JP3406341B2 - New peptides, their production methods and applications - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel peptide, a process for producing the same, and a use for lowering blood pressure thereof.

[0002]

2. Description of the Related Art First, the present inventors have conducted heat denaturation treatment on fish meat, hydrolyzed it with neutral or alkaline protease to deactivate the enzyme, and then separated the ACE to obtain ACE inhibitory activity. Was obtained (Japanese Patent Application No. 4-72227).

[0003]

DISCLOSURE OF THE INVENTION Peptide α is used in the present invention.
The objective was to isolate each peptide having a strong ACE inhibitory activity from -1000.

[0004]

In the present invention, an aqueous solution of peptide α-1000 is treated with a polystyrene resin, and a fraction having the highest ACE (angiotensin converting enzyme) inhibitory activity is separated from the eluate. Succeeded in
Then, the fraction having a strong inhibitory activity was chromatographed and further subjected to reverse phase HPLC to obtain a peptide having a strong ACE inhibitory activity, Thr-Tyr or / and Gly-Tr.
It was successful in isolating p.

That is, according to the present invention, α-1000, which is a peptide obtained by treating fish meat with an alkaline protease, is treated with a polystyrene resin, which is then eluted with an aqueous ethanol solution having an ethanol concentration of 0 to 20% to produce an ethanol fraction. The target peptide can be obtained by separating the fractions having a particularly high hydrophilicity, the fraction having a little bitterness, and the fraction having a strong ACE activity from these fractions.

Among the ethanol fractions thus obtained, the fraction eluted with a 0% ethanol aqueous solution, that is, the unadsorbed fraction (AF-1 fraction) and the fraction eluted with a 10% ethanol aqueous solution. (AF-2 fraction) is
It was confirmed that they are all unique in terms of action and effect, and they are novel substances that have not been published in the literature. Furthermore, Thr-Tyr or / and Gly-Trp were isolated from the AF-1 fraction, and these peptides were also isolated. It was confirmed to be a new substance.

These peptides have almost no bitterness, are naturally occurring and safe, and are excellent as an active ingredient of an antihypertensive agent or an active ingredient of an antihypertensive functional food.

Peptide of the present invention, AF-1 fraction, Thr
All of -Tyr and / or Gly-Trp are novel compounds that have not been published in the literature, and can be supplied from animal or plant proteins other than fish meat, and can also be used as peptides obtained by chemical synthesis.

Next, the peptide α-1000 will be described.

Peptide α-1000 is produced from seafood as a raw material. First, this is treated with a meat mining machine, a deboner or the like to separate the quality of fish meat. The raw materials are preferably as fresh as possible. The separated fish meat is 10k
It may be divided into about g g of surimi and used as it is for the next treatment, but it is rapidly frozen by blowing cold air at -20 to -50 ° C, for example, about -30 ° C, and stored at -20 to -25 ° C. Alternatively, it may be appropriately used if necessary.

Examples of seafood include sardines, horse mackerels, tuna,
Red fish such as skipjack, saury, mackerel; flounder, Thailand, kiss,
White fish such as conifer, cod, herring, yellowtail; cartilage fish such as shark, ray; freshwater fish such as smelt, carp, char, yamame; deep-sea fish such as shark, anglerfish, shrimp, crab, octopus, amy etc. it can.

After the meat is extracted, the seafood is crushed by a crusher or the like, water is added in an amount of 1/2 to 20 times, preferably 10 to 10 times the weight of the raw material, and then heat-treated. Therefore, it deactivates the autodigestive enzyme and kills various bacteria, and heat-denaturates the protein to increase the efficiency of the subsequent enzymatic reaction. As the heating condition, all the conditions can be used as long as such a condition is exhibited,
For example, the temperature is 65 ° C. or higher for 2 to 60 minutes, preferably 80 ° C. or higher for 5 to 30 minutes.

Then, the pH is adjusted to an appropriate value of the proteolytic enzyme to be used by adding an alkaline agent such as aqueous ammonia or an aqueous solution of sodium (potassium hydroxide) (for example, in the case of alkaline protease, pH is 7.5 or more, preferably Is 8 or more) and the temperature is suitable for the enzyme (2 depending on the enzyme used, but 2
0 to 65 ° C, 35 to 60 for alkaline protease
C., preferably 40 to 55.degree. C.), and a proteolytic enzyme is added and treatment is carried out for 30 minutes to 30 hours (30 minutes to 25 hours in the case of alkaline protease, preferably 1 to 17 hours).

