JPH06239897A - New hydrophilic peptide - Google Patents

Abstract

PURPOSE:To obtain a new peptide having ACE inhibitory activity, usable as a nutritive preparation or specific health food, also advantageously usable as a hypotensive agent. CONSTITUTION:This new peptide comprises a fraction which can be obtained by ODS treatment of a peptide produced by alkali protease treatment of fish meat followed by eluting the product with an aqueous 0-20% ethanol solution.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel peptide and its hypotensive use.

[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 separate a peptide having a strong ACE inhibitory activity from -1000.

[0004]

In the present invention, an aqueous solution of peptide α-1000 was treated with ODS resin, and a fraction having a strong ACE (angiotensin converting enzyme) inhibitory activity was successfully separated from the eluate. As a result of further research, the present invention has been completed.

That is, according to the present invention, α-1000, which is a peptide obtained by treating fish meat with an alkaline protease, is subjected to ODS treatment, and this is followed by ethanol concentration 0-99.
By eluting with a 5% aqueous solution of ethanol to obtain an ethanol fraction, and by separating a highly hydrophilic fraction, a fraction with little bitterness, and a fraction with a strong ACE activity from these fractions,
The target peptide is obtained.

Of the ethanol fractions thus obtained, the fraction eluted with a 0% aqueous ethanol solution, that is, the unadsorbed fraction (Y-1 fraction) and the fraction eluted with a 10% aqueous ethanol solution. It was confirmed that each of the components (Y-2 fraction) was a novel substance that was unique in terms of physical properties, structure, and action and was not published in the literature.
-2100 and α-2200.

[0007] These peptides are hydrophilic, have no bitterness or fishy odor, have high activity, are safe from their natural origin, and are useful as an active ingredient of an antihypertensive agent or an active ingredient of an antihypertensive functional food. It is excellent.

The peptide according to the present invention is a novel substance which has not been published in the literature and can be produced from animal and plant proteins other than fish meat, but the peptide α-1000 described above is used.
Can also be advantageously manufactured.

Therefore, 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,
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 filtration with a vibrating 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 liquid, pasty or powdery peptide thus obtained is α-1000.

The physicochemical properties of peptide α-1000 (spray dry powder) are shown in Tables 3, 4 and 5 below.

[0020]

[Table 3]

[0021]

[Table 4]

[0022]

[Table 5]

The aqueous solution of the peptide α-1000 obtained here is passed through a peptide-adsorbing resin such as ODS resin, washed with water and the like, and then eluted with various concentrations of ethanol or the like to elute and flow to obtain ACE from each fraction. The desired peptide is obtained by taking a fraction with a strong inhibitory activity. Then, of these ethanol fractions, the fraction obtained by eluting with an ethanol aqueous solution having an ethanol concentration of 0 to 20%
Particularly strong ACE inhibitory activity and reduction in bitterness were observed, and the target peptide was successfully obtained.

Each of the peptides of the present invention can be used as an ACE inhibitor by one or more of them, 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 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 and Examples.

[0027]

[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 and added with an equal amount of water, which is 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 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. After this was concentrated under reduced pressure according to a conventional method, UHTST sterilization was performed according to a conventional method to obtain α-1000 (liquid) product, which was further spray-dried according to a conventional method to obtain α-1000 having a particle size of 60 mesh. Get (powder) product,
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 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.

[0033]

[Table 6]

[0034]

[Table 7]

[0035]

Example 1 Peptide α-1000 obtained in Reference Example
Dissolve 5 g of (powder) with 500 ml of deionized water, OD
Pour onto S resin (YMC ODS-AQ 120-S50) column (3.5 × 13 cm) to adsorb the peptide,
Wash with deionized water, then 0%, 10%, 25%, 5
Elution was performed with 500 ml of 0% and 99.5% ethanol aqueous solutions to obtain Y-1, Y-2, Y-3, Y-4, and Y-5 fractions, respectively. ACE inhibitory activity I of these fractions
Sensory evaluations for C ₅₀ and bitterness are shown in Table 8.

