JPH08269088A - New peptide and its utilization - Google Patents

Abstract

PURPOSE: To obtain a new peptide having an angiotensin converting enzyme- inhibiting activity and useful for antihypertensives, health foods, cosmetics, animal feeds, etc., by separating and purifying the peptide from the protease decomposition product of a bovine K-casein glycomacropeptide. CONSTITUTION: A new peptide having an amino acid sequence of the formula. Since the peptide has an angiotensin converting enzyme-inhibiting activity and exhibits an antihypertensive action, the peptide is added to various medicines such as antihypertensives in the form of injections, sugar coated tablets, tablets or capsules, beverages or foods, refreshing beverages, fruit juices, fermented beverages, jellies or ice creams, and is useful for health foods used for treating and preventing hypertension, for cosmetics for imparting a vasodilating action, for animal feed additives, etc. The peptide is obtained by separating a K-casein glycomacropeptide from a whey protein concentrate obtained from milk, treating the separated K-casein glycomacropeptide with a protease such as pepsin, and subsequently subjecting the decomposition products to a gradient elution treatment with a column chromatography, etc., to fractionate the decomposition products.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel peptide. The present invention also relates to an angiotensin converting enzyme inhibitor containing the novel peptide as an active ingredient. Furthermore, the present invention relates to an antihypertensive agent containing this novel peptide as an active ingredient.

[0002]

2. Description of the Related Art Angiotensin converting enzyme (ACE)
Is an enzyme that acts on the renin-angiotensin system in the regulation of blood pressure. Angiotensinogen having a molecular weight of about 57,000 is converted to angiotensin I by renin which is transferred from the kidney into blood vessels. This angiotensin I is an inactive form showing almost no vasoconstrictor action,
It is converted to active angiotensin II by ACE. About this angiotensin II, the contractile effect of arterial peripheral capillaries [Furchgott, R. et al., J. PharmacolEx
p.Ther., vol.108, p.129, 1953], increased vascular permeability [Gim
brone, MA and Alexander, RW, Science, vol.189,
p.219, 1975], Promotion of adrenergic neurotransmission in the sympathetic nerve [Bell, C., Circ.Res., vol.31, p.348,
1972], Accelerating the release of catecholamines from the adrenal medulla and promoting the secretion of aldosterone from the adrenal cortex [Biro
n, P. et al., J.Clin.Invest, vol.44, p.1171, 1965]
It is known that the effects such as the above are brought about, which causes an increase in circulating blood flow and an increase in blood pressure. In addition, bradykinin, which has a blood pressure-lowering effect due to a vasodilatory effect, is decomposed by ACE, resulting in an increase in blood pressure. On the other hand, an ACE inhibitor is known to have an effective antihypertensive effect because it inhibits the above-mentioned action of ACE, and is useful as an active ingredient of an antihypertensive agent.

In recent years, due to an increase in adult diseases and an increase in diseases due to aging, the above-mentioned substances having a blood pressure lowering action have been in the limelight, and some of them have been provided as pharmaceuticals. However, it is said that diseases associated with hypertension are strongly affected by diet, and as a substance having a preventive effect on hypertension, there is great expectation for a substance having utility as a food material.

As an ACE inhibitor contained in food, a protein-degrading peptide derived from sardine muscle (manufactured by Nippon Suisan Kaisha, Ltd.) was isolated from krill by ion exchange chromatography, gel filtration chromatography and reverse layer chromatography. ACE inhibitors [Yukio Kawamura et al., Micronutrients Research, vol.7, p.37, 1990], oligopeptides derived from corn protein [JP-A No. 2-240027] and the like are known.

