JP2003113199A - New hydrophilic peptide y-2 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new peptide comprising a fraction obtained by performing alkaline protease treatment of fish meat, ODS treatment of the obtained peptide and elution of the resultant by using 0% ethanol solution and then 10% ethanol solution. SOLUTION: This new peptide α-2200 (fraction Y-2) is a peptide having 200-10,000 molecular weight, discolored and decomposed at 138 deg.C, having almost no bitterness, excellent in ACE inhibitory activity, usable as nutrient preparations and specified health foods and also advantageously applicable to hypotensive drugs.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel hydrophilic peptide Y-2 and a method for producing the same.

[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 The object of the present invention was to separate a peptide having a strong ACE inhibitory activity from the peptide α-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.
The peptide of interest was obtained by eluting with 5% ethanol aqueous solution to obtain an ethanol fraction, and separating a fraction having particularly high hydrophilicity, a fraction having little bitterness, and a fraction having a strong ACE inhibitory activity from these fractions. Is obtained.

Among the ethanol fractions thus obtained, the fraction eluted with a 0% ethanol aqueous solution, that is, the unadsorbed fraction (Y-1 fraction) and the fraction eluted with a 10% ethanol aqueous 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, and are safe in their natural origin, and are useful as an active ingredient of an antihypertensive agent or an active ingredient of an antihypertensive functional food. 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 by using seafood as a raw material. First, this is treated with a meat mining machine, a deponder 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 bonito, saury, mackerel; whitefish such as flounder, Thailand, kiss, konoshiro, cod, herring, yellowtail; cartilage fish meat such as shark, ray; freshwater fish such as smelt, carp, char, yamame; deep sea fish such as shark, monkfish In addition, shrimp, crab, octopus, aphids and the like can be used as appropriate.

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 in microorganisms in addition to plants and animals, and in addition to renin, trypsin, chymotrypsin, papain, promelein, bacterial protease, filamentous fungal protease, actinomycete protease and the like can be widely used. These enzymes are usually
Although commercially available products are used, solid or liquid enzyme-containing substances such as unpurified enzyme, enzyme-containing culture solution, and koji can also be used depending on the purpose. The amount of enzyme added may be about 0.1% to 5.0%.

Then, after performing a neutralization treatment if necessary,
The temperature is kept at 70 ° C. (preferably 80 ° C.) or higher for 2 to 60 minutes (preferably 5 to 30 minutes) to inactivate the enzyme and to make the subsequent separation good. After the heat deactivation treatment, roughly separate by filtration with a vibe screen,
If necessary, after performing a jet treatment, centrifuge treatment,
Float and precipitate are removed.

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, centrifuged or filtered again to obtain a peptide stock solution.
The peptide stock solution thus obtained was sterilized (UHTST
After other conventional methods), fill the product into a container (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 in a container such as a bag ( α-10
00 (powder)). These products are
Store 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 1 and 2 below.

(Table 1) Physicochemical properties of peptide α-1000 (powder) (A) Molecular weight: 200 to 10,000 (by Sephadex G-25 column chromatography) (B) Melting point: Colored at 119 ° C ( Decomposition point) (C) Specific optical rotation [α] _D ²⁰ = -22 ° (D) Solubility in solvent: readily soluble in water; hardly soluble in ethanol, acetone, hexane. (E) Distinction between acidic, neutral and basic: neutral pH 6.0-
8.0 (10% solution) (F) Appearance, components: White powder: Water content 5.14% (vacuum heating drying method); Protein 87.5% (Kjeldahl method, nitrogen-protein conversion coefficient 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: It is a peptide derived from fish meat, which is obtained by deactivating the autolyzing enzyme by heating and hydrolyzing it with a protease. (J) Amino acid composition: As shown in Table 2.

[0021] Analytical method: Automatic amino acid analysis method (however, for cystine
After the oxidation of performic acid, it was hydrolyzed with hydrochloric acid and measured. High-performance liquid chromatography was used for tryptophan. )

The aqueous solution of 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.

The peptide of the present invention can also be used as an ACE inhibitor, for example, as a hypotensive 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 it is 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 an ordinary 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.

[0026]

[Reference Example 1] Fresh sardines were processed with a deboner 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, 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.

[0028] 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 the analysis of the components and the measurement of the amino acid composition, the results shown in Tables 3 and 4 below were obtained.

(Table 3) Component analysis of peptide α-1000 (liquid) ────────────────────────────────── ─ Analytical test item Result Analytical method ────────────────────────────────── Protein 24.8% Kjeldahl method (* 1 ) Fat 0% Soxhlet extraction method Ash 1.6% Direct ash method Fiber 0% Henneberg Stomann improvement method Sugar 0% (* 2) Energy 99kcal / 100g (* 3) Amino nitrogen 1.2% Banslike method Iron 0.11mg / 100g o-phenanthroline absorptiometry Calcium 4.5mg / 100g Atomic absorptiometry Sodium 211mg / 100g Atomic absorptiometry Heavy metal (as Pb) 1.9ppm Sodium sulfide colorimetric arsenic (as As ₂ O ₃ ) 3.7ppm DDIC-Ag Absorptiometry Histamine Not detected High performance liquid chromatograph Law ────────────────────────────────── notes * 1. Nitrogen / protein conversion factor: 6.25 * 2. Calculation formula: 100- (water + protein + lipid + fiber + ash) * 3. Energy conversion coefficient: protein, 4; lipid, 9; carbohydrate (fiber + sugar), 4

[0033] Analytical method: Automatic amino acid analysis method (however, for cystine,
After oxidation with formic acid, hydrolysis was performed and measurement was performed. High-performance liquid chromatography was used for tryptophan. )

[0034]

Example 1 Peptide α-1000 obtained in Reference Example 1
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 carried out with 500 ml each of 0% and 99.5% ethanol aqueous solutions, and Y-1, Y-2, Y-3 and Y-4 were respectively eluted.
And Y-5 fractions were obtained. All of these fractions showed excellent ACE inhibitory activity. For flavor, Y-
No. 1 had no bitterness, and Y-2 had almost no bitterness. Then, it was recognized that the bitterness tended to become stronger as Y-3 and Y-4.

