JPH02154693A - Novel functional peptide and utilization thereof - Google Patents

Abstract

PURPOSE:To obtain the subject medicine utilizable for remedy and prevention of lipemia, diabetes mellitus, arteriosclerosis, etc., by carrying out simultaneous autolysis and proteolytic enzyme treatments of fishes and shellfishes and synthesizing a functional peptide with 500-5,000 molecular weight. CONSTITUTION:Fishes and shellfishes are subjected to the meat collecting treatment, etc., to separate the fish and shellfish meat and the separated fish and shellfish meat is ground using a crusher, etc. To the resultant ground fish and shellfish meat, water is added and a proteolytic enzyme is simultaneously added thereto followed by simultaneous autolysis and proteolytic enzyme treatments under mild stirring at about 20-60 deg.C and about pH 3-9. A reaction solution prepared by termination of the enzyme reaction of the above-mentioned ground meat is filtered using a vibrating screen, etc., to obtain a raw peptide solution and the resultant raw peptide solution is subsequently purified by filtration, etc., thereof using an active carbon to obtain a functional peptide with 500-6,000 molecular weight. As the concrete examples of the proteolytic enzymes, pepsin, resin, trypsin, etc., are exemplified and the amount thereof added is suitably 0.05-1wt.% based on the above-mentioned treating solution.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel peptide having one functionality and its use.

More specifically, the present invention relates to functional peptides obtained by simultaneously subjecting seafood to autolysis treatment and proteolytic enzyme treatment, and having a molecular weight of 500 to 6,000.

Furthermore, one aspect of the present invention relates to a hyperlipidemia treatment and prevention agent, a diabetes treatment and prevention agent, or a blood pressure lowering and vasodilator agent containing this functional peptide as an active ingredient.

(Prior Art and Problems) In general, it is common practice to hydrolyze seafood with acids, alkalis, proteolytic enzymes, etc. to obtain tasty hydrolysates.

In addition, the present inventors were able to obtain a new peptide by first autolyzing seafood and then treating it with proteolytic enzymes (Japanese Patent Application No. 6O-22
6834).

Various tests were conducted on the new peptide obtained, and it was found that it had excellent functionality. There were problems, such as the fact that the purification process was quite difficult industrially.

Regarding various peptides obtained from seafood,
Many functional peptides F (New
Food Industry Vol, 29.
No. 4 (1987) pp. 29-43),
The emergence of new functional peptides is expected.

(Means for Solving the Problems) As a result of intensive research in search of superior functional peptides, the present inventors have found that by simultaneously subjecting seafood to autolysis treatment and proteolytic enzyme treatment, a peptide with a molecular weight of 500 to 6 ,
,000 new functional peptides were successfully obtained.

The present invention is produced by simultaneously subjecting seafood to autolysis treatment and proteolytic enzyme treatment, and has a molecular weight of 500 to 6.
, 000 functional peptides.

Furthermore, the functional peptide of the present invention is a therapeutic and preventive agent for hyperlipidemia, a therapeutic and preventive agent for diabetes, a hypotensive and vasodilator, an agent for treating and preventing obesity, and a therapeutic and preventive agent for arteriosclerosis. These are the active ingredients of each.

The functional peptide of the present invention is produced using seafood as a raw material, and first, it is processed using a meat extractor, deboner, etc. to separate the fish flesh.

It is preferable that the raw materials be as fresh as possible. The separated fish meat may be divided into about 10 kg of surimi and used as is for the next processing, but at -20 to -50°C, for example -30°C.
℃, quickly freeze by blowing cold air to -20 to -2
It may be stored at 5°C and used as needed.

Seafood includes red fish such as sardines, horse mackerel, tuna, bonito, saury, and mackerel; flounder, sea bream, and kisu.