Further, it is known that many ACE inhibitors are present in the degradation products derived from the milk protein casein. That is, a peptide of human β-casein amino acid sequence 39-52 residues [Kohmura, M. et al., Agric. Biol. Chem.,
vol.53, p.2107, 1989], a peptide of human kappa casein with an amino acid sequence of 43 to 52 residues [Kohmura, M. et al., Agric.Bi.
ol.Chem., vol.54, p.835, 1990], the amino acid sequence of bovine α _S1 casein, a peptide with residues 23 to 34 and 194 to 199.
[Maruyama, S. et al., Agric.Biol.Chem., Vol.51, p.2
557, 1987], the amino acid sequence of bovine β-casein 177 to 183.
Residue peptide [Maruyama, S., Agric.Biol.Chem., Vol.
51, p.1581, 1987], a peptide with an amino acid sequence of 25 to 34 residues of bovine κ casein called Casoxin C [Mamoru Totsuka and Shuichi Uenogawa, Bulletin of the Japanese Society of Animal Science, vol.63, p.867, 1992]. Is. Further, as an ACE inhibitor-containing composition obtained by hydrolyzing bovine casein with a proteolytic enzyme, it is disclosed that an enzymatic hydrolyzate of bovine α-casein or bovine β-casein is effective in preventing hypertension. 2-167052 publication]. Alternatively, it has been disclosed that a bovine κ-casein glycomacropeptide, which is a peptide having an amino acid sequence of 106 residues or less, produced by chymosin degradation of bovine κ-casein has an effect of suppressing an increase in blood pressure [JP-A-6-345664]. .

[0006]

DISCLOSURE OF THE INVENTION The inventors of the present invention conducted extensive research to find a substance having an ACE inhibitory activity that can be used as a food material, and found that bovine κ contained in cheese whey and the like. A novel peptide having ACE inhibitory activity was found in a protease degradation product of casein glycomacropeptide. Then, in animal experiments, they found that this peptide had an action of lowering blood pressure, and completed the present invention. Therefore, an object of the present invention is to provide a novel peptide having ACE inhibitory activity. Another object of the present invention is to provide an ACE inhibitor containing this peptide as an active ingredient. Another object of the present invention is to provide a blood pressure lowering agent containing this peptide as an active ingredient.

[0007]

Means for Solving the Problems In order to find a substance having an ACE inhibitory activity which can be used as a food material, the present inventors investigated the ACE inhibitory activity of κ casein glycomacropeptide obtained from sweet cheese whey. As a result, a fraction having ACE inhibitory activity was found in the proteolytic enzyme digest of κ-casein glycomacropeptide. When the active substance of the fraction having this ACE inhibitory activity was isolated and identified, it was found to be a peptide having the following amino acid sequence (I). Ile-Ala-Ser-Gly-Glu-Pro (I)

The amino acid sequence of this peptide corresponded to the amino acid sequence 20 to 25 residues of kappa casein glycomacropeptide and the amino acid sequence 125 to 130 residues of kappa casein.

Further, it was confirmed that the peptide having the following amino acid sequence (I) has a blood pressure lowering action. Ile-Ala-Ser-Gly-Glu-Pro (I)

When the peptide of the present invention is used in foods, cosmetics, feeds or pharmaceuticals, a purified product may be used. However, even if the peptide of the present invention has low purity, the peptide of the present invention has a strong ACE inhibitory activity. A crude fraction containing a peptide may be used, or a digested product of κ-casein glycomacropeptide with pepsin or papain may be used as it is. Moreover, the thing obtained by chemical synthesis can also be used. The peptide can be easily synthesized by using a commonly used solid phase method or the like as a method for chemically synthesizing the peptide, for example, using a peptide synthesizer manufactured by Applied Biosystems. Further, using the nucleotide sequence corresponding to the amino acid sequence of the peptide of the present invention, DN
It is also possible to synthesize A and produce the peptide of the present invention by genetic engineering. The nucleotide sequence of bovine κ casein is already known [Alexander, LJ et al., Eur. J. Biochem.,
vol.178, p.395, 1988]. Therefore, ATT GCT AGT GGT GAG CCT is suitable as a nucleotide sequence used when the peptide of the present invention is produced by genetic engineering.

