JP2012170438A - Beans for food, method for producing the same, and food using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing beans for food having excellent ACE inhibiting activity, high in safety with no risk of a side effect and toxicity, holding the shape of beans as they are, high in digestion absorbability so as to be ingestible as a daily meal and to enable an ingesting person to obtain an antihypertensive effect without being burdened with further ingesting something other than the meal, utilizing the whole edible parts without producing waste to effectively utilize resources in conformity with zero emission, and attaining mass production.SOLUTION: The beans for food contain a peptide that contains Ile-Tyr produced by the action of a peptide bond hydrolase introduced inside. The total content of the peptide composed of 2-20 amino acid is twice or more of the content of the peptide before introducing the peptide bond hydrolase, and the shape before introducing the peptide bond hydrolase is maintained.

Description

  The present invention introduces a peptide bond hydrolase inside, contains a high concentration of peptides by hydrolyzing peptide bonds while maintaining the original shape, food beans having excellent antihypertensive action, The production method and a food using the same. Furthermore, the present invention relates to food beans that can inhibit the formation of a bitter component due to the action of a peptide bond hydrolase and can efficiently take an antihypertensive component as a daily meal, a production method thereof, and a food using the same.

  Although the subjective symptoms of high blood pressure itself are not so clear, it is said that high blood pressure is the main risk factor for heart disease and stroke, which is one of the top causes of death in Japanese people. It is said that there are an estimated 40 million or more hypertensive patients in Japan, and more than half of the elderly are hypertensive. As we enter an aging society in the future, there is a need for further blood pressure control, There are high expectations for food materials and their extracts that have an effect of preventing hypertension.

  One enzyme that plays an important role in the regulation of blood pressure is angiotensin I converting enzyme (ACE). The renin / angiotensin system for pressurization and the kallikrein / kinin system for hypotension play important roles in the blood pressure regulation system. In the renin-angiotensin system, angiotensinogen secreted from the liver is converted into angiotensin I by renin secreted from the kidney, and angiotensin I is further converted into angiotensin II by ACE. Angiotensin II contracts blood vessels to increase blood pressure. In the kallikrein-kinin system related to hypotension, bradykinin produced by kallikrein acting on kininogen relaxes blood vessels and lowers blood pressure, but bradykinin is degraded by ACE. Blood pressure rises. Therefore, an ACE inhibitor, which is a substance that inhibits or suppresses ACE enzyme activity, has attracted attention as an effective antihypertensive component.

  As for the use of ACE inhibitory substances from food materials, it is known that peptides, which are enzyme degradation products of proteins in food materials, have ACE inhibitory activity. As a food material in which the enzyme degradation product of the protein contained has ACE inhibitory activity, sardines (for example, Patent Document 1 and Non-Patent Document 1), Skipjack (Patent Document 2, Non-Patent Documents 2 and 3), Milk Casein (Patent Document) 3, Non-Patent Literature 4), corn (Patent Literature 4, Non-Patent Literature 5), soybean (Patent Literature 5, Non-Patent Literature 6) and the like have been reported.

  ACE-inhibiting peptides can be prepared by hydrolyzing proteins contained in food materials with proteolytic enzymes. In producing ACE-inhibiting peptides, the food materials are used in order to efficiently react the raw material proteins with the enzymes. A pretreatment such as pulverization, protein extraction from a food material, or a liquid food material is used. Furthermore, after degrading the protein, it goes through processing steps such as extraction and concentration of the target ACE-inhibiting peptide. Finally, it is in the form of drinks and tablet confections that are easy to ingest, including the amount necessary for daily intake. Is provided by.

  However, in these production methods, it is necessary to dispose of the solvent used for extraction of the target component and components other than the target component, which increases the processing cost and wastes resources. Furthermore, since ACE inhibitory peptides are provided as drinks and tablet confections, in order to take them daily, they must be taken separately from the meal.

  The present inventors have already developed a functional food (Patent Document 6) that generates a peptide inside a food material, but there is no mention of a food having an antihypertensive action.