As the proteolytic enzyme, any enzyme can be used alone or as a mixture as long as it is an enzyme capable of decomposing a protein under neutral or alkaline conditions. Its origin can be sought from microorganisms in addition to plants and animals. Pepsin, renin, trypsin, chymotrypsin, papain, bromelain, bacterial protease, filamentous protease,
Actinomycetes protease and the like can also be widely used. As these enzymes, commercially available ones are usually used, but unpurified enzymes, enzyme-containing culture solutions, solid or liquid enzyme-containing substances such as koji, may also be used according to the purpose. You can The amount of enzyme added is 0.1% to 5.0%
It may be a degree.

Next, if necessary, neutralization treatment is performed, and then 7
A temperature of 0 ° C. (preferably 80 ° C.) or higher is maintained for 2 to 60 minutes (preferably 5 to 30 minutes) to inactivate the enzyme and to allow good separation to be performed later. After the heat deactivation treatment, the product is roughly separated by a hive screen or the like, and if necessary, treated with a jetter, and then subjected to ultracentrifugation to remove suspended matters and precipitates.

Next, the mixture is filtered using a filter aid such as diatomaceous earth (for example, Celite), and the filtrate is treated with activated carbon (0.0
5 to 20% W / V, preferably 0.1 to 10% W / V used, 20 to 65 ° C, preferably 25 to 60 ° C, 15 minutes to
4 hours, preferably 30 minutes to 2 hours), deodorizing, decolorizing and purifying.

This is concentrated under reduced pressure (0 to 50 ° C.) or any other conventional method (up to about 30 B ×), and if necessary, again (ultra) centrifuged or filtered to obtain a peptide stock solution. The peptide stock solution thus obtained was sterilized (UH
After TST and other conventional methods), it is filled in a container to obtain a product (α-1000 (liquid)). If desired, the product can be further concentrated or diluted in reverse, or powdered to about 60 mesh by a conventional method such as spray drying or freeze drying, and then filled into a container such as a bag ( α
-1000 (powder)). Store these products refrigerated or frozen.

The liquid, pasty or powdery peptide thus obtained is α-1000.

The physicochemical properties of peptide α-1000 (spray dry powder) are shown in Tables 2, 3 and 4 below.

[0020]

[Table 2]

[0021]

[Table 3]

[0022]

[Table 4]

The aqueous solution of the peptide α-1000 obtained here is passed through a peptide adsorbing resin such as a giant reticulated polystyrene-based resin such as a polystyrene-based resin, washed with water and the like, and then subjected to elution flow with various concentrations of ethanol or the like. ,
A fraction with a strong ACE inhibitory activity was taken from each fraction, separated and purified by size exclusion chromatography, and then reversed phase H
Each single peptide can be isolated by performing chromatography by PLC.

For each peptide, Thr-Tyr or / and Gly-Trp could be confirmed by an amino acid analysis system and a protein sequencer.

It was confirmed that each of these peptides has a high ACE inhibitory activity.

Each peptide of the present invention may be one kind or two kinds.
Depending on the species, it can be used as an ACE inhibitor, for example, as a blood pressure lowering agent or a peptide for food for specified health use for the purpose of lowering blood pressure. Therefore, the present peptide is used as a food such as a seasoning and a food for nutritional enrichment, or as an additive for animal feed, and because of the above-mentioned unique physiological activity, it can be used as a medicine for the prevention or treatment of hypertensive diseases. As or as infusions, health foods,
It can be widely used as a clinical nutrition food.

When used as a food, the peptide may be added as it is, or may be used in combination with other foods or food ingredients according to a conventional method. When used as a medicine, it can be administered orally or parenterally. In the case of oral administration, for example, tablets, granules, powders, capsules and powders can be prepared according to a conventional method, and in the case of parenteral administration, for example, injection preparations, drops, suppositories, etc. It can be used as an agent or the like.

Hereinafter, the present invention will be described in more detail with reference to Examples and Examples.

[0029]

[Reference Example] Fresh sardines were processed with a bodyner and minced. The minced fish meat is divided into 10 kg of surimi, which is rapidly frozen at -30 ° C or lower, crushed by a crusher, added with an equal amount of water, and then sent to a tank at 10 ° C at 100 ° C.
After heating for a minute, the autolyzing enzyme was inactivated and heat denatured.
Then, aqueous ammonia was added to adjust the pH to 10.0.