Next, a method for producing the peptide of the present invention, which is particularly useful, will be described. As a method for obtaining a κ-casein glycomacropeptide useful as a raw material, for example, a method in which cheese whey powder is dissolved, heated and then cooled, and ethanol-precipitated supernatant is fractionated with an anion exchange resin [Saito.T. Et al., J. Dairy Sci., Vol.
74, p.2831, 1991], using the effluent obtained during the preparation of rennet casein curd as a raw material, adjusting the pH of this effluent to an acidic range to remove the formed precipitate, and then obtaining the resulting supernatant. [JP-A-63-284199], a dairy raw material containing κ-casein glycomacropeptide
After adjusting the pH to less than 4, use a membrane with a molecular weight cutoff of 10,000 to 50,000 and perform ultrafiltration to cut the permeate to a molecular weight cutoff of 50,000.
A method of concentrating using the following membrane [JP-A-2-276542], a whey protein-containing solution is heated and then frozen to obtain a frozen product, which is then thawed, followed by whey protein and a supernatant. Method of separating and separating and treating the supernatant and recovering the supernatant
A κ casein glycomacropeptide can be produced by any method such as [JP-A-3-294299], but here, the κ casein glycomacropeptide obtained by any method may be used.

This κ-casein glycomacropeptide is hydrolyzed with a proteolytic enzyme. The proteolytic enzyme used is preferably pepsin, but papain can also be used. It is also possible to prepare the peptide of the present invention by acid hydrolysis. When pepsin is used, pepsin may be added to the κ casein glycomacropeptide at a weight ratio of 1/10 to 1/10000. The enzyme reaction is usually carried out at 30 to 40 ° C, but it may be in the temperature range where the enzyme activity is recognized. The pH range suitable for the enzymatic reaction is preferably 1 to 5, but it is more preferably pH 1 to 4 because the reaction takes a considerable time at pH 4 or higher. As a buffer solution used for the enzyme reaction, a citrate buffer solution, an acetate buffer solution, a potassium chloride-hydrochloric acid buffer solution, (glycine + salt)
A hydrochloric acid buffer solution or the like can be used, and the pH can be conveniently adjusted with an acid solution such as a hydrochloric acid solution before use. The reaction time is 10 minutes or longer, preferably 1 to 48 hours. If the reaction time is less than 10 minutes, ACE inhibitory activity
20% or less, and ACE inhibitory activity of 60 after 48 hours
%, But when the reaction time is long, problems such as microbial contamination and a decrease in pepsin activity are unfavorable.
In some cases, pepsin can be added again during the reaction. It is convenient to stop the enzyme reaction by heating it at 100 ° C for 5 minutes to inactivate the enzyme. However, cold acetone is added for filtration and the pH is adjusted to 7.0 with a basic substance such as sodium hydroxide. You may perform the process which adjusts. The obtained solution is filtered to remove the precipitate, if necessary, and further concentrated under reduced pressure to dryness, freeze-dried or spray-dried. Alternatively, it may be dried powder after being concentrated by a reverse osmosis membrane, an electrodialysis membrane, a thin-film descending concentration, or the like. The crude fraction containing the peptide of the present invention thus obtained is 10 to 1
It exhibits ACE inhibitory activity on the order of 00 μg / ml, and can be sufficiently used as a food material. In addition, when an acute toxicity test was conducted, it was found that the peptide of the present invention showed an oral dose of 1 g / kg or more, and was highly safe.

The use form of the peptide of the present invention is not particularly limited, and it can be used as a solution, powder, granule, tablet, etc., can be added to various foods and drinks, cosmetics, feed, and can be used as a raw material for pharmaceuticals. Since the peptide of the present invention is produced in milk, it can be safely used. The dose of the peptide of the present invention is preferably 150 μg / kg / day or more. When the dose is 150 μg / kg / day or less, the hypotensive effect is weakened.

