JP2005097269A - Composition containing mung bean protein-decomposed material having angiotensin-converting enzyme inhibitory activity and active oxygen-removing activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein-decomposed material having a high angiotensin-converting enzyme (ACE) inhibitory activity and active oxygen-removing activity (SOD like activity), having extremely high safety, excellent in absorbability, having a good flavor, without having a smell, having a good platability and obtained inexpensively, and a method for producing the same. <P>SOLUTION: This mung bean protein-decomposed material-containing composition is obtained by hydrolyzing the protein derived from the mung beans with a protease and having the angiotensin-converting enzyme inhibitory activity and active oxygen-removing activity. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to a mung bean protein having a high angiotensin converting enzyme (hereinafter referred to as ACE) inhibitory activity and an active oxygen removing activity, which is naturally derived, extremely safe, excellent in absorbability, good in flavor, and can be produced at low cost. The present invention relates to a decomposed product-containing composition, a method for producing the same, and a food containing the composition. More specifically, the flavor of the mung bean protein, which is a by-product after extracting starch from the mung bean, is hydrolyzed with a protease, and insoluble matter is removed by a separation process such as filtration and centrifugation, and exhibits excellent ACE activity and SOD-like activity. The present invention relates to a good mung bean proteolysate-containing composition, a method for producing the same, and a food containing the composition.

Angiotensin converting enzyme (ACE) is mainly present in lungs, vascular endothelial cells, and renal proximal tubules, and C of angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu). It is an enzyme that releases dipeptide His-Leu from the terminal and produces angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) having a strong pressor action. In addition, this enzyme has an action of inactivating bradykinin, which is an antihypertensive substance in vivo, and is strongly associated with an increase in blood pressure. Therefore, substances that inhibit ACE activity are considered effective for the prevention and treatment of hypertension.

Substances having an ACE inhibitory action are roughly classified into two types, that is, synthetic products or natural products. For example, a synthetic product of captopril, which is a proline derivative, and its analogs inhibit strong angiotensin converting enzyme activity and have been confirmed to have a blood pressure lowering effect (see Patent Document 1). Examples of naturally-occurring products include protein degradation products of animals and microorganisms (see Patent Document 2) and the like, and examples of identified peptides and peptide mixtures include laver-derived peptide mixtures (see Patent Document 3). And a peptide of Leu-Lys-Pro (see Patent Document 4).

On the other hand, in-vivo active oxygen is originally produced as a bactericidal and tumoricidal substance in order to protect the living body. However, since there is no selective toxicity, it may act on normal cells, and excess of active oxygen is known to cause various damages to the living body. As an example, active oxygen generates hydroxyl radicals in the presence of a metal ion catalyst, but hydroxyl radicals cause peroxidation of blood LDL, and the oxidized LDL acts on vascular endothelial cells to form a thrombus. . As a result, it becomes easy to develop a disease such as hypertension, arteriosclerosis, or diabetes, which is called a lifestyle-related disease (see Non-Patent Document 1).

Superoxide dismutase (hereinafter referred to as SOD) is an antioxidant enzyme that exists widely and in large amounts in the living body and is important as an active oxygen removing component in the living body. SOD catalyzes the disproportionation reaction of superoxide anion, which is a kind of active oxygen, and this reaction reduces the superoxide in cells to 1 / 100,000 (see Non-Patent Document 2).
As a factor capable of reinforcing the SOD-like action in the body, there is SOD itself or an SOD-like substance. Since SOD is not readily available in large quantities, most of the means to reinforce the SOD-like action are ingestion of SOD-like active substances. For example, intake of vitamin C, vitamin E, and catechins can be mentioned.

However, these substances are not stable enough to remove active oxygen in the living body due to problems such as stability and absorbability, and therefore, development of better SOD-like substances has been desired. Examples of peptides and peptide mixtures that exhibit SOD-like activity so far include hexapeptides obtained by the decomposition of wheat gluten (see Patent Document 5), peptide mixtures obtained by enzymatic degradation of plant proteins (see Patent Document 6), and the like. It has been reported.

Thus, such ACE enzyme inhibitors and SOD-like active substances have been used in foods for specific health use, health foods, and the like as preventive factors for diseases such as hypertension, arteriosclerosis and diabetes. In order to exhibit both functions of ACE inhibitory activity and SOD-like activity, means for achieving the object by combining and blending materials can be considered. However, from the viewpoint of labor and know-how, it is possible to have both ACE inhibitory activity and SOD-like activity with one type of material, or to add more functionality at the same time than preparing many materials. Material is required.