JP 2003-192607 A JP2001-112470 WO95 / 28425 JP 06-087886 A JP 2002-053595 A JP2008-187908

Japanese Journal of Nutrition and Food, 52, 271-277. (1999) Bioscience Biotechnology and Biochemistry, 56, 1541-1545. (1992) Bioscience Biotechnology and Biochemistry, 57, 695-697. (1993) Agricultural and Biological Chemistry, 51, 1581-1586. (1987) Agricultural and Biological Chemistry, 55, 1313-1318. (1991) Soy Protein Research, 6, 73-77. (2003)

  The problem of the present invention is that it has excellent ACE inhibitory activity, has no risk of side effects and toxicity, is highly safe, and has high digestibility and absorbability despite maintaining the shape of beans. Therefore, it is possible to provide food beans and foods that can obtain an antihypertensive effect without incurring the burden of ingesting anything other than meals. In addition, the entire edible portion can be used without any waste to effectively use resources, and simple and inexpensive food beans can be obtained in line with recent zero emissions. Another object of the present invention is to provide a method for producing food beans that enables mass production. Furthermore, when protein is enzymatically decomposed, bitterness components are also produced, but the generation of bitterness components is suppressed, and not only the original shape but also the taste is not impaired, providing high-product-value beans and foods that can be ingested without discomfort There is to do.

  In order to solve such problems, the present inventors have conducted research on a method capable of promoting the enzymatic reaction by maintaining the original shape of beans and introducing a peptide bond hydrolase into the beans. As a result, it was found that the peptide-bonded hydrolase easily passes through the seed coat of beans by introducing fine scratches on the seed coat of beans before introducing the enzyme into the inside. And the content of the peptide comprised by 2-20 amino acids containing Ile-Tyr produced | generated by the effect | action of a peptide bond hydrolase in the beans is 2-20 before the introduction of a peptide bond hydrolase. It has been found that food beans having an excellent ACE inhibitory activity can be obtained by accelerating the peptide bond hydrolase reaction so that the content of the peptide composed of amino acids is twice or more. Furthermore, sodium salts such as sodium chloride and sodium glutamate, phosphatidic acid, lysophosphatidic acid, or exopeptidase are introduced into the beans together with the peptide bond hydrolase, thereby generating a bitter component due to the peptide bond hydrolase reaction. The present inventors have found that food-grade beans that can be suppressed and that the original taste of beans is not impaired in terms of not only the shape but also the taste, and the present invention has been completed.

  That is, the present invention contains a peptide containing Ile-Tyr produced by the action of a peptide bond hydrolase introduced therein, and the total content of peptides composed of 2 to 20 amino acids is peptide bond The present invention relates to a food legume characterized by being at least twice the content of a peptide before introduction of a hydrolase and having a shape before introduction of a peptide bond hydrolase.

  The present invention also relates to a method for producing the above-mentioned food beans, a seed coat damage process for damaging the seed coat of beans, an enzyme contact process for contacting a peptide bond hydrolase with beans damaged in the seed coat, reduced pressure or pressure treatment Step of introducing a peptide bond hydrolase into the inside, and the peptide bond hydrolase introduced inside the peptide bond so that the content of peptides composed of 2 to 20 amino acids is more than doubled The present invention relates to a method for producing edible beans, comprising an enzyme reaction step for decomposing and an enzyme deactivation step for deactivating peptide bond hydrolase by heating.

  The present invention also relates to a food product obtained by drying or freezing the food beans.

  The food beans of the present invention and foods obtained using the same have excellent ACE inhibitory activity, have no risk of side effects and toxicity, are highly safe, and retain the shape of the beans as they are. Regardless, it is highly digestible and absorbable and can be ingested as a daily meal, and the ingestor does not bear the burden of ingesting anything other than meals, and can obtain an antihypertensive effect. In addition, the method for producing food beans of the present invention is based on the recent zero emission without producing waste, and by using all edible parts for effective use of resources, it is simple and inexpensive for food use. Beans can be obtained and mass production is possible. Furthermore, the food beans and foods of the present invention suppress the occurrence of bitter components that occur when proteins are enzymatically decomposed, do not impair the taste as well as the original shape, and can be ingested by consumers without discomfort. Is expensive.