[0014]

[Reference Example 1] 150 g of cheese whey powder was mixed with a 12% (w / v) concentration of trichloroacetic acid solution, shake-extracted at 4 ° C for 1 hour, and then centrifuged (9,000 xg, 15 minutes),
The supernatant was collected and cooled on ice. The supernatant was neutralized with 1N sodium hydroxide, dialyzed against distilled water with a dialysis membrane (Viskase Sales Corp.), and then centrifuged (9,000 xg, 15 minutes).
Then, the supernatant was collected and freeze-dried. In this way, 7 g of 81% pure κ casein glycomacropeptide was obtained.

[0015]

Test Example 1 The κ-casein glycomacropeptide prepared in Reference Example 1 was subjected to a digestion test using 5 types of proteolytic enzymes. The combination of enzyme and buffer solution is as follows. (1) Proteinase K (manufactured by Sigma) and 0.01M Tris-
Hydrochloric acid buffer (pH 7.5) (2) Actinase E (Nacalai Tesque) and 20 mM calcium chloride containing 0.1M Tris-hydrochloric acid buffer (pH 7.4) (3) Pepsin (Sigma) and 0.2M citrate buffer (pH
3.0) (4) Trypsin (manufactured by Sigma) and 0.1M Tris-HCl buffer containing 20 mM calcium chloride (pH 8.0) (5) Papain (manufactured by Sigma), 3.3 mg sodium cyanide and 3.3 mg 0.1M phosphate buffer containing EDTA Liquid (pH 7.0)

Kappa casein glycomacropeptide was dissolved in 10 ml of each buffer solution to a concentration of 1 mg / ml, 5% (w / w) concentration of enzyme was added, and the mixture was sufficiently stirred. Each sample was subjected to enzyme digestion in the presence of a small amount of toluene at 37 ° C for 24 hours. Then, add 3 volumes of cold acetone to the reaction mixture, and add 1 at -20 ℃.
After standing for a time, the solution was filtered and freeze-dried to obtain a test sample. Then, 1 mg of each test sample is dissolved in 200 μl of distilled water, and 15 μl of this test sample solution is diluted with 150 μl of distilled water.
It diluted to 1 and measured the ACE inhibitory activity. Table 1 shows the results.

[0017]

[Table 1] ─────────────────────────── Enzyme ACE inhibitory activity ──────────────── ──────────── Proteinase K 0 (%) Actinase E 0 Pepsin 97 Trypsin 0 Papain 5 κ Casein glycomacropeptide 0 ───────────────── ──────────

The ACE inhibitory activity was measured as follows. First, 1 unit of ACE (derived from rabbit lung, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 2 ml of 50% glycerol solution to prepare an ACE enzyme solution (A). In addition, hippuric acid-histidyl leucine 53.688 mg was added to a borate buffer solution (p
H 7.8) dissolved in 10 ml and then pH adjusted with aqueous sodium hydroxide solution.
A substrate solution (B) adjusted to 8.3 was prepared.

150 μl of the test solution in a medium-sized test tube with a screw,
Substrate solution (B) 100 μl and ACE enzyme solution (A) 10 μl
l, add 60 ml of ethyl acetate to each test tube with a glass pipette to stop the reaction after reacting for 60 minutes at 37 ℃, shake with a vortex mixer for 15 seconds. It was extracted by. 1,000r after extraction
The upper layer (ethyl acetate) obtained by centrifugation at pm for 5 minutes was transferred to another test tube with a small screw, and ethyl acetate was evaporated to dryness under a nitrogen gas spray at 80 ° C. The sample thus evaporated to dryness was treated at 80 ° C for 10 minutes, returned to room temperature, 3.0 ml of 0.9% sodium chloride solution was added, and the mixture was shaken with a vortex mixer for 15 seconds to dissolve, and immediately the ultraviolet absorbance at 228 nm was measured. It was measured. Then, the ACE inhibitory activity was calculated by the following formula. ACE inhibitory activity (%) = {(E _C −E _S ) / (E _C −E
_B)} × 100 E _S: absorbance of the sample where the sample solution 150μl was added E _C: test solution absorbance of control of distilled water was added 150μl instead of E _B: distilled water 150μl instead of the test solution was added, the reaction Absorbance of sample whose reaction was stopped immediately after starting

As shown in Table 1, pepsin has the strongest ACE inhibitory activity of various proteolytic enzyme digests.
It was also found slightly in papain. Therefore, a substance having an ACE inhibitory activity in the pepsin digest was searched for.