On the other hand, as described above, the blood pressure lowering action due to the ACE inhibitory action and the blood pressure lowering action due to the SOD-like action are different in the mechanism of action, so that a better effect can be expected by trying to adjust the blood pressure from both sides. Until now, most reports on the blood pressure lowering action of proteolysates and peptides are related to the ACE inhibitory action, and there are few reports on trials of adjusting blood pressure in terms of both ACE inhibition and active oxygen suppression by SOD-like activity. As an example of trying to adjust blood pressure from both sides, there is a report of a low-molecular peptide-containing product obtained by hydrolyzing a white pearl barley powder acetic acid-treated product with a protease (see Patent Document 6), but the effect is not always satisfactory. I can't say that. In addition, there are reports of oligopeptides derived from livestock, fish residues (see Patent Document 7), cultures obtained by inoculating and culturing legume soup (see Patent Document 8), etc. In the production of these materials There is a disadvantage that the manufacturing process is complicated because decalcification treatment and fermentation are necessary. There are also problems with the flavor of fermentation, the flavor of livestock and fish.

JP-A-8-40896 Patent No. 3110075 Japanese Patent No. 3272621 Japanese Patent No. 3040389 Japanese Patent No. 2835504 Japanese Patent No. 3108059 JP2001-211185 JP 2002-121141 A Current Therapy, Vol. 16, no. 1, pp. 105-109, 1998 Supervised by Kazutomo Imabori and others, "Biochemistry Dictionary 3rd Edition", Tokyo Chemical Doujin, p. 743

  The present invention solves the above-mentioned problems, and has a composition containing a proteolysate, which is extremely safe, excellent in absorbability, good in flavor and odorless, good in taste and inexpensive, and its An object is to provide a manufacturing method.

  In view of the above situation, the present inventors have conducted intensive studies, and as a result, the composition containing a mung bean protein hydrolyzate obtained by hydrolysis with a protease using mung bean protein obtained from mung bean has excellent ACE enzyme inhibition. It has been found that it has an action and SOD-like activity, and the present invention has been completed.

That is, the first of the present invention relates to a mung bean proteolysate-containing composition having angiotensin converting enzyme inhibitory activity and active oxygen removal activity, which is obtained by hydrolyzing a mung bean-derived protein with a protease.
In a preferred embodiment, the present invention relates to the composition described above, wherein the ratio of peptides having a molecular weight of 132.1 to 576.6 in all peptides which are mung bean proteolysates is 10% or more.
The second of the present invention relates to a method for producing a composition containing a mung bean proteolysate having an angiotensin converting enzyme inhibitory activity and an active oxygen removing activity, characterized by hydrolyzing a mung bean-derived protein with a protease.
3rd of this invention is related with the foodstuff containing the composition in any one of the said.

The present invention has a ACE inhibitory activity and an SOD-like activity, is extremely safe, excellent in absorbability, good in flavor and odorless, good in taste and inexpensive, and a composition containing a proteolysate Products and methods of manufacturing the same can be provided.

The raw material containing protein derived from mung bean used in the present invention (hereinafter referred to as “raw material”) is extracted from mung beans directly with acid or alkali, extracted from mung bean juice with water, or obtained by crushing mung beans themselves. Is obtained. For example, a by-product containing a protein derived from mung bean from a Harusame manufacturing plant can be used, but is not particularly limited thereto. The form can be used in any form such as liquid, powder and paste. In addition to the protein derived from mung bean, saccharides, fibers, salt, moisture, fats and oils and the like may be contained. The weight content of the protein in the raw material (hereinafter referred to as “crude protein content”) is not particularly limited, but is preferably 15% by weight or more, more preferably 30% by weight in the sense that the effect is easily obtained. That's it.