To this was added a 0.1% solution of a commercially available alkaline protease product, and the mixture was kept at 45 ° C. for 17 hours for enzymatic decomposition. The enzyme was then inactivated by boiling for 15 minutes.

This was passed through a vibrator screen (150 mesh), treated with a diector at 5000 rpm, and then treated with a Sharpless centrifuge (15000 rpm),
The diatomaceous earth was used as a filter aid, and the filtered solution was used as a peptide stock solution.

1% W of activated carbon was added to the peptide stock solution obtained above.
/ V was added, the mixture was stirred at 30 ° C. for 60 minutes and then filtered to obtain a filtrate. This is concentrated under reduced pressure (20 ° C.) according to a conventional method, and then subjected to UHTST sterilization according to a conventional method to obtain α-1.
000 (liquid) product is obtained, which is further spray-dried according to a conventional method to obtain α-1000 (powder) having a particle size of 60 mesh.
The products were obtained and each of these products was stored frozen.

Peptide α-10 thus prepared
00 (powder) was subjected to gel filtration using a Sephadex G-25 column under the following conditions. As a result, the results shown in FIG. 3 were obtained, and the molecular weight was found to be 200 to 10,000. Column size: Diameter 16 x 950 mm, Eluent:
0.1 M phosphate buffer (pH 7.0), fraction: 2 ml / tube, flow rate: 10 ml / h, molecular weight marker: bacitracin (molecular weight 1450).

Α-1000 (liquid) obtained above
Had a water content of 73.6% (vacuum heating drying method), exhibited a pale yellow color, and its 10% solution had a pH of 7.5, and no fishy odor or bitterness was observed. As a result of analyzing the components and measuring the amino acid composition, the results shown in Tables 5 and 6 below were obtained.

[0035]

[Table 5]

[0036]

[Table 6]

[0037]

Example 1 Peptide α-1000 obtained in Reference Example
Dissolve 5 mg (powder) in 100 ml deionized water, run on an AMBERLITE XAD-2 resin column (3.5 x 13 cm) to adsorb the peptide, wash with deionized water, then 0 % And 10% ethanol aqueous solution (500 ml each) were eluted to obtain active AF-1 and AF-2 fractions, respectively. The ACE inhibitory activity IC ₅₀ of these fractions is shown in Table 7 below.
The AF-1 fraction was obtained from 3.5 g of fish meat protein in an amount of 1.4 g, and the molecular weight distribution measured by GPC was an oligopeptide having a molecular weight of about 200 to 1000 centered on di- and tripeptides.

[0038]

[Table 7]

The amino acid compositions of the obtained AF-1 and AF-2 fractions are shown in Table 8 below. For reference, the amino acid composition of α-1000 and F-3 (pepsin digestion product of sardines, ODS treatment, 25% ethanol elution fraction reported by Kyoda et al.) Is also shown.

[0040]

[Table 8]

As is clear from the above results, compared with the amino acid composition of the F-3 fraction, AF-1 has a low content of hydrophobic amino acids such as leucine and amino acids having a bitter taste such as arginine, and glutamic acid and alanine. It has been shown that the content of amino acids having acidic and hydrophilic amino acids such as umami and sweetness is high, and that the amino acid ratio of AF-2 has a stronger hydrophobic component with hydrophobic amino acid residues in the resin column. It strongly suggests the correlation between the hydrophobic amino acid residue and the bitterness component.

AF-1 and AF separated in this way
The -2 fraction was subjected to a sensory evaluation by a panel of 10 trained people, and the results shown in Table 9 below were obtained. As a control, the same sensory evaluation was performed on α-1000.

[0043]

[Table 9]

As is clear from the above results, powder product α-
Compared with 1000, AF-1 had almost no bitterness and fishy odor, improved umami and sweetness, and a clear reduction in aftertaste. Further, AF-2 had a weak umami, and a bitterness and fishy smell were recognized. Thus, the fraction according to the present invention, combined with the strength of ACE inhibitory activity, can be advantageously used for the purpose of lowering blood pressure as a food or a composition for oral administration.