[0021]

[Reference Example 2] 5% (w / w) was added to 10 ml of a solution of κ-casein glycomacropeptide dissolved in 0.1N hydrochloric acid so as to have a concentration of 1 mg / ml.
w) A concentration of pepsin was added, and the mixture was allowed to stand at 37 ° C for 24 hours for digestion, and then heated at 100 ° C for exactly 5 minutes to stop the enzymatic reaction. Then, the reaction solution is filtered, freeze-dried and κ
To obtain a pepsin digest of casein glycomacropeptide,
This was used as a sample.

90% of 10 ml of Butyl Toyopearl (manufactured by Tosoh)
After washing with methanol and then with distilled water, the column (1.7
× 12 cm). Next, the above sample was dissolved in distilled water to a concentration of 10 mg / 5 ml and added to the column. Then, after passing 200 ml of distilled water through the column, stepwise elution was carried out with 100 ml of each 10% to 100% methanol solution prepared in 10% increments. For each of the eluted fractions, methanol in the fractions was concentrated under reduced pressure and freeze-dried. Then, the ACE inhibitory activity was measured by the same method as in Test Example 1. ACE inhibitory activity was observed.
Therefore, this non-adsorbed fraction was further purified.

[0023]

[Reference Example 3] 9 mg / 5 ml of the non-adsorbed fraction obtained in Reference Example 2
What was dissolved in distilled water to a concentration was used as a sample. 10 g of Licloprep RP18 (manufactured by Merck) was washed with 90% methanol and then with distilled water, and then packed in a column (1.7 × 12 cm). Then add the above sample to the column and add 200 mL of distilled water.
After flowing ml, 100 ml of a 10% to 100% methanol solution prepared in 10% increments was used for stepwise elution. About each of the eluted fractions, methanol in the fractions was concentrated under reduced pressure and freeze-dried, and then A was prepared by the same method as in Test Example 1.
When the CE inhibitory activity was measured, a strong ACE inhibitory activity of 90% or more was observed in the methanol elution fraction having a concentration of 10% to 20%. In addition, 50% to 15% ACE inhibitory activity was also observed in the 30% to 60% concentrated methanol elution fraction. Therefore,
The 10% -60% concentration of the methanol elution fraction was collected and
The inhibitory activity crude fraction was used.

[0024]

[Reference Example 4] The ACE activity crude fraction obtained in Reference Example 3 was
Dissolve in distilled water to give a concentration of mg / 5 ml,
Rechromatography with RP18 (Merck) was performed. And 5%, 10%, 12%, 14%, 16%, 18%, 20
Stepwise elution was carried out with methanol solutions having a concentration of 30% and 30%. For each of the eluted fractions, methanol in the fractions was concentrated under reduced pressure, freeze-dried, and then the ACE inhibitory activity was measured by the same method as in Test Example 1, and the result was 5%.
A strong ACE inhibitory activity was observed in the methanol-eluted fraction of concentration, the methanol-eluted fraction of 10% concentration, and the methanol-eluted fraction of 12% concentration. Therefore, these fractions were further purified.

[0025]

[Reference Example 5] For the active fraction obtained in Reference Example 4, OD
HPLC using P-50 (Asahi Pack, 7.6 x 250 mm)
Purified in. Column contains 0.05% trifluoroacetic acid 10
% Acetonitrile aqueous solution (A) and equilibrated with 0.05% trifluoroacetic acid-containing 60% acetonitrile aqueous solution as the eluent (B), and gradient elution was carried out so that the acetonitrile of the eluent (B) was 50% in 30 minutes. .
The flow rate was 0.5 ml / min. As a result, a strong ACE inhibitory activity was observed in the fractions with a retention time of 10 to 18 minutes, so this fraction was collected and further purified.