White fish such as whitefish, cod, herring, and yellowtail; cartilaginous fish meat such as sharks and rays; freshwater fish meat such as smelt, carp, char, and yamame; deep sea fish meat such as merganser shark and monkfish, as well as shrimp, crab,
Octopuses, mysids, various shellfish, etc. can also be used as appropriate.

In the present invention, after harvesting the meat, the seafood meat is crushed using a crusher or the like, and at the same time, water is added and a proteolytic enzyme is added.
Heat the enzyme to an appropriate temperature (approximately 20 to 60 degrees Celsius, depending on the enzyme used), adjust the pH to an appropriate value (approximately pH 3 to 9), and perform autolysis and proteolytic enzyme treatment while stirring gently. be carried out simultaneously for 2 to 20 hours, preferably 3
In order to stop the treatment after ~5 hours, the treatment solution was heated for ~10 ~
Boil for 15 minutes.

For autolytic enzymes, seafood is kept without being heated until it is treated with enzymes.

Furthermore, as the protease, any enzyme that can decompose proteins can be used alone or in combination. Its origin can be found in microorganisms as well as animals and plants, and in addition to pepsin, renin, trypsin, chymotrypsin, papain, and promelain, bacterial proteases, filamentous fungal proteases, and actinobacterial proteases are also widely available. Commercially available enzymes are usually used, but unpurified enzymes and culture fluids containing enzymes are used.

Solid or liquid enzyme-containing substances such as koji can also be used as needed depending on the purpose.

The amount of protease varies depending on the degree of purification of the enzyme and also varies depending on the treatment time, but 0.05
It is sufficient to use about 1%, preferably 0.1%.

The enzyme reaction termination treatment solution is filtered using a vibroscreen or the like, treated with a jetter if necessary, and then heated using a Sharpless centrifuge or the like to
Centrifuge at 0 rpm.

After concentrating this by vacuum concentration etc. to about 30 Bx), centrifugation is performed again to obtain a peptide stock solution.

The peptide stock solution thus obtained can be purified by activated carbon filtration, diatomaceous earth filtration, etc., and used as it is as a functional peptide stock solution.

The functional peptide stock solution obtained here can be further purified with alcohols, purified with ion exchange resin, etc., but these purifications are not particularly necessary.
It has been purified to the extent that it can be used as an active ingredient in various compositions.

The functional peptide stock solution obtained here can also be powdered by freeze-drying or spray-drying, with or without the addition of dextrino.

The functional peptide of the present invention is novel and has not been published in any literature, and was named ESP-2.

The physicochemical properties of ESP-2 are as follows.

1. Elemental analysis value C; 55.22%, H: 5.83%, N; 7.
31%, ○; 3L64% 2, Molecular weight The molecular weight was determined to be 500 to 6,000 by Sephadex G-50 column chromatography.

The molecular weight distribution map obtained by gel filtration is shown in FIG.

3. Melting point: 145℃ decomposition 4. Specific optical rotation [αko♂0=19° 5. UV spectrum as shown in Figure 2.

6. IR spectrum as shown in Figure 3.

7. Solubility in solvents Water, methanol, oMsO#Soluble in polar solvents,
Insoluble in non-polar solvents such as chloroform and hexane 8, Color reaction Ninhydrin reaction 10 Biuret reaction 10 Copper-Folin reaction 10 Phenol sulfuric acid reaction 9 Distinction between basic, acidic and neutral slightly acidic, PH 6, 21 (10% solution) 10. Color and shape of substance Yellowish white powder (freeze-dried product) 11. Moisture content 3.75% (normal pressure drying method) 12. Salinity 3.97% (measured as CΩ by potentiometric titration method) 13.
, total nitrogen and amino dysmorphic nitrogen T-N: 14.04% (microkaelsol method) amino dysmorphic-N: 2.22% (formol method) The functional peptide of the present invention has various physiologically active functions. ing.

