JP4295886B2 - Peptide purification method - Google Patents

Description

【００３３】
【発明の効果】
魚肉蛋白質を加水分解して得られたアンギオテンシン変換酵素阻害ペプチド含有溶液を、平均細孔直径３nm以下の活性炭を用いて処理する本発明の精製方法により、ＡＣＥ阻害ペプチド含有溶液からＡＣＥ阻害活性を低下させることなく苦みや臭いを除くことができ、血圧降下剤又は血圧降下食品として有用なペプチドが収得できる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for purifying an angiotensin converting enzyme inhibitory peptide that is useful as a blood pressure lowering agent or a blood pressure lowering food.
[0002]
[Prior art]
Angiotensin-converting enzyme is present mainly in lungs, vascular endothelial cells, and renal proximal tubules, and acts on angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu). It is an enzyme that cleaves and releases a dipeptide (His ⁹ -Leu ¹⁰ ) from the C terminus of I to produce angiotensin II having a strong pressor action.
[0003]
This enzyme also has an action of degrading and inactivating bradykinin, which is an in vivo antihypertensive substance, and is strongly involved in the pressor system. Conventionally, if the activity of angiotensin converting enzyme is inhibited, it is thought that it works on hypotension and is clinically effective for the prevention and treatment of hypertension.
Recently, since captopril, a proline derivative, was synthesized and antihypertensive activity was confirmed, synthetic researches on various angiotensin converting enzyme inhibitory substances have been active, and acquisition from natural products has also been attempted.
This is because a natural product-derived angiotensin converting enzyme inhibitor is obtained from foods or food materials, and is expected to be a low-toxic and highly safe antihypertensive agent.
However, a peptide obtained by hydrolyzing a protein derived from a natural product with an enzyme contains a large amount of a bitter taste peptide, which limits its use as an angiotensin converting enzyme inhibitor.
[0004]
As such measures, b) a method for applying a leucine aminopeptidase and carboxypeptidase peptide (Yasushi Sato, Yoshiaki Sekiguchi, Chiba charity, Katsuhiro Ikai, Noka, 43, 286, (1969) , S.Arai, M. Yamashita, H. Kato, M. Fujimaki, Agr. Biol. Chem., 34 , 729, (1970)), b) A method in which α-chymotrypsin is allowed to act on peptides to form plastin (M. Fujimaki, M. Yamashita , S. Arai, H. Kato, Agr. Biol. Chem., 34 , 483, 1325 (1970)), c) a method in which angiotensin converting enzyme-inhibiting peptide is adsorbed on a synthetic adsorbent and eluted with an organic solvent 4-341193), d) a method of adsorbing and removing bitter peptides on a hydrophobic adsorbent (Japanese Patent Laid-Open No. 4-1909797) and the like.
However, the methods (i) and (b) change the primary structure of the peptide, and the methods (c) and (d) are not industrially effective due to the poor removal efficiency of bitter peptides. Therefore, a new method that can efficiently remove bitter peptides without changing the primary structure of the peptide is desired.
[0005]
Therefore, in purifying the peptide solution containing an angiotensin converting enzyme inhibitor obtained by hydrolyzing a protein in an aqueous medium with a proteolytic enzyme, the present applicant also disclosed (1) hydrolysis reaction. After removing insoluble matter from the solution, the peptide concentration is adjusted to 10% by weight or more, (2) the solution is brought into contact with the synthetic adsorbent, and the non-adsorbed fraction is collected, and (3) the synthetic adsorbent. A method for recovering a non-adsorbed fraction by washing with water or an aqueous salt solution was disclosed.
[0006]
[Problems to be solved by the invention]
However, although the bitterness could be removed by purification with the above synthetic adsorbent, the odor derived from the material remained, and the peptide recovery rate was slightly poor at about 70 to 75%.
Therefore, the present inventors searched for other means for purifying peptides and focused on activated carbon that is usually used for removing colored components and impurities. However, purification of angiotensin converting enzyme-inhibiting peptides with activated carbon often fails to remove bitter peptides or decreases angiotensin converting enzyme-inhibiting activity, and it is necessary to investigate the performance aspects of activated carbon more deeply. It was considered.