[0026]

Reference Example 6 For the active fraction obtained in Reference Example 5, H using a Superiorex ODS (Shiseido, 4.6 × 150 mm) was used.
Purified by PLC. The column was equilibrated with a 0.05% trifluoroacetic acid aqueous solution (A), and a 60% acetonitrile aqueous solution containing 0.05% trifluoroacetic acid was used as an eluent (B), and the eluent (B) contained 50% acetonitrile. Gradient elution was performed as follows. The flow rate is 0.5 ml
I went at / min. As a result, a single peak was observed as shown in FIG. 1, and this was fractionated to determine the amino acid sequence.

[0027]

[Reference Example 7] Regarding the single peak obtained in Reference Example 6,
To determine the amino acid sequence, an amino acid sequencer (Type 473A, manufactured by Applied Biosystems) was used, and 1 μg of the sample was dot blotted on a PVDF membrane for analysis. As a result, the main body of the substance having ACE inhibitory activity is
It was found to be a peptide having the following amino acid sequence. Ile-Ala-Ser-Gly-Glu-Pro

[0028]

Reference Example 8 A peptide was synthesized in order to confirm the ACE inhibitory activity of the peptide whose amino acid sequence was confirmed in Reference Example 7. Peptide synthesizer for chemical synthesis of peptides
(Model 430A, manufactured by Applied Biosystems)
-Moc method. That is, 0.5 mmol of Boc-1
-Cln-O-CH ₂ -PAM the constituent amino acids of the resin and 2mmol performed by filling the peptide synthesizer synthesis, to obtain a resin bound peptide of interest. This resin 1.
The peptide bound to 5g was cleaved with trifluoromethanesulfonic acid in the presence of thioanisole and ethanedithiol, precipitated with diethyl ether, dissolved with 10% acetic acid, and replaced with 10% acetic acid. Purification by passing through an exchange resin (Bio-Rex MSZ 1-X8) yielded 140 mg of peptide. This peptide is further added to Aquapac RP-300
Reversed-phase HPLC (manufactured by Applied Biosystems)
With 0.1% trifluoroacetic acid / water and 0.1% as eluents.
Purification using% trifluoroacetic acid / acetonitrile afforded 30 mg of peptide white powder. Then, the ACE inhibitory activity was measured by the same method as that shown in Test Example 1. The results are shown in Table 2. The ACE inhibitory activity of the peptide purified in Reference Example 6 was also measured.

[0029]

[Table 2] ────────────────────────────────── ACE inhibitory activity ───────── ───────────────────────── Ile-Ala-Ser-Gly-Glu-Pro peptide (Reference Example 6) 100 (%) Ile-Ala -Ser-Gly-Glu-Pro peptide (Reference Example 7) 97 ───────────────────────────────────

[0030]

[Test Example 2] The blood pressure improving effect of the peptide having the following amino acid sequence (I) was confirmed. Ile-Ala-Ser-Gly-Glu-Pro (I) SHR / NCri rats (purchased from Charles River Japan) that spontaneously develop 4-week-old hypertension were divided into the groups shown below, and animal experiments were conducted. . Group A: Amino acid sequence (I) peptide was administered at 300 μg / day B group: Amino acid sequence (I) peptide was administered at 30 μg / day C group: Amino acid sequence (I) peptide was administered at 3 μg / day D group: Peptide was not administered

CE-containing no casein, each group consisting of 5 animals
It was bred for 6 weeks using 2 (CLEA) as a feed. Each test sample was forcibly administered twice a day, and drinking water was freely ingested.
Then, the blood pressure after 6 weeks was measured. Table 3 shows the results.

