JPS6287058A - Novel peptide - Google Patents

Abstract

NEW MATERIAL:A compound produced by treating fish or shellfish with a proteinase, fractionating the decomposition product with an alcohol and subjecting the product to ion exchange resin treatment. It has the following characteristics. Appearance, white powder; elemental analysis (%), C 41.5, H 6.7, N 16.9, O 34.9; molecular weight, 500-10,000; melting point, 173 deg.C; solubility, soluble in polar solvent such as water, methanol, etc., and insoluble in non-polar solvent such as hexane; color reaction, positive to ninhydrin reaction and biuret reaction; etc. USE:Seasoning, nutrient, health food, clinical nutrient food, transfusion, medicine, etc. PREPARATION:Crushed dish of shellfish is treated with a proteinase such as pepsin after the control of water-content, temperature and pH. The treated product is fractionated with an alcohol (preferably methanol, etc.) and treated with an ion exchange resin (preferably strongly acidic cation exchange resin).

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to a peptide, and more specifically, the present invention relates to a novel peptide that has not been published in any literature, and is applicable to protein-related technical fields, nutritional supplements, etc. .

It is widely used in technical fields such as nutritional foods, seasonings, and pharmaceuticals, as well as in the technical field of the fisheries industry for the effective use of marine products.

(Prior art) Seafood meat peptides are contained in extract components obtained by hydrolyzing seafood with acids, alkalis, enzymes, etc., but only useful peptides are purified in large quantities from these extract components. A method for extraction and separation has not yet been developed.

Moreover, the present invention not only has excellent taste and is useful for nutritional enrichment, but also has many medicinal effects in addition to hypotensive action, and has unique physical properties and structure as described later. The peptide.

This is completely new and has not been described in any literature.

In other words, the peptide according to the present invention has not been previously developed and is completely unknown in terms of both physical properties and effects.

(Object of the Invention) The arteriosclerotic preventive effect of unsaturated fatty acids from seafood, such as sardines, mackerel, and saury, has recently attracted attention. Therefore, the present inventors got the idea that useful physiologically active substances may exist in other components of seafood, and focused on peptides in seafood meat.

In addition to having a liver-strengthening effect, peptides from fish and shellfish meat not only have many physiological activities, but also have new findings that can be fully utilized for their taste and nutritional enhancement properties. The present invention has been made for the purpose of widespread and effective use.

(Disclosure of the Invention) In order to achieve the above object, the present inventors conducted intensive research to produce a large amount of highly pure peptide, and as a result,
The present invention was realized based on the discovery that a target peptide can be produced by allowing a proteolytic enzyme to act on seafood, and that this peptide can be isolated and purified in large quantities with high purity. It is completed.

Therefore, according to the present invention, peptides are produced by treating seafood with proteolytic enzymes.

This is then separated and purified by alcohol fractionation and ion exchange resin treatment to obtain the target peptide.

It was confirmed that the peptide thus obtained was unique not only in physical properties and structure but also in its action and effect, and was a novel substance that had not been described in any literature, and was named DAN-100.

This peptide is produced using seafood as a raw material, and first, it is processed using a meat extractor, deboner, etc. to separate the fish meat quality. It is preferable that the raw materials be as fresh as possible. Divide the separated fish meat into about 10 pieces of surimi,
You can use it as is for the next process, but the
℃0 e.g. -30'C.

Quickly freeze by blowing cold air to -20 to -25℃
You may also save it in a file and use it as needed.

Seafood includes red fish such as sardines, horse mackerel, tuna, bonito, saury, and mackerel; white fish such as flounder, sea bream, kisu, yellowtail, cod, herring, and yellowtail; cartilaginous fish such as shark and ray; smelt, carp, char, Freshwater fish meat such as yamame trout; merganser shark,
In addition to deep-sea fish meat such as monkfish, shrimp, crab, octopus, mysids, etc. can be used as appropriate.

In the present invention, after harvesting the meat, the seafood meat is crushed using a crusher or the like, water is added, and the mixture is heated to an appropriate enzyme temperature (approximately 20 to 60°C, depending on the enzyme used), and the pH is also adjusted to an appropriate value (pl+3 9), add proteolytic enzyme, and treat for 1 to 30 hours. If you wish, after adding water to the crushed seafood meat, heat it to about 40-60℃ in a stirring tank.
Hold for 5 hours to allow autolysis2. Then add enzyme'
It may be added and decomposed.