[0007]
[Means for Solving the Problems]
However, as a result of intensive studies to solve such problems, the present inventor has obtained an angiotensin-converting enzyme-inhibiting peptide-containing solution obtained by hydrolyzing fish protein with an protease in an aqueous medium, with an average pore diameter of 3 nm. It was found that by treating the following activated carbon with 40 to 100% by weight of the peptide-containing solution , the bitterness and odor can be removed without reducing the inhibitory activity, and the present invention has been completed.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
In the present invention, fish protein is first hydrolyzed with an protease in an aqueous medium to obtain a solution containing an angiotensin converting enzyme inhibitory peptide (hereinafter referred to as ACE inhibitory peptide) .
As the proteolytic enzyme in the present invention, known proteolytic enzymes such as thermolysin, pepsin, trypsin, chymotrypsin, and proteolytic enzymes produced by microorganisms can be used.
The aqueous medium in the present invention is not particularly limited, and examples thereof include water, alcohols such as ethanol and methanol, hydrochlorides such as sodium, potassium, magnesium and calcium, sulfates and carbonates.
[0009]
To hydrolyze fish protein with an enzyme, the formulation differs depending on the properties of the fish meat protein. However, in the case of poor solubility, the fish protein is mixed with hot water and homogenized with strong stirring, and then the solid content is 0.1-50. After preparing a solution or suspension of about wt%, the enzyme is added to the fish protein in an amount of 0.001 to 50 wt%, preferably 0.1 to 15 wt%, and 5 to 90 ° C., preferably It hydrolyzes by standing or stirring under a reaction condition of 20 to 70 ° C. for 1 minute to 3 days until the decomposition rate becomes 5% or more to obtain an ACE inhibitory peptide-containing solution. The decomposition rate is expressed as% amino acid nitrogen relative to total nitrogen. The measuring method is based on Journal of Agricultural and food Chemistry 24 No. 6 1091093 (1976).
[0010]
In the present invention, the ACE-inhibiting peptide-containing solution obtained above is purified by using activated carbon having an average pore diameter of 3 nm or less, preferably 1 to 3 nm. .
The ACE-inhibiting peptide-containing solution may enter the purification step using the activated carbon as it is, but it is preferable to first recover the liquid component by separating and removing insolubles from the hydrolysis product generated above. For removing insoluble materials, any of ordinary solid-liquid separation methods such as centrifugation, filtration, and decantation is employed. It is preferable that the liquid after removing the insoluble matter is concentrated so that the ACE inhibitory peptide concentration is 1 wt% or more, preferably 5 to 30 wt%. As a concentration method, a vacuum concentration method such as a flash method or a centrifugal thin film method, an ultrafiltration membrane concentration method, a reverse osmosis membrane concentration method, or the like is adopted.
[0011]
Next, the ACE inhibitory peptide-containing solution is brought into contact with activated carbon having an average pore diameter of 3 nm or less, and the non-adsorbed fraction is collected.
The average pore diameter of the activated carbon is required to be 3 nm or less, and if it exceeds 3 nm, bitterness and odor cannot be removed, which is inappropriate. The average pore diameter is preferably 1 to 3 nm. If it is less than 1 nm, bitterness and odor may not be sufficiently removed. Such average pore diameter is measured by a nitrogen gas adsorption method. As the measuring device, for example, “ASAP2400” manufactured by Mitchies is used.
[0012]
Examples of the activated carbon having a specific average pore diameter in the above include activated carbon activated with an oxidizing gas such as water vapor, carbon dioxide, and air, and preferably activated carbon activated with water vapor. Although it does not restrict | limit especially as an activation method, 750-1000 degreeC water vapor | steam is sprayed on carbide | carbonized_materials, such as wood, a coconut husk, and coal whose average particle diameter is about 0.001-10 mm, and is 30-48 hours. Activated carbon activated with water vapor is prepared by activating to a certain extent.
Specifically, “Shirakaba AW50”, “Shirakaba A”, “Shirakaba M”, “Shirakaba C”, “Shirakaba P”, “Shirakaba CW”, “Shirakaba PHC”, “Morshibon” manufactured by Takeda Pharmaceutical Co., Ltd. NGP _X ”,“ granular white birch WHA ”and the like.