[0032]

[Table 3] Numerical values are shown as mean value ± standard deviation. In group A and group B, a significant blood pressure lowering effect was observed.

[0033]

Example 1 According to the method described in JP-A-2-276542, 50 g of κ casein glycomacropeptide was obtained from 1 kg of whey protein concentrate (WPC). 50 g of this κ-casein glycomacropeptide was dissolved in 1,250 g of water, pH was adjusted to 1.5 with hydrochloric acid, 0.5 g of pepsin (manufactured by Sigma) was added, and the enzyme reaction was carried out at 37 ° C. for 15 hours. The enzymatic reaction was stopped by heating at 100 ° C. for 5 minutes, filtered through filter paper (manufactured by Advantech), and then lyophilized to obtain 37 g of a pepsin digest of κ casein glycomacropeptide. Next, 37 g of this digest was dissolved in 1,850 g of distilled water and added to a column (20 cm × 20 cm) packed with 5 kg of Biosil C18HL (manufactured by Bio-Rad) equilibrated with distilled water, and the column was washed with distilled water. After that, it was eluted with a 60% aqueous solution of methanol. Then, this eluate fraction was concentrated under reduced pressure and freeze-dried to obtain the active fraction.
I got 12g. Furthermore, 300 mg of this active fraction was dissolved in 1 ml of a 2% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid and equilibrated with a 2% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid, and a TSKgel ODS-120T column (55 mm x 60 c
m, manufactured by Tosoh) containing 0.1% trifluoroacetic acid 5
Gradient elution was performed with a 0% aqueous acetonitrile solution.
Then, a single peak at a concentration of acetonitrile of about 10% where ACE inhibitory activity was observed was collected. This operation was repeated and the collected fractions were concentrated with a centrifugal dryer and freeze-dried to obtain 1.8 g of the peptide.

Then, Superiorex ODS (4.6 mm ×
150 mm, manufactured by Shiseido), the purity of this peptide was 93%. In addition, when the amino acid was analyzed by an amino acid sequencer, this peptide was a peptide having the following amino acid sequence (I). Ile-Ala-Ser-Gly-Glu-Pro (I) The present inventors named the peptide having the above amino acid sequence (I) as kappa caseinosine.

[0035]

EXAMPLE 2 0.5mmol of Boc-l-Cln-O- CH 2
-A PAM resin and 2 mmol of constituent amino acids were packed in a peptide synthesizer (430A type, manufactured by Applied Biosystems) for synthesis to obtain a resin having the desired peptide bound thereto. The peptide bound to 1.5 g of this resin was cleaved with trifluoromethanesulfonic acid in the presence of thioanisole and ethanedithiol, precipitated with diethyl ether, dissolved with 10% acetic acid, and replaced with 10% acetic acid as a strong base. Anion exchange resin (Bio-Rex MSZ1-X8)
It was purified by passing through a column to obtain 140 mg of the peptide. The peptide was further purified by reverse phase HPLC on Aquapac RP-300 (manufactured by Applied Biosystems) using 0.1% trifluoroacetic acid / water and 0.1% trifluoroacetic acid / acetonitrile as eluents. 30 mg of white powder of peptide was obtained. When this peptide was subjected to amino acid analysis with an amino acid sequencer, it was a peptide having the following amino acid sequence (I). Ile-Ala-Ser-Gly-Glu-Pro (I)

[0036]

[Example 3] Method of Saito et al. [J. Dairy Sci., Vol.74, p.2]
831, 1991], 13 g of κ-casein glycomacropeptide was obtained from 1 kg of cheese whey powder. 13 g of this κ-casein glycomacropeptide was dissolved in 0.33 liters of citrate buffer (pH 3.0), and 0.01 g of pepsin (manufactured by Sigma) was added,
The enzymatic reaction was carried out at 32 ° C for 48 hours. The enzymatic reaction was stopped by adding 1.95 liters of cold acetone, then concentrated to 0.5 liter with an evaporator and freeze-dried to obtain 11 g of an active fraction. When the ACE inhibitory activity of this active fraction was measured by the same method as in Test Example 1, the ACE inhibitory activity was 100% at 100 μg / ml.
Were present.