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. These enzymes are usually commercially available.

Solid or liquid enzyme-containing substances such as unpurified enzymes, enzyme-containing culture solutions, and koji can also be used as needed depending on the purpose.

When using a protease solution with a concentration of 0.1%, when processing red meat such as sardines as fish meat, p1
14.1. Approximately 17 hours at 45℃, or PH5,9,48
Enzyme treatment is completed in about 4 hours at °C.

After neutralization, the enzyme is then kept at a temperature of 80° C. or higher for 5 to 30 minutes to deactivate the enzyme. After heat inactivation treatment, filtration with a vibroscreen etc., and if necessary, a rejector treatment, and then using a Sharpless centrifugal brooder etc., for example, io
,ooo~30. Centrifuge at OOOrpm.

After concentrating this in a conventional manner to about 30 Bx), centrifugation is performed again to obtain a peptide stock solution.

In addition to being used as a purified peptide raw material, the peptide stock solution obtained in this way can be used as it is or concentrated (diluted if necessary) to be used as seasonings, nutritional supplements, health foods, clinical nutritional foods, etc.
@Can be used in liquids, drinks, etc.

Next, the peptide stock solution is decolorized by adding activated carbon and stirring, and then filtered. Add alcohol to the filtrate and let stand.

Methanol is alcohol.

Ethanol, propatool, isopropatool.

Butanol and the like are preferred, but other alcohols may also be used. Alcohol should be used when cold, 0-1
(0°C), use the same amount to 30 times or more of the peptide stock solution. After cold alcohol is added, the mixture is left in a cold room for 30 minutes to 3 hours or more to form a precipitate.

The obtained precipitate is made into a solution by adding water and subjected to chromatogelaf treatment, but as in the case of the peptide stock solution, the precipitate may be used as it is for the various purposes mentioned above.

The solution is treated with an ion exchange resin, and the resin used is a cation exchange resin, in particular a strongly acidic cation exchange resin. For example, the above solution is injected into a column packed with these resins, washed with water, and then eluted with an alkaline aqueous solution such as dilute aqueous ammonia to obtain an eluate.

After removing ammonia from the obtained eluate by a method such as vacuum concentration, it is concentrated if necessary and powdered by a conventional method such as freeze-drying to obtain the basic peptide of interest. As described above, the eluate may be concentrated or diluted and used for various purposes.

The peptide thus obtained was a novel and extremely useful peptide unknown in the literature, and was named DAN-100.

The physicochemical properties of peptide DAN-100 are as follows.

1. Elemental analysis value C: 41.5%, H: 6.7%, N: 16.9%.

○; 34.9% 2. Molecular weight Estimated to be 10,000 or less and 500 or more by Sephadex G-25 column chromatography.

3. Melting point 173℃ 4. Specific rotation [α]D 23° 5. UV spectrum: No unique absorption observed.

6. IR spectrum As shown in the drawing 7. Solubility in solvents Soluble in polar solvents such as water, methanol, DMSO, etc.
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 Weak basic, pH 9,60 (10% Solution) 10. Color and shape of substance White powder 11. Moisture content 4.01% (normal pressure drying method) 12. Salinity 8.19% (measured as CΩ by potentiometric titration method) 13.
, total nitrogen and amino dysmorphic nitrogen T-N: 16.81% (microkale pool method) amino dysmorphic-N: 2.46% (formol method) In addition to the taste effect, the peptide according to the present invention has the following effects. It has various physiologically active effects: growth promoting effect, blood pressure lowering effect, anemia preventive effect, anti-ulcer effect, strong liver effect, and
Improves diabetes, obesity, hyperlipidemia, arteriosclerosis, etc.

Therefore, this peptide is used as a food additive such as seasonings and nutritionally fortified foods, or as an animal feed additive.Due to the above-mentioned unique physiological activity, this peptide is also used as a medicine for the prevention and, in some cases, treatment of various diseases. It can also be widely used as an infusion, health food, clinical nutritional food, etc.