[0045]

Example 2 The AF-1 fraction obtained in Example 1 has physicochemical properties as shown in Tables 10 and 11 below and, as is clear from the above, a relatively high A value.
It showed CE inhibitory activity, and a significant improvement in color and taste was observed as compared with α-1000. Such a peptide is a novel one which is unknown in the literature and is extremely useful.

[0046]

Table 10 Physicochemical properties of AF-1 fraction (A) Molecular weight: 200 to 1000 (ASAHIPAK
(By GS-320 high performance liquid chromatography) (B) Melting point: Colored and decomposed at 95 ° C. (C) Specific optical rotation [α] _D ²⁰ = −17 ° (D) Solubility in solvent: It is readily soluble in water, but hardly soluble in ethanol, acetone, and hexane. (F) Appearance of substance: White to pale yellow powder. Component (G): water content 5.3% (normal pressure heating and drying method); protein 88.7% (Kjeldahl method, nitrogen / protein conversion coefficient 6.
25); lipid 0% (Soxhlet extraction method); ash 4.8
% (Direct ashing method). (H) UV spectrum: FIG. 5 (I) IR spectrum: FIG. 6 (J) Amino acid composition: Table 11

[0047]

[Table 11]

[0048]

Example 3 Further separation of AF-1 fraction, which is a deionized water fraction (unadsorbed fraction), eluted by Amberlite XAD-2 column chromatography was performed by HPLC. First, since it is considered that this fraction contains a large amount of polar or hydrophilic peptides, G using Asahipak GS-320 capable of separation in multiple modes was used.
PC analysis was performed. AF-1 250ml as sample liquid
Was concentrated to 10 ml and used as a mobile phase.
When the analysis was performed using mM ammonium acetate (pH 6.7) / acetonitrile (80/20), the values A to F were consistent with the absorbance at 220 nm, which is the peptide bond absorption.
Six highly active peptide fractions (FIH) were obtained (Fig. 7).

Each of the 6 highly active peptide fractions A to F was concentrated to dryness and subjected to reverse phase HPLC using a μ Bondapaic C18 column. Elution is by eluent:
(A) 0.05% trifluoroacetic acid, (b) 0.05%
Using 10% acetonitrile in trifluoroacetic acid (a)
From (b) to (80 minutes) with a linear gradient, 2
It was monitored at a wavelength of 20 nm and separated into numerous fragments. Further, several kinds of fractions were isolated by performing size exclusion chromatography and reverse phase chromatography of the peak fractions. The ACE inhibitory activity of the fraction was measured, and amino acid analysis of the active fraction was performed. As a result, two fractions (FIH
-A3 and FIH-B6) were confirmed as peptides.

[0050]

Example 4 Amino acid analysis was carried out using a Shimadzu LC-6A amino acid analysis system after hydrolysis with hydrochloric acid by a conventional method. Column: Shim-pack ISC-0
7 / s 1504Na (0.40 cm × 15 cm), column temperature: 55 ° C., eluent: A 0.2 N sodium citrate pH 3.20 (containing 7% ethanol), B 0.2 M
Boric acid / 0.6N sodium citrate pH 10.0 program gradient, flow rate: 0.3 ml / min, detection: o-phthalaldehyde (oPA) method. The amino acid sequence of the ACE inhibitory peptide is ABI (Applied Bi
ossystems Inc. ), A 477A / 120A type protein sequencer manufactured by KK).

The peptide obtained in the present invention is Thr-T
yr or / and Gly-Trp.

[0052]

[Example 5] ACE inhibitory activity was measured by ACE (E manufactured by Sigma)
C. 3.4.5.1; from rabbit lung) 2.5 mU, synthetic substrate Hippuryl-histid manufactured by Protein Research Institute
Using yl-L-leucine 12.5 mH, the method of Matsui et al. (T. Matui, H. Matsufuj
i and Y. Osajima: Biosci. Bi
otech. Biochem . , 56 , 517 (199
2)) was measured. That is, the generated L-hi
convert stidyl-L-leucine into TNP, 4
Absorbance at 16 nm was measured. The absorbance in the test solution is E
B, the value when water was added instead of the test solution was EC, the value in the test solution when the reaction stop solution was added in advance and the reaction was EB ₂ , and the value in water was EB ₁ , and the inhibition rate ( %) = ((E
C-EB ₁ )-(EB-EB ₂ ) / (EC-EB ₁ )) ×
Expressed as 100. The ACE inhibitory activity IC ₅₀ value of the sample is
The protein concentration of the sample required to inhibit the ACE enzyme activity by 50% (inhibition rate) is shown.