As is clear from the test examples described later, the functional peptide of the present invention can be used to treat and prevent hyperlipidemia, treat and prevent diabetes, lower blood pressure and dilate blood vessels, treat and prevent obesity,
It shows excellent effects in the treatment and prevention of arteriosclerosis,
It can be widely used as a medicine for prevention or treatment of these diseases, an infusion or nutritional food, and a health food.

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 the conventional method, and in the case of parenteral administration, for example, injection preparations, drips, semi-doses, etc. It can be used as a percentage.

Furthermore, when the functional peptide of the present invention is orally administered in combination with egg yolk, it exhibits extremely significant medicinal effects.

As the egg yolk, in addition to chicken eggs, eggs of quail, duck, and local birds are mainly used, but eggs of other animals can also be used as appropriate. As the egg yolk, the egg yolk itself may be used as it is, or it may be diluted with a solvent such as water or physiological saline and used as an egg yolk liquid, and the dilution rate may be determined as appropriate.
Approximately 10 to 50% to achieve both economy and effectiveness.
Conveniently, it is an egg yolk solution.

When administering, add 5 to 5 functional peptides to egg yolk (liquid).
It may be used after being dissolved at about 0%, the two may be simply mixed together, or both may be placed in a capsule and mixed within the body.If egg yolk is used in this way, it can be administered orally. It is also possible to obtain the desired medicinal effect.

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, > 3,000 ■/-
Subcutaneous, >5,0001g/kg orally: both rats)
.

The functional peptide of the present invention has a molecular weight of about 0.1 to 6,000, although it varies depending on the type, administration method, patient's symptoms, age, etc.
+ag/)cg/day, and is preferably administered 1 to 4 times a day.When taken by healthy people for preventive purposes, there are no particular restrictions on dosage, frequency of administration, etc. Also, if necessary, it can be used in combination with other drugs.

Hereinafter, the present invention will be explained in more detail using test examples, production examples, and preparation examples.

Test Example 1 Functional peptide ES prepared from sardines according to Production Example
Using P-2, rats were divided into the following three groups and the blood pressure lowering effect was tested.

1) Intravenous injection group 2) Oral administration group dissolved in physiological saline 3) 30% egg yolk aqueous solution oral administration group.

Rats were purchased from Has: 5HR (spontaneous hypertensive rats) for 10 weeks from Kinsei Hoshino Experimental Animals Co., Ltd.
Those that had been pre-reared for a week were used.

Administration was performed as follows. After administration, blood pressure was measured over time using a non-invasive tail artery blood pressure device (PS-IOO manufactured by Riken Kaihatsu Co., Ltd.) and the results shown in Table 1 were obtained. Ta.

Intravenous administration: 1n of functional peptide ESP-2 dissolved in physiological saline to give a dose of 117μ/kg.
+u/animal was administered to rats.

Oral administration: (a) Sardine peptide dissolved in physiological saline was administered at a dose of 6 mΩ/animal so that the dose of functional peptide ESP-2 was 4000 μ/kg.

(b) The dose of functional peptide ESP-2 is 4000■
Sardine peptide was dissolved in 30% egg yolk solution and administered at a concentration of 6 ml/kg/animal.

The following can be seen from Table 1.

A clear blood pressure lowering effect was observed when administered intravenously.

2) When the functional peptide ESP-2 was dissolved in egg yolk and administered orally, blood pressure was significantly lowered.

Test Example 2 In this test example, the functional peptide ESP-2 was dissolved in egg yolk, and the relationship between dosage and blood pressure reduction was investigated using N1fedip.
Ina was used as a positive control sample, and the test was conducted using SHR rats in the same manner as in Test Example 1.

A predetermined amount of sardine peptide was dissolved in egg yolk water to give a 30% egg yolk solution, and the solution was orally administered.

After administration, a non-invasive tail artery blood pressure device (P manufactured by Riken Kaihatsu Co., Ltd.) was used.
Blood pressure was measured over time using a blood pressure sensor (S-100).