The crude protein content here was calculated by multiplying the amount of nitrogen by a conversion factor. The amount of nitrogen was measured by the semi-micro Kjeldahl method. That is, about 50 mg of a sample was weighed in a Kjeldahl flask, and the weighed value was recorded. 10 g of potassium sulfate and 1 g of copper sulfate were mixed to form a decomposition accelerator, and 1 g of the mixture was added to the flask. Thereto was added 5 ml of concentrated sulfuric acid and left overnight. Thereafter, the flask was gradually heated, and after the liquid became transparent and no carbide was observed on the inner wall of the flask, the heating was stopped. After cooling, 20 ml of distilled water was added and mixed well, then ice-cooled, and the flask was connected to a distillation apparatus previously washed with steam. A receiver for receiving the distillate was charged with 10 ml of 0.1N sulfuric acid and 2-3 drops of indicator (mixed solution of methyl red and methylene blue reagent solution), and the lower end of the cooler of the distillation apparatus was immersed in this liquid. From a funnel connected to a distillation apparatus, 20 ml of 40% sodium hydroxide was added to the flask and distilled through steam for 6 to 7 minutes. The lower end of the cooler was separated from the liquid surface, the portion was washed with a small amount of water, and titrated with 0.1N sodium hydroxide. Moreover, the sample measured by the same method without adding a sample was used as a blank. The amount of crude protein was calculated by the following formula.
Crude protein content (% (w / w)) = {([B] − [A]) × F × 1.4007 × 6.25 / [C]} × 100
In the above formula, “A” represents the amount of 0.1N sodium hydroxide required for titration when the sample was added (ml), and “B” represents the amount of 0.1N sodium hydroxide required for the titration of the control (ml). ing. “C” represents a sample weight (mg), and “F” represents a factor of sodium hydroxide used for titration. “1.4007” corresponds to a nitrogen amount (mg) corresponding to 1 ml of 0.1N sulfuric acid, and “6.25” was used as a protein amount conversion coefficient from the nitrogen amount.

  Proteases that can be used to hydrolyze mung bean-derived proteins used in the present invention are proteases and peptidases that hydrolyze proteins. subtilis, Bacillus sp. Any of enzymes derived from microorganisms such as Bacillus such as thermolysin, animal origin such as pepsin and pancreatin, and plant-derived enzymes such as papain and bromelain can be used. In order to obtain a relatively good flavor, the protease used is preferably an acidic protease derived from Aspergillus. Enzymes can be used by combining one or two or more of purified products and crude products, and there is no problem even if they are used in combination. Regarding the reaction conditions for the hydrolysis (reaction temperature, pH, time, amount of enzyme used), conditions are generally set in accordance with the optimum action conditions of the protease used, but are not particularly limited thereto. From the viewpoint of good efficiency, the temperature is preferably 10 ° C. to 80 ° C., the pH is 2 to 11, the reaction time is 2 to 48 hours, and the amount of enzyme used is preferably 10 to 30,000 units per gram of crude protein. Further, the reaction temperature is 30 to 60 ° C., the pH is 3 to 8, the reaction time is 4 to 20 hours, and the enzyme amount is more preferably 100 to 7000 units per gram of crude protein.

The proportion of peptides having a molecular weight of 132.1 to 576.6 in all peptides, which are mung bean proteolysates obtained by protease treatment, is preferably 10% or more. When the proportion of the peptide having a molecular weight of 132.1 to 576.6 is less than 10%, the expected effects (ACE inhibitory action and SOD-like activity) of adding the composition to foods can be obtained. It may be difficult. In view of the inhibitory action of ACE converting enzyme and the effect of SOD-like activity, it is more preferably 40% or more. From the viewpoint of excellent absorbability, it is more preferably 50% or more, and still more preferably 60% or more. After protease treatment, insoluble matter is removed from the obtained mung bean proteolysate by filtration or centrifugation. The obtained supernatant is dried by a method such as concentration under reduced pressure, lyophilization, spray drying or the like to obtain the desired product. The form of the present mung bean proteolysate may be any of liquid, powder and paste.

The ratio of peptides having a molecular weight of 132.1 to 576.6 in all peptides that are mung bean proteolysates and the molecular weight distribution of mung bean proteolysates were analyzed by the following methods.
Molecular weight distribution is measured by Superdex, a gel filtration column.
It was measured by high performance liquid chromatography using “Peptide HR10 / 30 (Pharmacia)”. A 30% aqueous acetonitrile solution containing 0.1% trifluoroacetic acid was used as the mobile phase, the flow rate was 0.3 ml / min, and detection was performed at room temperature (25 ° C.) for 80 minutes by ultraviolet absorption at 220 nm. The molecular weight distribution was shown as an integration chart using a data processor (D-2500 Chromat-Integrator) manufactured by Hitachi, Ltd. The molecular weight was determined by preparing a standard line using oligopeptides “Ala-Pro = 186.2” and “Substance P = 1347.7” with known molecular weights. The area of the peptide having a molecular weight of 132.1 to 576.6 was determined from the integration chart, and the ratio to the total area of the entire peptide was determined.

