JPH05271297A - New peptide alpha-1000 - Google Patents

Abstract

PURPOSE:To obtain the subject new peptide derived from fish meat, having specific physicochemical characteristics, having angiotensinase-inhibitory activity, thus useful for e.g. hypotensive agents by heating fish meat to deactivate autodigestive enzyme followed by hydrolysis with a protease. CONSTITUTION:Fresh sardine is specifically treated and the meat is taken and ground. The ground meat is then rapidly frozen at <=-30 deg.C followed by grinding with a grinder. Thence, an equal amount of water is added to the resulting meat powder and the mixture is fed to a tank where the mixture is heated at 100 deg.C for 10min to deactivate autodigestive enzyme, thus thermally denaturating the meat powder. The resultant mixture is adjusted in pH value to 10.0 by addition of ammonia water followed by addition of alkali protease to carry out enzymolysis under residence at 45 deg.C for 17hr followed by boiling for 15min to deactivate the enzyme. Thence, the decomposition product is put to gel filtration and purified, thus obtaining the objective neutral new peptide alpha-1000 which has the following characteristics: (1) molecular weight: 200-10000 (determined by column chromatography); (2) decomposable at 119 deg.C, resulting in discoloration; (3) specific rotatory power [alpha]<20>D=-22 deg.; (4) readily soluble to water; and (5) insoluble in ethanol, acetone, and hexane.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to peptides, and more particularly, the present invention relates to novel peptides which have not been published in the literature. The present invention also relates to a method for producing the same, particularly to an ACE inhibitor, an antihypertensive agent, or a specific food or drink having hypotensive properties.

[0002]

2. Description of the Related Art Generally, it is common practice to hydrolyze fish and shellfish with an acid, an alkali, a proteolytic enzyme or the like to obtain a tasting hydrolyzate.

The inventors of the present invention were able to obtain a novel peptide, DAN-100, by first subjecting the seafood to autolysis and then treating it with a proteolytic enzyme. 3-54958).

[0004]

DISCLOSURE OF THE INVENTION The present invention has been made with the focus on the useful physiological activity of fish meat, with the aim of developing a novel peptide that has hitherto been unknown, and further developing a useful use of the novel peptide. It is a thing.

[0005]

Means for Solving the Problems The present invention has been made to achieve the above object, and as a result of earnest research to develop a superior novel functional peptide, it is possible to self-digest fish and shellfish. After heat treatment to prevent
A molecular weight of 200-
Not only was it successful for the first time to obtain 10,000 novel peptides, but this peptide also excels in angiotensin-converting enzyme (Angiotensin-converter).
The present invention has been completed on the basis of these new findings.

Therefore, according to the present invention, seafood is first heated to inactivate its autolyzing enzyme, and then this heat-denatured fish meat is treated with a neutral or alkaline protease to form a peptide, The desired peptide can be obtained by separating, filtering, purifying, concentrating and sterilizing.

The peptides according to the present invention are
N-100) is fundamentally different from N-100) and heat-denatures pre-formed fish meat, and does not utilize alcohol fractionation and ion exchange resin treatments for separation and purification, and its physical properties and structure are unique. Not only is it more unique in that it also has an excellent ACE inhibitory action, hypotensive action, or antihypertensive action, and such a peptide is not described in the literature and is therefore a novel substance. It was confirmed that this was designated as α-1000.

This peptide is produced from fish and shellfish 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 material is preferably as fresh as possible. The separated fish meat may be divided into about 10 kg of surimi and used as it is for the next treatment.
It is also possible to blow cold air at -20 to -50 ° C, for example, about -30 ° C to quick-freeze it, store it at -20 to -25 ° C, and use it as needed.

The seafood includes 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.

In the present invention, after meat is collected, the seafood is crushed by a crusher or the like, and the amount is 1/2 amount to the weight of the raw material.
After 20 times the amount of water, preferably an equivalent amount to 10 times the amount of water, it is heat-treated to inactivate the autodigesting enzyme and kill other bacteria, and at the same time heat denaturate the protein to carry out an enzymatic reaction. Increase efficiency. As the heating condition, all the conditions can be used as long as such a function is exhibited, for example, 65 ° C. or higher, 2 to 60 minutes,
It is preferably 80 ° C. or higher and 5 to 30 minutes.

Then, an alkaline agent such as aqueous ammonia or an aqueous solution of sodium (potassium hydroxide) is added to adjust the pH to an appropriate value for the proteolytic enzyme used (for example, in the case of alkaline protease, pH is 7.5 or more, preferably pH 7.5 or more). 8 or more), and the temperature is suitable for the enzyme (depending on the enzyme used, 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 in combination as long as it is an enzyme capable of degrading 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.

Then, 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 filtration with a high-broth screen or the like, optionally 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 Bx), 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 thus obtained peptide in liquid, paste or powder form is a novel one which is unknown in the literature and is extremely useful, and was named α-1000.

The novel peptide α-1000 according to the present invention
The physicochemical properties of (spray dried powder) are as shown in Tables 3, 4, and 5 below.

[0018]

[Table 3]

[0019]

[Table 4]

[0020]

[Table 5]

The peptide α-1000 according to the present invention has no unpleasant fishy odor or bitterness, which is a characteristic of peptides derived from fish meat, can be easily pulverized, and becomes a white powder that is easy to handle. It can be used extensively.