In an example of the food beans of this invention, it is a figure which shows the chromatogram at the time of isolating Ile-Tyr, and MS chromatogram. In an example of the beans for foodstuffs of this invention, it is a figure which shows MS spectrum at the time of identifying Ile-Tyr. In an example of the food beans of this invention, it is a figure which shows the chromatogram at the time of identifying Ile-Tyr, and MS chromatogram. It is a figure which shows the blood pressure change of the rat which administered long-term example of the food beans of this invention.

  As the beans to be applied to the food beans of the present invention, soy beans, red beans, green beans, lima beans, peas, safflower beans, broad beans, cowpeas, chickpeas, mung beans, lentils, Any of peanuts and the like may be used. Of these, soybean is preferable because it has a high protein content, abundant food experience, good taste of the composition after decomposition, a large amount of cultivation, and a low price. Examples of soybeans include yellow soybeans, red soybeans, and black soybeans, with yellow soybeans being particularly preferable.

  These soybeans can be used after absorbing water. As a water absorption amount, it is preferable that it is 0-130 mass% with respect to the mass of soybean, for example.

  The peptide bond hydrolase can be used without particular limitation as long as it is an enzyme that hydrolyzes peptide bonds of proteins and peptides. Exopeptidase that cuts 1-2 amino acids from the ends of proteins and peptides, and cleaves the central part. Any endopeptidase may be used and may or may not be purified. Examples of peptide bond hydrolases include proteolytic enzymes (digestive enzymes) derived from animal digestive systems such as peptidase, pepsin, trypsin, chymotrypsin, and elastase in the narrow sense, proteolytic enzymes derived from the genus Aspergillus, and Bacillus ( Proteolytic enzymes derived from the genus Bacillus, proteolytic enzymes derived from the genus Rhizopus, proteolytic enzymes derived from the genus Penicillium, proteolytic enzymes derived from microorganisms such as the proteolytic enzymes derived from the genus Mucor, Examples include plant-derived proteolytic enzymes such as papain, bromelain and ficin. As the peptide bond hydrolase, a proteolytic enzyme derived from a microorganism and a proteolytic enzyme derived from a plant are preferable, and a proteolytic enzyme derived from the genus Bacillus is more preferable.

  The amount of the peptide bond hydrolase introduced into the beans may be 0.001 to 2.0 g, preferably 0.01 to 0.5 g, per 100 g of beans. Moreover, nutrient components, such as a seasoning, salt, an organic acid, a vitamin, and a mineral, can also be contained in an enzyme liquid.

  In order to increase the contact efficiency of the peptide bond hydrolase to the substrate, an enzyme other than the peptide bond hydrolase may be used in combination as long as the function of the peptide bond hydrolase is not inhibited. Examples thereof include plant tissue degrading enzymes such as cellulase, hemicellulase and pectinase, lipolytic enzymes such as lipase, and amylolytic enzymes such as amylase. These enzymes may or may not be purified.

  In addition, β-glucosidase can be used to decompose glycosides contained in beans to produce aglycone, increase the content of isoflavones, etc., and develop an antioxidant effect or the like. Examples of β-glucosidase include cellulase, amylase, pectinase, hemicellulase, and cyclodextrin producing enzyme. The amount of β-glucosidase introduced into the beans is preferably 0.01 to 0.5 g with respect to 100 g of beans.

