whey hydrolyzate-free tablet" produced by the same method except that maltodextrin was added instead of the whey hydrolyzate was used. The formulation of the test food per grain is as shown in Table 3.

(4) Measurement Blood pressure was measured on the subject using a non-invasive blood pressure monitor. Specifically, the test food was administered once before the start of intake of the test food, at 6 weeks after the start of intake, and at 12 weeks after the start of intake of the test food. In addition, at 6 weeks and 12 weeks, the test food was taken 30 minutes before the measurement.

(5) Evaluation and analysis A value (difference, hereinafter referred to as "amount of change in blood pressure") obtained by subtracting the measured value before the start of ingestion of the test food from the measured value at each measurement time was evaluated. The changes in blood pressure were compared between groups using the unpaired t-test.

(6) Results a. Subjects The number of subjects to be analyzed was 51 in the test food group (17 men, 34 women) and 53 in the control food group (20 men, 33 women). The (average value±standard deviation) was 60.7±5.7 years for the test food group and 61.2±5.6 years for the control food group. The blood pressures before the start of ingestion of the test food are as shown in Table 4, and no difference between groups was observed in systolic blood pressure and diastolic blood pressure.

B. Blood pressure The results for systolic blood pressure are shown in FIG. 1, and the results for diastolic blood pressure are shown in FIG. The change in blood pressure (mean ± standard deviation) at each measurement time was generally lower in the test food group, and a significant difference in systolic blood pressure was observed between the groups on the 85th day of ingestion (P<0. .05).

(7) Discussion It was found that a whey degradation product containing GTWY and WY has an action of lowering blood pressure. Considering that the subjects in this human test are healthy subjects, it was expected that the hypotensive effect of the whey degradation product containing GTWY and WY would be more significantly exerted on subjects having a tendency to hypertension. Furthermore, considering together with the result of Example 2 (that the contribution of the ACE inhibitory effect of GTWY was large to the ACE inhibitory effect of the whey decomposition product), GTWY was also considered to have a blood pressure lowering action.

Claims (6)

A composition for lowering blood pressure, comprising a milk protein enzymatic degradation product as an active ingredient, wherein the milk protein enzymatic degradation product comprises a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1). .. The composition according to claim 1, wherein the milk protein enzymatic degradation product is a whey protein enzymatic degradation product. The composition according to claim 1 or 2, wherein the whey protein enzymatic degradation product is ingested in an amount of 0.01 to 100 g (solid content conversion) per day for a human. A composition for lowering blood pressure, comprising a peptide having the amino acid sequence of GTWY (SEQ ID NO: 1) as an active ingredient. The composition according to claim 1 or 4, wherein the peptide is ingested in an amount of 0.01 to 100 mg (solid content equivalent) per day for a human. The composition according to any one of claims 1 to 5, which is in the form of a food product.

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