In particular, as is clear from the examples described below, the peptide α-1000 according to the present invention has excellent AC.
Since it was confirmed to have E-inhibitory activity, it can be used as an ACE inhibitor, for example, a blood pressure-lowering agent or a peptide for food for specified health use for the purpose of lowering blood pressure. Therefore, the 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 is used as a pharmaceutical drug for the prevention or treatment of hypertensive diseases. It can also be widely used as an infusion, a health food, a clinical nutrition food, and the like.

When used as a food, the peptide can be added as it is, or can 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.

[0025]

Example 1 Fresh sardines were treated with a bodyner and minced. The minced fish meat was divided into 10 kg of surimi, which was rapidly frozen at -30 ° C or lower, crushed by a crusher and added with an equal amount of water, which was then sent to a tank and heated to 100 ° C at 10 ° 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.

A 0.1% solution of a commercially available alkaline protease product was added thereto, 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 vibro 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.

The 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 as described 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 6 and 7 below were obtained.

[0031]

[Table 6]

[0032]

[Table 7]

[0033]

Example 2 Vitamin C 20 g, granulated sugar 50 g,
Example 1 was added to 30 g of an equal mixture of corn starch and lactose.
50 g of the peptide α-1000 obtained in 1. was added and mixed well. The mixture is divided into 100 equal parts and packed in a bag, 1.5g per bag
100 stick-shaped nutritional health foods were produced.

[0034]

Example 3 The ACE inhibitory activity of peptide α-1000 was tested and confirmed as follows. First, the liquid sample and the powder sample of α-1000 manufactured in Example 1 were prepared. For these, the conventional method (Lieberm
ACE inhibitory activity was measured using a modified method of an). As a control, captopril (manufactured by Sankyo Co., Ltd.) was used.

As a result, peptide α-1000 (liquid)
An IC ₅₀ value of 0.87 mg / ml was obtained for the same and 0.73 ml / ml for the same (powder). The IC ₅₀ value of captopril was 5.48 ng / ml.

As is clear from the above results, the peptide of the present invention has a remarkable ACE inhibitory activity, and it has been confirmed that it has low toxicity and safety as described below. Therefore, it can be used as an ACE inhibitor. Yes, ACE
It can be widely used for the prevention or treatment of various diseases caused by (angiotensin converting enzyme),
For example, it can be advantageously used as a hypotensive agent for the prevention or treatment of hypertension.

Since the functional peptide of the present invention is of natural origin and is also a food, it has no or very low toxicity and is extremely safe (LD ₅₀ > 3,000 mg).
/ Kg subcutaneously,> 5,000 mg / kg oral: all rats).

The functional peptide of the present invention varies depending on its type, administration method, patient's condition, age, etc.
It is 1 to 6000 mg / kg / day, and is preferably administered 1 to 4 times a day. When taken by a healthy person for the purpose of prevention, there are no particular restrictions on the dose, the number of doses, etc. If necessary, it can be used in combination with other drugs.

[0039]

Example 4 10 g of the peptide α-1000 (liquid) obtained in Example 1, 9 g of sodium chloride, 5 chlorobutanol
g, and 1 g of sodium hydrogen carbonate were dissolved in 1000 ml of distilled water, and this was dispensed into two 500 ml infusion bottles to obtain an antihypertensive infusion solution.

[0040]

Example 5 A tablet was produced according to the following formulation. (1) Peptide α-1005050 g obtained in Example 1, (2) Lactose 90 g, (3) Corn starch 29 g, (4) Magnesium stearate 1 g.

First, (1), (2) and (3) (however, 1
7g) were mixed and granulated with the paste prepared from (3) (however 7g). To the obtained granules, (3) (however, 5 g) and (6) were added and mixed well, and this mixture was compressed by a compression tableting machine to obtain peptide α-100 per tablet.
1000 tablets containing 10 mg of 0 were produced.

[0042]

INDUSTRIAL APPLICABILITY According to the present invention, a novel peptide α-1000 derived from fish meat was obtained. α-1000 can be used not only as a food or drink itself or as an additive for peptide strengthening but also as an excellent ACE inhibitory activity, and thus can be used as a food for specified health purposes for suppressing or preventing an increase in blood pressure. Moreover, it can 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.

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[Procedure amendment]

[Submission date] December 10, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction content]

[0018]

[Table 3]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12N 9/99 C12P 21/06 8214-4B

Claims (6)

[Claims] 1. A novel peptide α-1000 having physicochemical properties shown in Tables 1 and 2 below. [Table 1] [Table 2] 2. The peptide α-1000 according to claim 1.
Angiotensin-converting enzyme inhibitor containing as an active ingredient. 3. The peptide α-1000 according to claim 1.
An antihypertensive agent containing 4. The peptide α-1000 according to claim 1.
A food for specified health use. 5. The peptide α- according to claim 1, wherein the fish meat is subjected to heat denaturation treatment, neutralized to alkaline protease treatment to be hydrolyzed to inactivate the enzyme, and then separated. 1000 manufacturing methods. 6. The method according to claim 5, further comprising filtration, purification, concentration and sterilization treatment.