[0013]
Furthermore, the pore volume of the activated carbon is preferably 0.01 to 0.8 cc / g, more preferably 0.1 to 0.7 cc / g. If the pore volume is less than 0.01 cc / g, odor and bitterness may not be completely removed, and if it exceeds 0.8 cc / g, ACE inhibitory activity may be reduced.
Moreover, the specific surface area of the activated carbon is preferably 800 to 1300 m ² / g, and more preferably 900 to 1200 m ² / g.
If the specific surface area is less than 800 m ² / g, the odor and bitterness may not be completely removed, and if it exceeds 1200 m ² / g, the ACE inhibitory activity may decrease and the yield may also decrease.
The pore volume and specific surface area are also values measured by the nitrogen adsorption method.
[0014]
The method for treating the ACE-inhibiting peptide-containing solution with the activated carbon may be either a batch-type treatment or a continuous column treatment, but the batch-type is preferred in terms of treatment efficiency. In the case of batch processing, after adjusting the concentration of the ACE inhibitory peptide-containing solution to 1 to 50% by weight (preferably 5 to 30% by weight), 40 to 100% by weight of activated carbon is used for the peptide. Is necessary . If it is less than 40 % by weight, bitterness and odor are not sufficiently removed, and if it exceeds 100% by weight, the ACE inhibitory activity is lowered and the yield is also lowered .
After adding the activated carbon, the mixture is stirred at 5 to 80 ° C. (preferably 30 to 70 ° C.) for about 30 to 120 minutes, and then the activated carbon is removed by filter paper, filter cloth, glass filter, centrifugation, etc. Collect it.
In order to prevent the activated carbon from leaking into the filtrate when filtering the activated carbon, the activated carbon may be filtered after forming a diatomaceous earth layer simultaneously with the activated carbon.
[0015]
When processing with a continuous column, activated carbon is used so that the linear velocity (LV) is 3 cm / hour or more and the space velocity (SV) is about 3 / hour or less in an aqueous solution of ACE inhibitory peptide solution having a concentration of 1 to 20% by weight. May be introduced into a column packed with.
[0016]
The activated carbon after the adsorption treatment with the above activated carbon is washed with water as necessary, and the non-adsorbed fraction is collected.
When elution of the bitter component adsorbed at the time of recovery is observed, an aqueous salt solution may be used. The aqueous salt solution is not particularly limited, and examples thereof include aqueous solutions of hydrochlorides such as sodium, potassium, magnesium, and calcium, and salts such as carbonates and sulfates. In particular, an aqueous solution of sodium chloride is practical. The use concentration of the aqueous solution is 1% by weight to a saturated concentration, preferably 5 to 20% by weight, and the use amount is preferably 1 to 3 times the charged volume of activated carbon.
[0017]
The ACE inhibitory peptide solution obtained by the purification method of the present invention is pulverized by a method such as concentration to dryness, freeze drying, spray drying, or vacuum drying, if necessary, and used as a pharmaceutical or health food (health supplement). Or, it has no bitterness and does not smell so it can be added to food as it is.
[0018]
When used as a pharmaceutical or health food, the peptide is used alone or in the form of a preparation prepared by mixing with a preparation carrier. As a pharmaceutical carrier, a substance that is commonly used in the pharmaceutical field and does not react with a peptide is used. Specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, carboxymethylcellulose calcium, methylcellulose, gelatin, Arabic Rubber, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, Refined lanolin, glycero gelatin, polysorbate, macrogol, vegetable oil, C, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, and the like.
Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, injections and the like. These preparations are prepared according to a conventional method.
The pharmaceuticals and health foods formulated as described above are used for the purpose of lowering blood pressure, preventing myocardial hypertrophy, and preventing strokes. Examples of health foods include the following.