  The peptide produced by the action of the peptide bond hydrolase is a peptide composed of 2 to 20 amino acids, including the dipeptide Ile-Tyr, has an ACE inhibitory function, and has an antihypertensive effect (hereinafter referred to as “hypertensive effect”). Also called anti-hypertensive peptide.) Specific examples of these antihypertensive peptides include Ile-Tyr, Pro-Trp, Tyr-Thr, Leu-Ala-Pro, Leu-Glu-Phe, Leu-Lys-Tyr, Tyr-Pro-Ser, Val. -Ala-Trp, Val-Lys-Pro, Asp-Thr-Lys-Phe, Gln-Val-Val-Phe, Ile-Thr-Pro-Leu, Leu-Glu-Phe-Leu, Pro-Ala-Gly-Tyr Pro-Arg-Val-Phe, Val-Val-Phe-Asp, Gly-Asp-Ala-Pro-Asn, Gly-Glu-Leu-Phe-Glu, Ile-Val-Phe-Asp-Ala, Pro-Ala -Gly-Tyr-Leu, Pro-Arg-Val-Phe-Leu, Va -Gln-Val-Val-Phe, mention may be made of the Val-Thr-Val-Pro-Gln and the like. Of these, dipeptide Ile-Tyr composed of isoleucine and tyrosine is particularly preferable because it has an extremely high ACE inhibitory activity and an antihypertensive effect.

  The food legumes of the present invention contain the dipeptide Ile-Tyr, and the antihypertensive peptide produced by the enzymatic action is equal to or more than the antihypertensive peptide originally contained in the beans, that is, the antihypertensive peptide originally contained in the beans. The total content of the hypertensive peptide and the antihypertensive peptide produced by the enzyme action is more than twice that of the antihypertensive peptide originally contained in beans. Specifically, the total content of antihypertensive peptides can be 0.001 to 100 mg as the antihypertensive peptide content in 100 g of beans, and an excellent antihypertensive effect can be obtained. ~ 100 mg is preferred.

  As the content of the antihypertensive peptide in the beans, a value measured by high-speed chromatography can be adopted.

  In addition to these antihypertensive peptides, peptides obtained by the action of the above peptide bond hydrolase include peptides having secondary anti-cholesterol action, antioxidant, anti-stress action, etc. In the food beans of the present invention, anticholesterol action, antioxidant and antistress action are also obtained.

  The food beans of the present invention preferably contain one or more selected from sodium chloride, sodium salt of amino acid, phosphatidic acid, lysophosphatidic acid, and exopeptidase. These substances can suppress the production of bitter components due to the action of the peptide bond hydrolase. These can be used alone or in combination of two or more. These are preferably introduced into the beans at the same time when the peptide bond hydrolase is introduced into the beans. These substances are preferably introduced in an amount of 0.05% by mass or more, and preferably 100% by mass or less, with respect to the peptide bond hydrolase introduced into the beans.

  Examples of the method for producing food beans include a seed coat damage process for damaging the seed coat of beans, an enzyme contact process for contacting a peptide bond hydrolase with beans damaged in seed coat, and a peptide bond hydrolase by depressurization or pressure treatment. Enzyme introduction step to be introduced inside, enzyme reaction step to break the peptide bond until the content of the peptide composed of 2 to 20 amino acids is doubled by the peptide bond hydrolase introduced inside, heating the enzyme And a method including an enzyme deactivation step for deactivation.

  The seed coat damage process that damages the seed coat of beans is performed to form fine scratches on the seed coat of beans to the extent that the shape of the beans is not impaired, and to facilitate the introduction of peptide-bond hydrolase into the beans. It is a process. As a method of forming fine scratches on the seed coat of beans, for example, a method of heating at 100 ° C. or higher is preferable, and a temperature of 120 ° C. or higher is more preferable. By such heating, the seed coat of beans can be uniformly damaged, and the enzyme can be uniformly introduced into the inside. The heating time can be appropriately selected in relation to the type of beans and the temperature. For example, in the case of soybeans, it can be 1 to 60 minutes at 100 ° C. or higher, 20 minutes at 120 ° C. or higher, and the like. It is preferable to adjust the heating temperature and time depending on the thickness of the seed coat and flexibility. Such heating can inactivate the enzymes inherent in the beans, facilitate control of the enzyme reaction in the subsequent process, and prevent discoloration. When the beans are in a dry state or have a low water content, heating in steam or water is preferred.