The measurement was repeated five times, the highest and lowest values were discarded, and the average of the three points was taken as the measured value, giving the results shown in Figure 4.

The following can be seen from Figure 4.

1) The amount of blood pressure reduction steadily increased as the dose increased.

2) The amount of blood pressure lowered increased until approximately 3 hours after administration, and the antihypertensive effect was still maintained even after 6 hours had passed after administration.

In the case of N1fedipine used as a control sample,
30 minutes after administration, the blood pressure decreased rapidly to 46.3 nu Hg, but 3 hours after administration, it decreased to 14.9 nynl.
The blood pressure decreased to 1 g, and 6 hours later, blood pressure in 4 out of 6 animals rose to the pre-administration level.

Test Example 3 2.4.8 and 16g/of functional peptide ESP-2
Each animal was dissolved in 20 ml of purified water and orally administered to each rabbit individually using a cannula.

Rabbit ears have prickly hair and blood vessels are aff! After making it easier to m, administer medication, and check for changes in the ear blood vessels! A vasodilation test was performed.

The rabbits were Std (New Zealand White breed), 14 to 15 weeks old, made of gold, weighing 0±0.5 kg, and were purchased from the Shizuoka Experimental Animal Agricultural Cooperative, and after being pre-reared for one week, they were healthy. In 6, animals were subjected to testing! The observation times were before administration, 20:30, 60:00, and 120 minutes, and each time the rabbit's ears were touched with a mat.

Thirty minutes after administration of 2 g, slight vasodilation was observed.

Test Example 4 Effect on hyperlipidemia and arteriosclerosis A group of 6 male Wistar rats weighing approximately 220 g were fed powdered feed (CA-1, manufactured by Nippon Talea) with 1 and 0.0 mg of cholesterol and cholic acid, respectively. A 5% mixture was ingested for 14 days, and at the same time, 500 and 1000+ ng/kg of the functional peptide ESP-2 and 200 ng/kg of the comparative drug clofibrate were each orally administered using a metal stomach tube. After the end of the administration period.

Blood and liver were collected and lipid content was measured as in the case of normal rats described above.

The results are shown in Table 2.

Test Example 5 Effect on diabetes: 0.01 on male Wistar rats weighing approximately 180 g
Streptozocin (manufactured by Upjohn) dissolved in M citrate buffer (pH 4, 5) was intravenously administered for 45 μ/day, and blood was collected from the tail vein one week after administration, and the blood glucose level was 270 to 330 μ/day. Animals within the range of ST
were considered Z-diabetic rats. These STZM urinary rats were divided into 6 rats per group, and powdered feed (CA-1, manufactured by Nippon Talea Co., Ltd.)
The mice were given a mixture of DAN-100 at a ratio of 1.25.2.5 or 5% at a rate of 40 g/animal/day for 7 days. After the administration period, blood and liver were collected and the glucose and lipid contents were measured in the same manner as in the case of normal rats described above.

The results are shown in Table 3.

Test Example 6 Effect on Obesity Male Zucker (hereditary obese) rats (fafa) weighing approximately 300 g were arranged in groups of 3 to 4 rats and fed powdered feed (CA).
-1, Nippon Talea) with 5 or 10% ESP-2
20g/animal/day a month of the mixture was ingested.

At the end of the administration period, blood and liver were collected to measure lipid content.

The results are shown in Table 4.

Production Example 1 Fresh sardines are treated with a deboner and the meat is harvested, 10 kg of the obtained sardine meat is crushed with a crusher, then 9 equivalents of water and Bunazyme (commercially available protease preparation brand name) Log are added and mixed well. The 6 reaction solution was kept at 45°C for 4 hours to perform autolysis and enzymatic decomposition, then neutralized to pH 7, and then 1
The enzyme was inactivated by boiling for 5 minutes.

This was filtered through a Vibroscreen (150 mesh), and the filtrate was treated with a diector at 500 rpm.