[0019]
B) Food containing saponin (food containing ginseng root, food containing sorghum)
B) Sugar-containing foods [Oligosaccharides (Fructooligosaccharide-containing foods, Isomalto-oligosaccharide-containing foods, Galacto-oligosaccharide-containing foods), Polysaccharides (Shiitake-containing foods, Mucopolysaccharides, Protein-containing foods, Chondroitin sulfate-containing foods, Mannentake (Reishi) ) Contained foods, chitin, chitosan-containing foods c) Mineral-containing foods (calcium-containing foods, alfalfa-containing foods, prune extract foods, β-carotene-containing foods d) Oil-containing foods Vitamin E-containing oils [wheat (wheat, pigeon) germ Oil, soybean germ oil, rice germ oil] food containing eicosapentaenoic acid, food containing soybean lecithin, food containing γ-linolenic acid (primrose oil, borage oil), food containing docosahexaenoic acid e) food containing food containing soybean protein, food containing soybean protein Casein, whey protein, koji processed food f) taurine oyster processed food, shijimi processed food, green Shellfish processed food 【0020】
Moreover, the following are mentioned as a foodstuff to which a peptide is specifically added.
(1) Agricultural and fishery products Harusame, Koshian, Konnyaku, Bread, Noodles (immediate noodles, pasta, raw noodles, dried noodles), rice cakes, cereal foods, processed soybean products (tofu, soymilk, natto, frozen tofu), processed fishery products , (Crab flavor) salmon, (fish meat) ham, (fish meat) sausage, (fish meat) wiener, sprinkles, tea sauce], canned (sea chicken, oil sardine, yakitori), retort food (curry, stew, spaghetti)
(2) Dairy milk, processed milk, lactic acid bacteria beverage, butter, cheese, condensed milk, powdered milk (3) confectionery cake, mousse, (powder) dessert, ice cream, rice cake, chocolate, gummy, candy, cookies, wafers, jelly ( 4) Seasoning miso, soy sauce, umami (flavor) seasoning, (powder) natural seasoning, sauce, dressing, grilled meat sauce, mirin, curry, stew, spice, spice, yogurt (5) beverage soft drink (carbonated beverage) , Fruit drinks, sports drinks, nutrition drinks), taste drinks (coffee, cocoa, wort), miso soup, soup
The dose (intake) of the ACE inhibitory peptide varies depending on the administration method, patient's symptoms, age, etc., but is usually 0.001 to 5000 mg at a time, preferably 0.01 to 2000 mg at 1 to 3 times a day. is there.
The above preparation or food may contain ACE inhibitory peptide in an amount of 0.01% by weight or more, preferably 0.5 to 100% by weight. These preparations or foods may contain other therapeutically valuable ingredients.
[0022]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
40 g of water was added to 50 g of skipjack tuna (protein content 70 wt%), homogenized sufficiently, adjusted to pH 7.0 with sodium hydroxide, and 0.6 g of thermolysin (1.7 wt% with respect to protein) was added. A time standing reaction was carried out (decomposition rate 28.6%). Thereafter, the mixture was boiled at 100 ° C. for 10 minutes and left standing to obtain a supernatant obtained by centrifugation at 3000 × g. To the residue, 80 ml of water was added and stirred, and then centrifuged again under the same conditions to obtain a supernatant. The supernatants were combined to obtain 140 ml of a supernatant having a peptide concentration of 7.1% by weight.
[0023]
The ACE-inhibiting peptide-containing solution prepared as described above was concentrated with a rotary evaporator to a peptide concentration of 10% by weight, and 100 ml of this solution was added to activated carbon (“Shirakaba A50W” manufactured by Takeda Pharmaceutical Co., Ltd., average pore diameter 2.35 nm). 4 g of a specific surface area of 1020 m ² / g, a pore volume of 0.60 cc / g, and a water content of 50%), and stirred at 60 ° C. for 1 hour. Further, 5 g of diatomaceous earth was added and stirred, followed by filtration and adsorption. Minutes were collected.
The recovered solution was subjected to peptide recovery, measurement of ACE inhibitory activity, and bitterness and odor sensory tests.
[0024]
Peptide recovery rate The peptide recovery rate after purification with respect to the peptides in the ACE-inhibiting peptide-containing solution before purification was measured by the Kjeldahl method.
[0025]
Measurement of ACE inhibitory activity The ACE inhibitory activity was measured by the following method according to the method of Cheung and Cushman [Biochemical Pharmacology 20 , 1637 (1971)].