[0037]

Example 4 According to the method described in JP-A-2-276542, 17 g of κ casein glycomacropeptide was obtained from 1 kg of cheese whey powder. After dissolving 17 g of this κ-casein glycomacropeptide in 65 liters of 0.1 M Tris-hydrochloric acid buffer (pH 7.5), 0.17 g of papain (manufactured by Sigma) was added, and 37
The enzymatic reaction was performed at 20 ° C. for 20 hours. The enzymatic reaction was stopped by adding 1.95 liters of cold acetone. Then, this reaction solution was concentrated to 0.5 liter with an evaporator and freeze-dried to obtain 15 g of a papain digest of κ-casein glycomacropeptide. The ACE inhibitory activity of this papain digest was measured by the method described in Test Example 2, and it was found that the ACE inhibitory activity was 9 at 100 μg / ml.
% Existed.

[0038]

Example 5 A trial preparation of a drink formulation containing the peptide obtained in Example 1 was made. Peptide of the present invention 150 mg, sucrose
3.2g, citric acid 53g, sodium citrate 53g, vitamin B ₂ 0.06g, vitamin C 0.37g, folic acid 0.03g, flavor 6.
7g, 5 times concentrated apple juice (134g) and water (3.5Kg) were mixed at 90 ° C for 10 seconds to heat sterilize the composition, then 5 to 10 ° C.
It was cooled to and stored in a sterile container. Next, this mixed solution was aseptically filled in a sterilized 350 ml container to manufacture a drink preparation for preventing hypertension.

[0039]

Example 6 A tablet containing the peptide obtained in Example 2 was manufactured as a trial. Peptide of the present invention 20 mg, lactose 33.3 mg, corn starch 16.4 mg, carboxymethyl cellulose calcium 12.8 mg and magnesium stearate 1.5 m
g was blended, and a tablet for suppressing hypertension was manufactured according to a conventional method.

[0040]

Example 7 An animal feed containing the active fraction containing the peptide of the present invention obtained in Example 3 was experimentally produced. An animal feed was produced by mixing 1 g of the active fraction containing the peptide of the present invention, 60 g of skim milk powder, 14.3 g of whey protein concentrate (WPC), 17.2 g of fat, 5.0 g of glucose, 2.5 g of vitamins and 2.5 g of minerals. .

[0041]

Example 8 A milk for preventing hypertension containing the peptide obtained in Example 1 was manufactured as a trial. 750 mg of the peptide of the present invention
, 1,195 g of skim milk, 263 g of whey powder, 119 g of vegetable oil and fat, and 5 g of vitamins / minerals were mixed to produce milk powder for preventing hypertension.

[0042]

EFFECT OF THE INVENTION The peptide of the present invention is a highly safe peptide derived from milk, and is used as an injection, a dragee, a tablet, a capsule or the like, or as a soft drink, fruit juice drink, fermented drink, jelly, ice cream and the like. It can be added to various foods and drinks and used for the purpose of treating and preventing hypertension. It can also be added to cosmetics to impart a vasodilatory effect. Furthermore, it can be added to animal feed and used for the purpose of treating and preventing hypertension in livestock and animals.

[Brief description of drawings]

FIG. 1 shows the results of HPLC in Reference Example 6.

Claims (3)

[Claims] 1. A peptide having the following amino acid sequence (I). Ile-Ala-Ser-Gly-Glu-Pro (I) 2. An angiotensin converting enzyme inhibitor containing the following amino acid sequence (I) as an active ingredient. Ile-Ala-Ser-Gly-Glu-Pro (I) 3. A hypotensive agent containing the following amino acid sequence (I) as an active ingredient. Ile-Ala-Ser-Gly-Glu-Pro (I)