[0036]

[Table 8]

[0037]

Example 2 The Y-1 fraction obtained in Example 1 was
It has physicochemical properties as shown in Tables 9 and 10 below, and as is clear from the above, the ACE inhibitory activity is somewhat low, but it is significantly larger in terms of color and taste than α-1000. Improvement was recognized. Such a peptide is a novel one which is unknown in the literature and is extremely useful, and was named α-2100.

[0038]

[Table 9]

[0039]

[Table 10]

[0040]

Example 3 The Y-2 fraction obtained in Example 1, that is, the 10% ethanol fraction eluted by ODS column chromatography has physicochemical properties as shown in Tables 11 and 12 below. Moreover, as is clear from the above, the ACE inhibitory activity was very high, and no particular problem was found regarding the bitterness. Such a peptide is a novel one which is unknown in the literature and is extremely useful, and was named α-2200.

[0041]

[Table 11]

[0042]

[Table 12]

[0043]

Example 4 The sensory evaluation of the Y-2 fraction was performed as follows. Evaluation is about three items of umami, bitterness, fishy smell,
When it was felt to be very strong, the score was set to 5 and a score of 6 was given, and the results shown in Table 13 below were obtained. From this result, it was revealed that the Y-2 fraction had a slightly weak taste but low bitterness and fish odor.

[0044]

[Table 13]

As described above, since the Y-2 fraction has no off-taste or off-flavor, it is suitable as a food material that is required to be tasteless and odorless.
The fraction was found to have extremely high applicability as a functional food material.

[0046]

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 is a diagram showing a gel filtration pattern of peptide α-1000.

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

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

FIG. 6 illustrates the IR spectrum of Y-1 fraction.

FIG. 7 illustrates the molecular weight HPLC pattern of the Y-2 fraction.

FIG. 8 illustrates the UV spectrum of the Y-2 fraction.

FIG. 9 illustrates the IR spectrum of the Y-2 fraction.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Yutaka Oshima Shima 2-24-26 Maimatsubara, Higashi-ku, Fukuoka

Claims (10)

[Claims] 1. A peptide α-1000 obtained from fish meat is added as O.
A novel peptide comprising a fraction obtained by adsorbing it on DS resin and then eluting it with an aqueous ethanol solution having an ethanol concentration of 0 to 20%. 2. The fraction Y-1 obtained by eluting the fraction according to claim 1 with an aqueous ethanol solution having an ethanol concentration of 0%,
And a fraction Y-2 obtained by elution with an ethanol aqueous solution having an ethanol concentration of 10%, The novel peptide according to claim 1. 3. Fraction Y-1 according to claim 2, which is a novel peptide α-2100 having the following physicochemical properties. (A) Molecular weight: 200 to 10,000 (ASAHIPA
KGS-320 by high performance liquid chromatography) (B) Melting point: Colored and decomposed at 65 ° 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 9.58% (normal pressure heating drying method); protein 73.94% (Kjeldahl method); lipid 0% (Soxhlet extraction method); ash content 5.85% (direct ashing method). (F) Amino acid composition; as shown in Table 1 below. [Table 1] 4. The fraction Y-2 according to claim 2, which is a novel peptide α-2200 having the following physicochemical properties. (A) Molecular weight: 200 to 10,000 (ASAHIPA
(By K GS-320 high performance liquid chromatography) (B) Melting point: Colored and decomposed at 138 ° 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. Component (E): water content 2.72% (normal pressure heating drying method); protein 87.25% (Kjeldahl method); lipid 0% (Soxhlet extraction method); ash content 0.20% (direct ashing method). (F) Amino acid composition; as shown in Table 2 below. [Table 2] 5. A hypotensive agent comprising the novel peptide according to claim 1 as an active ingredient. 6. A hypotensive functional food, comprising the novel peptide according to claim 1 as an active ingredient. 7. Fraction Y- according to claim 2 or claim 3.
An antihypertensive agent comprising 1 to peptide α-2100 as an active ingredient. 8. The fraction Y- according to claim 2 or claim 3.
A functional blood pressure-lowering food, which comprises 1 to peptide α-2100 as an active ingredient. 9. Fraction Y- according to claim 2 or claim 4.
An antihypertensive agent comprising 2 to peptide α-2200 as an active ingredient. 10. Fraction Y according to claim 2 or claim 4.
-2 or peptide α-2200 as an active ingredient, a hypotensive functional food.