The following method was used to measure the ACE inhibitory activity of the mung bean protein degradation product.
Hipril histidyl leucine (Bz-Gly-His-Leu) was dissolved in 0.1 M borate buffer (pH 8.3, containing 0.3 M sodium chloride) to a concentration of 5 mM to obtain a substrate solution. Add 40 μl of sample solution (dissolved in 0.1 M borate buffer, pH 8.3) to 0.2 ml of substrate solution, incubate at 37 ° C. for 10 minutes, and then add 40 μl of ACE, 70 mU / ml (derived from bovine lung). The mixture was further reacted at 37 ° C. for 30 minutes. The reaction was stopped by adding 0.25 ml of 1M hydrochloric acid, 1.7 ml of ethyl acetate was added, the stopper was tightly sealed, and the mixture was vigorously stirred for 20 seconds to extract the hypophosphoric acid produced by the reaction. Thereafter, the mixture was centrifuged at 1500 g for 10 minutes, 1.4 ml of an ethyl acetate layer was collected, and ethyl acetate was evaporated. 1 ml of distilled water was added thereto and stirred well, allowed to stand for 15 minutes, and then stirred again to dissolve. Absorption at 228 nm of the extracted hyprilic acid was measured. For the control, the borate buffer was used instead of the sample solution. Moreover, what added distilled water instead of the enzyme solution about each was made into the blank. The ACE inhibition rate was determined from the absorbance at 228 nm. The concentration of the sample crude protein when the ACE inhibition rate was 50% was defined as IC ₅₀ (μg / ml). The ACE inhibition rate was calculated by the following formula.
Inhibition rate (%) = {1- (AB) / (CD)} × 100
In the above formula, “A” represents the absorbance when the sample solution was added, “B” represents the absorbance of the blank, “C” represents the absorbance of the control, and “D” represents the absorbance of the blank.

The SOD-like activity of the mung bean protein degradation product was measured by the following method.
3mM xanthine solution (pH 10.2, 0.05M sodium carbonate buffer included), 3mM EDTA, 0.15% BSA 0.1ml each and sample solution (pH 10.2, 0.05M carbonate buffer included) 2.5ml Mix and incubate at 25 ° C. for 5 minutes. Thereto, 0.1 ml of xanthine oxidase (0.4 units / ml derived from buttermilk) and 0.1 ml of 0.75 mM nitroblue tetrazolium chloride were added and reacted at 25 ° C. for 20 minutes. Absorbance at 560 nm was measured by adding 0.1 ml of 6 mM cupric chloride. What added 0.05M sodium carbonate buffer (pH 10.2) instead of the sample was set as a control, and what added distilled water instead of the enzyme for each sample was used as a blank. The concentration of the sample crude protein when the superoxide anion elimination rate was 50% was defined as IC ₅₀ (μg / ml). The superoxide anion elimination rate was calculated using the following formula.
Erasure rate (%) = {1- (AB) / (CD)} x 100
In the above formula, “A” represents the absorbance when the sample solution was added, “B” represents the absorbance of the blank, “C” represents the absorbance of the control, and “D” represents the absorbance of the blank.

  Proteins, fibers (including plant fibers), starches, sugars, lipids, minerals, vitamins and the like can be added to the mung bean proteolysate composition of the present invention in addition to the mung bean proteolysate. In this case, the ratio of the mung bean proteolysate in the composition is preferably 5 to 99% by weight.

As food containing the mung bean proteolysate composition of the present invention, grains such as rice porridge, bread, thickening, processed products of beans, processed livestock products such as sausage and ham, processed fishery products such as seaweed, chikuwa, yogurt, Dairy products such as soy milk, egg processed products such as pudding and steamed rice cakes, confectionery products such as biscuits and rice crackers, cooked and processed food products such as frozen croquettes and fried shrimp, beverages such as juice and cocoa powder, cup noodle seasonings, soy sauce and sauce And other seasonings. These products may take the form of normal foods as well as healthy foods and drinks and foods for specified health use, as well as liquids, solids, blocks, powders, semi-fluids, etc. included.