The ACE inhibitory activity IC ₅₀ of each isolated peptide is shown in Table 12 below.

[0054]

[Table 12]

As is clear from the above results, the fraction FIH
-A3, FIH-B6 (peptide Thr-Tyr, Gl
y-Trp) has IC ₅₀ values of 288 μM and 86 μM, respectively.
It is M and has excellent ACE inhibitory activity.

Both peptides according to the present invention have hydrophilic amino acids at the N-terminals, have no bitterness, and have excellent activity. Therefore, these peptides are
Among the ACE-inhibiting active peptides, it can be said that it is a naturally-occurring peptide having a good taste, no bitterness or fishy odor, and having hydrophilicity, and can be widely used in various applications.

[0057]

INDUSTRIAL APPLICABILITY Since each novel peptide obtained by the present invention has almost no bitterness, it can be used not only as a food or drink itself or as an additive, but also has an excellent ACE inhibitory activity, which suppresses an increase in blood pressure as a health food. Or, it can be used for prevention, and can also be advantageously used as a medicine by formulating into various dosage forms as an ACE inhibitor or an antihypertensive agent.

[Brief description of drawings]

FIG. 1 illustrates the UV spectrum of peptide α-1000.

FIG. 2 illustrates the IR spectrum of peptide α-1000.

FIG. 3 illustrates the molecular weight distribution of peptide α-1000 by gel filtration.

FIG. 4 illustrates the molecular weight HPLC pattern of AF-1 fraction.

FIG. 5 illustrates the UV spectrum of the AF-1 fraction.

FIG. 6 illustrates the IR spectrum of AF-2 fraction.

FIG. 7: Asahipak GS-32 of AF-1 fraction.
9 is a graph showing an elution curve at 0 column.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yutaka Oshima Shima 2-24-26 Maimatsubara, Higashi-ku, Fukuoka City (58) Fields investigated (Int.Cl. ⁷ , DB name) C07K 5/06 C07K 14/46 A23L 1 / 305 C12P 21/00-21/06 CA (STN) REGISTRY (STN)

Claims (3)

(57) [Claims] 1. A peptide A having the following physicochemical properties:
F-1 fraction. Physicochemical properties of AF-1 fraction (A) Molecular weight: 200 to 1000 (ASAHIPAK
(By GS-320 high performance liquid chromatography) (B) Melting point: Colored and decomposed at 95 ° C. (C) Solubility in solvent: It is readily soluble in water, but hardly soluble in ethanol, acetone, and hexane. (D) Appearance of substance: White to pale yellow powder. (E) component: water content 5.3% (normal pressure heat drying method); protein 88.7% (Kjeldahl method); lipid 0% (Soxhlet extraction method); ash content 4.8% (direct ashing method). (F) It has ACE inhibitory activity. (G) Peptide α-100 having the following physicochemical properties
This is a fraction obtained by adsorbing 0 on a polystyrene resin and then eluting it with water. Physicochemical properties of peptide α-1000 (powder) (A) molecular weight 200 to 10,000 (by Sephadex G-25 column chromatography) (B) melting point: 119 ° C. (decomposition point) (C) specific optical rotation [Α] _D ²⁰ = -22 ° (D) Solubility in solvent: Easily soluble in water: hardly soluble in ethanol, acetone, and hexane. (E) Distinction between acidic, neutral and basic: Neutral pH 5.0 to 8.0 (10% solution) (F) Appearance, component: White powder; Water content 5.14% (vacuum heating drying method); Protein 87.5% (Kjeldahl method, nitrogen / protein conversion factor 6.
25); lipid 0% (Soxhlet extraction method); ash 5.0
% (Direct ashing method). (G) UV spectrum: As shown in FIG. (H) IR spectrum: As shown in FIG. (I) Characteristics: A peptide derived from fish meat, which is obtained by deactivating the autolytic enzyme by heating and hydrolyzing it with a proteolytic enzyme. 2. A hypotensive agent comprising the peptide AF-1 fraction according to claim 1 as an active ingredient. 3. A hypotensive functional food comprising the peptide AF-1 fraction according to claim 1 as an active ingredient.