Enzyme substrate; Bz (benzyl) -Gly-His-Leu
(86 mg solution in 8 ml water and 8 ml phosphate buffer)
Enzyme: Rabbit lung acetone powder (Sigma)
(1 g of crushed supernatant in 10 ml of 50 mM phosphate buffer and then centrifuged)
100 μl of the above enzyme substrate, 12 μl of the enzyme solution and the peptide of the predetermined concentration of the present invention were mixed, and the whole was made up to 250 μl with water, followed by reaction at 37 ° C. for 30 minutes.
[0026]
The reaction was terminated with 250 μl of 1N HCl. Ethyl acetate (1.5 ml) was added to the reaction mixture, and the mixture was stirred with Vortex for 15 seconds, and then centrifuged.
1.0 ml was taken out from the ethyl acetate layer, the ethyl acetate was distilled off, 1 ml of distilled water was added thereto to dissolve the residue, and the ultraviolet absorption ₂₂₈ nm value (OD ₂₂₈ ) of the extracted hippuric acid was measured. .
ACE inhibition rate the OD ₂₂₈ of when reacted without peptide as 100%, a reaction time of 0 minutes peptide concentration IC ₅₀ for the time the OD ₂₂₈ of the inhibition rate of 50% determined as 0% when the (μgPro / ml) Activity was displayed.
Bitter sensory test The powder obtained by freeze-drying the recovered liquid was evaluated by 10 panelists.
The evaluation was calculated by adding 10 points when no bitterness was felt, 5 points when slightly feeling bitterness, and 0 points when feeling bitterness strongly, and totaling the evaluation scores of 10 panelists.
Odor sensory test The powder obtained by freeze-drying the treatment liquid was evaluated by 10 panelists.
The evaluation was calculated by adding 10 points when no odor was felt, 5 points when feeling a little odor, and 0 points when feeling strong odor, and totaling the evaluation points of 10 panelists.
[0027]
Example 2
The same treatment as in Example 1 was performed except that the amount of activated carbon added in Example 1 was changed to 6 g.
[0028]
Example 3
As the activated carbon in Example 1, activated carbon (“Shirakaba P” manufactured by Takeda Pharmaceutical Co., Ltd., average pore diameter 2.05 nm, specific surface area 1000 m ² / g, pore volume 0.58 cc / g, water content 3%) The same treatment as in Example 1 was conducted except that was used.
[0029]
Example 4
In Example 1, thermolysin was replaced with pepsin of the same weight, and an experiment was conducted. The working conditions of pepsin were adjusted to pH 1.6 with hydrochloric acid, and the reaction temperature was 37 ° C. for 5 hours. Thereafter, the same processing was performed and the evaluation was made in the same manner as in Example 1.
[0030]
Example 5
In Example 1, it processed similarly and evaluated similarly except having used chicken instead of the bonito.
[0031]
Comparative Example 1
In Example 1, activated carbon activated by an aqueous zinc chloride solution (“Carborafine” manufactured by Takeda Pharmaceutical Co., Ltd., average pore diameter 3.65 nm, specific surface area 1450 m ² / g, pore volume 1.19 cc / g, water content The rate was 3% and the same evaluation was performed in the same manner as in Example 1 except that 4 g was used.
The results of Examples 1 to 5 and Comparative Example 1 are summarized in Table 1.
[0032]

[0033]
【The invention's effect】
The ACE inhibitory activity is reduced from the ACE inhibitory peptide-containing solution by the purification method of the present invention in which an angiotensin converting enzyme inhibitory peptide-containing solution obtained by hydrolyzing fish protein is treated with activated carbon having an average pore diameter of 3 nm or less. It is possible to remove bitterness and odor without causing the peptide to be useful as a blood pressure lowering agent or blood pressure lowering food.

Claims (1)

The concentration of the peptide in the angiotensin-converting enzyme-inhibiting peptide-containing solution obtained by hydrolyzing fish protein with an protease in an aqueous medium is adjusted to 5 to 30% by weight, and then the average pore diameter is 3 nm or less . The activated carbon activated with water vapor having a pore volume of 0.01 to 0.8 cc / g and a specific surface area of 800 to 1300 m ² / g is treated with 40 to 100% by weight based on the peptide in the solution containing the angiotensin converting enzyme-inhibiting peptide. And a method for purifying the peptide.