  It was confirmed that the mung bean proteolysate of the present invention has DPPH radical scavenging activity and glutathione production promoting action in skin fibroblasts (derived from normal human adults) as an active oxygen removing action other than SOD-like activity. Yes.

  Examples of the present invention will be described below.

Production Example 1
The dried product of mung bean protein discharge liquid, which is a by-product obtained in the production process of Harusame, was pulverized with a mill, and tap water was added and dispersed so that the crude protein content was 6% by weight. After adjusting this to pH 3 with hydrochloric acid, 5,000 units of acidic protease (trade name: Denapsin, Nagase ChemteX) was added per gram of crude protein and reacted at 40 ° C. for 16 hours with stirring. After the reaction, the enzyme was inactivated by heating at 80 ° C. to 85 ° C. for 20 minutes. The enzyme reaction solution was centrifuged, and the supernatant was freeze-dried to obtain a sample of mung bean proteolysate (Sample 1). The molecular weight distribution of Sample 1 measured by the method described above is shown in FIG. The proportion of peptides having a molecular weight of 132.1 to 576.6 in this sample 1 was 59.7%.

Example 1
When the inhibition rate of ACE activity of Sample 1 obtained in Production Example 1 was measured by the method described above, it showed a high inhibitory activity of IC ₅₀ = 35 μg / ml. Further, when the SOD-like activity of Sample 1 obtained in Production Example 1 was measured by the method described above, it was found that IC ₅₀ = 0.3 mg / ml and excellent SOD-like activity.

Production Example 2
The mung bean protein dispersion obtained in the same manner as in Production Example 1 was adjusted to pH 7 with sodium hydroxide, and then alkaline protease (trade name: Biolase SP-15FG, Nagase ChemteX) was added at 10,000 units per gram of crude protein. The mixture was reacted at 40 ° C. for 16 hours with gentle stirring. The enzyme was inactivated by heating at 80 to 85 ° C. for 20 minutes. The enzyme reaction solution was centrifuged, and the supernatant was freeze-dried to obtain a sample of mung bean proteolysate (sample 2). The molecular weight distribution of Sample 2 measured by the method described above is shown in FIG. The proportion of peptides having a molecular weight of 132.1 to 576.6 in Sample 2 was 61.1%.

Example 2
Sample 2 obtained in Production Example 2 showed high ACE activity inhibitory activity (IC ₅₀ = 38 μg / ml) as in Sample 1. Sample 2 obtained in Production Example 2 showed excellent SOD-like activity (IC ₅₀ = 0.35 mg / ml) in the same manner as Sample 1.

Example 3
10 g of mung bean proteolysate obtained from Examples 1 and 2 was dissolved in 100 ml of water to obtain an aqueous solution. These solutions were subjected to sensory evaluation (flavor evaluation) by 10 panelists. The evaluation method was scored for each item, and the final evaluation was shown as an average value of the scores of 10 people. Items and scoring criteria are shown below.
Odor: odorless-5, only -4, slightly strong-3, strong-2, very strong-1.
Hue: good -5, not bad -4, don't care -3, care -2, very care -1.
Flavor: Delicious-5, as it is delicious-4, general-3, not good-2, bad-1.
Aftertaste: Delicious-5, Remain-delicious-4, General-3, Not good-2, Bad-1.
Bitterness: None -5, not much felt -4, slightly felt -3, bitter -2, very bitter -1.
Table 1 shows the flavor evaluation results of Samples 1 and 2.

表１より本発明の緑豆蛋白分解物含有組成物は無臭の上、風味も優れることが分かった。

From Table 1, it was found that the mung bean protein hydrolyzate-containing composition of the present invention was odorless and excellent in flavor.

  Examples of utilization of the present invention include not only normal foods but also health foods and drinks, foods for specified health use, and the like.

Diagram showing the molecular weight distribution of Sample 1 Diagram showing the molecular weight distribution of Sample 2

Claims (4)

A mung bean proteolysate-containing composition having an angiotensin converting enzyme inhibitory activity and active oxygen removing activity, obtained by hydrolyzing a mung bean-derived protein with a protease. The composition according to claim 1, wherein the ratio of peptides having a molecular weight of 132.1 to 576.6 in all peptides of the mung bean proteolysate is 10% or more. A method for producing a mung bean proteolysate-containing composition having angiotensin converting enzyme inhibitory activity and active oxygen removal activity, wherein a protein derived from mung bean is hydrolyzed with a protease. A food containing the composition according to claim 1 or 2.

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