When used as a food, the peptide can be added as it is, or used in combination with other foods or food components, as appropriate in accordance with conventional methods. Moreover, 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 conventional methods, and in the case of parenteral administration, for example, injection preparations, drips, and suppositories can be prepared. It can be used as an agent, etc.

Since the peptides of the present invention are of natural origin, they have no or very low toxicity and are extremely safe (LD
SO > 3,000mg/Kg subcutaneously, ) 5,000mg
/Kg oral: both rats).

Example 1 Fresh sardines were processed with a deboner and the meat was harvested.

The harvested fish meat was divided into 10 kg of surimi. After pulverizing this with a pulverizer, an equal amount of water was added and heated to 45 to 48°C, and this was transferred to a stirring tank and kept at the same temperature for 2 to 3 hours to cause autolytic decomposition. The pH was then adjusted to 4.1.

Add 0 to Bunazyme (commercially available protease preparation brand name)
．． A 1% solution was added and kept at 45°C for 17 hours to perform enzymatic decomposition. The enzyme was neutralized and then boiled for 15 minutes to inactivate the enzyme.

This was filtered through a Vibroscreen (150 mesh), and the filtrate was treated with a jetter at 500 rpm, and then treated with a Sharpless centrifuge.15. OOOrpm)
.

The mixture was heated and concentrated at room temperature until it reached 20 Bx, and then subjected to Sharpless centrifugation treatment (15,000 rpm) again to obtain a peptide stock solution (DAN-No. 2).

Example 2 The veptide stock solution (pH 6,22) obtained above was heated at 184oC.
n+Q was taken, 100 g of activated carbon was added thereto, stirred for 60 minutes, and then filtered to obtain 1500 ml of filtrate (pH 6,12).

Take 100 mρ of the filtrate and add 2000 mρ of cold methanol to it.
mQ was added and the mixture was left in a cold room for 60 minutes to form a precipitate.

Water was added to the resulting precipitate to obtain a solution (E).

Example 3 Take 12 liters of solution (E) and add it to Dowex 5011 ()! ”) packed column (φ5
x 15 cm).

Then, wash the resin with 2000mQ of water and
-N840H250+++Q was used for elution, and the resulting eluate was removed with ammonia by a conventional method and then lyophilized to obtain a white powder of peptide DAN-100.

Application example 1 1 kg of the peptide stock solution DAN-No2 obtained in Example 1 was added to 500 g of soy sauce, 200 g of liquid sugar, and 500 g of mirin, stirred, and aged in an aging tank for 5 days, then sterilized.
By bottling, a soy sauce seasoning with excellent flavor was obtained.

Application Example 2 50 g of the peptide DAN-100 obtained in Example 3 was added to 20 g of vitamin C, 50 g of granulated sugar, and 30 g of an equal mixture of corn starch and lactose and thoroughly mixed.

The mixture was divided into 100 equal parts and packed into bags to produce 100 bags of stick-shaped nutritious health food of 1 bag i, sg.

Application Example 3 Peptide DNA-100 obtained in Example 3 was continuously orally administered (3-10 g/Kg body weight, 3- After 27 days), the liver was decapitated, the liver was immediately opened, the liver was collected, and the liver wet weight was measured.
Compared to the control group (normal diet only), all groups had a growth rate of 3.
-14% (average value of 10 animals) was obtained.

As is clear from the above results, this peptide showed a remarkable liver regenerating effect and was found to be extremely useful as a liver tonic. The dosage varies depending on the patient's symptoms and age, but it is 0.1 to 1000 mg/kg/day once a day.
Administer 4 times.

Application example 4 (1) Peptide DAN-100Log obtained in Example 3
(2) Sodium chloride 9g
(3) Chlorobutanol 5g
(4) Sodium hydrogen carbonate 1g
Dissolve all ingredients in distilled water 1000o+Q and add 500o
The mixture was dispensed into two mQ drip bottles to obtain a nutrient infusion.

[Brief explanation of drawings]

The figure illustrates the IR spectrum of peptide DAN-100 according to the present invention.

Claims (1)

[Claims] A novel peptide obtained by treating seafood with a proteolytic enzyme, fractionating the resulting product with alcohol, and then treating it with an ion exchange resin.