Processed in a Sharpless centrifuge at 15,000 rpm
m), concentrated by heating at room temperature until it reached 20Bx, and then subjected to Sharpless centrifugation treatment (15,00 rpm) again.

The obtained clear liquid was concentrated four times using a vacuum concentrator, and the concentrated liquid was filtered with activated carbon and then diatomaceous earth to obtain a slightly yellowish and transparent solution of the functional peptide ESP-2.

Production Example 2 The functional peptide ESP-2 obtained in Production Example 1 was directly freeze-dried to obtain a yellow-white functional peptide ESP-2.

Production Example 3 5% dextrin was added to the functional peptide ESP-2 solution obtained in Production Example 1 and spray-dried to obtain functional peptide E.
SP-2 dry powder was obtained.

Production Example 4 Frozen sardines (lokg) were crushed, and equal amounts of water and pepsin (
8 g of commercially available) was added, mixed well, and kept at 45°C for 3 hours to perform autolysis and enzymatic decomposition. Adjust the reaction solution to pH 7
and boiled for 15 minutes to inactivate the enzyme.

After cooling the reaction solution, it was centrifuged, and the separated solution was concentrated 5 times by ultrafiltration, and the concentrated solution was filtered with activated carbon and then diatomaceous earth to obtain a slightly yellowish and transparent functional peptide ESP-
A solution of 2 was obtained.

Preparation Example 1 (1) Freeze-dried powder of ESP-2 (Production Example 2) 1
0g (2) Sodium chloride 9g (3
) Chlorobutanol 5g (4) Sodium hydrogen carbonate 1g All ingredients in distilled water 100%
The mixture was dissolved in 100O and dispensed into 1000 brown ampoules, which were replaced with Nitken gas and sealed to produce an injection.

Preparation Example 2 (1) Spray-dried powder of ESP-2 (It Preparation Example 3)
10g (2) Egg yolk powder 3g (
3) Lactose 87g (4) Cornstarch 29g (5) Magnesium stearate Ig (1), (2),
(3) and 17 g of corn starch were mixed together and granulated with a paste made from 7 g of corn starch. 5 g of corn starch and (5) were added to the granules, and the resulting mixture was compressed into 2 tablets using a compression tablet machine. 1000 pieces were manufactured.

Preparation example 3 ESP-2 solution (manufacturing example 4) 20 kg, egg yolk 30 kg
, add water to 10kg of granulated sugar to make a total of 100kg
This was mixed well.

Next, the product was sterilized at 90 to 95°C for 15 to 30 seconds, and the temperature of each product was rapidly cooled to about 70°C, and 100g each was filled into pre-sterilized brown bottles, immediately sealed, and the drink was 100g.
0 pieces were manufactured.

[Brief explanation of the drawing]

Figure 1 shows the molecular weight distribution of functional peptide ESP-2 by gel filtration, Figure 2 shows the UV spectrum of ESP-2, and Figure 3 shows the IR spectrum of ESP-2. , FIG. 4 is a graph illustrating the hypotensive effect of ESP-2. Agent Patent attorney 1) Parent Male

Claims (1)

[Scope of Claims] 1. A functional peptide with a molecular weight of 500 to 6,000, which is obtained by simultaneously subjecting seafood to autolysis treatment and proteolytic enzyme treatment. 2. A composition for treating and preventing hyperlipidemia, which contains the functional peptide of claim 1 as an active ingredient. 3. A composition for the treatment and prevention of diabetes, which contains the functional peptide of claim 1 as an active ingredient. 4. A composition for lowering blood pressure and dilating blood vessels, which contains the functional peptide of claim 1 as an active ingredient. 5. A composition for the treatment and prevention of obesity, which contains the functional peptide according to claim 1 as an active ingredient. 6. A composition for treating and preventing arteriosclerosis, which contains the functional peptide according to claim 1 as an active ingredient.