[0035]

Reference Example 2 The Y-1 fraction obtained in Example 1 was
It has physicochemical properties as shown in Tables 5 and 6 below and is superior in ACE inhibitory activity to α-1000,
Significant improvements were observed in terms of color and taste. Such a peptide is a novel substance which has not been published in the literature and is extremely useful, and was named α-2100.

(Table 5) Physicochemical properties of Y-1 (A) Molecular weight: 200 to 10,000 (ASAHIPA
KGS-320 by high performance liquid chromatography) (FIG. 4) (B) Melting point: colored at 65 ° C. (decomposition point) (C) Specific rotation: [α] _D ²⁰ = −11 ° (D) Dissolved in solvent Properties: easily soluble in water, but 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 of substance: White to pale yellow powder. (G) component: moisture 6.58% (normal pressure heating drying method); protein 73.94% (Kjeldahl method, nitrogen / protein conversion coefficient 6.25); lipid 0% (Soxhlet extraction method); ash content 9.85 % (Direct ashing method) (H) UV spectrum: FIG. 5 (I) IR spectrum: FIG. 6 (J) Amino acid composition: Table 6

[0037] Analysis method: Amino acid automatic analysis method

[0038]

Example 2 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 7 and 8 below. In addition, the ACE inhibitory activity is very high (IC ₅₀ (mg protein / ml): 0.015, whereas α-1000 is 0.263), there is almost no bitterness, and the taste is a particular problem. Was not recognized. Such a peptide is a novel substance that has not been published in the literature and is extremely useful, and was named α-2200.

(Table 7) Physicochemical properties of Y-2 (A) Molecular weight: 200 to 10,000 (ASAHIPA
(By K GS-320 high performance liquid chromatography)
(FIG. 7) (B) Melting point: Colored at 138 ° C. (decomposition point) (C) Specific optical rotation [α] _D ²⁰ = −40 ° (D) Solubility in solvent: It is easily soluble in water, Almost insoluble in ethanol, acetone and hexane. (E) Distinction between acidic, neutral and basic: neutral pH 5.0 to
8.0 (10% solution) (F) Appearance of substance: White to pale yellow powder. Component (G): Water content 2.72% (normal pressure heating drying method); Protein 87.25% (Kjeldahl method, nitrogen / protein conversion coefficient 6.25); Lipid 0% (Soxhlet extraction method); Ash 0.20 % (Direct ashing method) (H) UV spectrum: FIG. 8 (I) IR spectrum: FIG. 9 (J) Amino acid composition;

[0040] Analysis method: Amino acid automatic analysis method

[0041]

Example 3 The sensory evaluation of the Y-2 fraction was carried out as follows. As for the evaluation, 3 items of umami, bitterness and fishy smell
When it was felt to be very strong, the score was set to 5 and a score of 6 was given (5: one that felt very strong → 0: one that did not feel anything), and the results shown in Table 9 below were obtained. From this result, Y-2
It was revealed that the fraction had a slightly weak taste but low bitterness and fishy odor.

[0042]

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

[0044]

INDUSTRIAL APPLICABILITY The novel peptide obtained according to the present invention has almost no bitterness, and therefore it can be used as a food or drink itself or as an additive, and has an excellent ACE inhibitory activity, so that it does not suppress an increase in blood pressure as a health food. 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 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.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI Theme Coat (reference) A61K 35/60 A61K 35/60 38/55 37/64 (72) Inventor Toshiro Matsui Jonan distinction office, Fukuoka City 3 -2-10 Tenmaki Building No. 503 (72) Inventor Yutaka Oshima Shima 2-24-26 F-term Maimatsubara, Higashi-ku, Ehime City (reference) 4B018 MD20 MD22 MD74 ME04 MF01 MF12 4C084 AA06 AA07 BA04 BA05 CA45 DC40 NA14 ZA42 ZC17 ZC20 4C087 AA04 BB29 CA03 CA22 CA23 CA45 NA14 ZA42 ZC17 ZC20 4H045 AA10 AA20 AA30 BA09 CA50 EA01 EA07 EA20 FA70 GA20 HA02 HA03 HA04

Claims (2)

[Claims] 1. A peptide α-2200 (fraction Y-2) 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). 2. A peptide α-1000 having the following physicochemical properties is adsorbed on an ODS resin, peptide α-2100 (fraction Y-1) is eluted with an ethanol aqueous solution having an ethanol concentration of 0%, and then the ethanol concentration is increased. The method for producing peptide α-2200 (fraction Y-2) according to claim 1, wherein the peptide α-2200 is eluted with a 10% aqueous ethanol solution. (A) Molecular weight: 200 to 10,000 (by Sephadex G-25 column chromatography) (B) Melting point: Coloring at 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 (F) Appearance, component: White powder: water content 5.14% (vacuum heating drying method); protein 87.5% (Kjeldahl method, nitrogen / protein conversion) Coefficient 6.
25); lipid 0% (Soxhlet extraction method); ash 5.0
% (Direct ashing method).