  In the seed coat damage process, it is preferable to perform a surface drying treatment for 10 minutes to 3 hours after the heat treatment or in place of the heat treatment under a reduced pressure of 100 Pa or less. By the surface drying treatment, it is possible to form a wound on the seed coat that maintains the shape of the beans and can effectively replace the air inside and the peptide bond hydrolase. The surface drying treatment is preferably performed at a temperature of -1 to -40 ° C.

  In the seed coat damaging step, a method of damaging the seed coat by a mechanical action such as stirring beans and causing them to collide with each other can also be adopted.

  In the seed coat damage process, it is preferable to perform a freeze / thaw process in which beans are frozen and thawed as a pre-process or post-process of the heat treatment at 100 ° C. or higher for 1 minute or longer. By performing the beans freezing and thawing step together with the superheat treatment step, it is possible to effectively form a wound on the seed coat of the beans.

  It is preferable that the seed coat damage process described above is a treatment process that is obtained through the subsequent processes and that does not greatly impair the shape and appearance of the beans having peptides generated therein.

  The freezing process for beans may be any method as long as it can freeze water contained therein. Examples of the freezing temperature include -1 to -40 ° C. Furthermore, it is preferable to dry the frozen beans because the enzyme is easily introduced into the beans having a hard seed coat. Examples of the method for drying frozen beans include steam heating of beans. These treatments can be performed using a freeze-drying apparatus, and specifically include surface drying for 10 minutes to 3 hours.

  A method for thawing frozen beans may be left at a temperature of 0 ° C. or higher, for example, 0 to 50 ° C., and slow thawing at a low temperature of 4 to 10 ° C. in a refrigerator is preferable. Thawing is preferably performed immediately before use, since frozen beans can be stored with quality preserved. It can also be thawed using running water or a microwave oven.

  The enzyme contact step of contacting a bean with damaged seed coat with a peptide bond hydrolase is a step of bringing a peptide bond hydrolase introduced into the bean into contact with the surface of the bean first, and is a liquid or powdered peptide bond hydrolase. Is directly immersed in an enzyme solution prepared by dissolving or dispersing in water, seasoning liquid, buffer solution, alcohol, thickening polysaccharide or the like, or liquid enzyme. It may be a method used in the enzyme introduction step, a pick-up dipped in the enzyme solution, or a method of applying and spraying the enzyme solution. In the case of a powder enzyme, a method in which the powder enzyme is directly sprinkled or sprayed on beans can also be used.

  The amount of enzyme to be used can be selected as appropriate. When a liquid enzyme is used directly, for example, a range of 0.001 to 1.0 g can be given with respect to 100 g of beans. The enzyme solution can be prepared, for example, by dissolving or dispersing the enzyme in the range of 0.01 to 3.0% by mass with respect to the solvent solution. The pH of the enzyme solution is preferably 4-10. For the pH adjustment, pH adjusting agents such as organic acids and their salts and phosphates can be used, and a pH-adjusted seasoning liquid or the like can also be used.

  In the enzyme contact step, a bitter component inhibitor comprising any one or more selected from sodium chloride, amino acid sodium salt, phosphatidic acid, lysophosphatidic acid, and exopeptidase, in order to suppress the formation of a bitter component of the bitter component. Is preferably brought into contact with beans. These bitter component inhibitors can be contained in the enzyme solution or sprinkled with the enzyme. The amount of the bitter component inhibitor used is preferably 0.05% by mass or more, and preferably 100% by mass or less, based on the peptide bond hydrolase.

  In the enzyme introduction step, the peptide-bond hydrolase is introduced into the beans by depressurizing or pressurizing the beans contacted with the enzyme in the enzyme contact step. For the depressurization treatment, an apparatus having a pressure-resistant sealed container and a vacuum pump can be used. Beans immersed in an enzyme solution or beans having an enzyme attached to the surface are placed in a pressure-resistant sealed container and left under reduced pressure. The decompression is preferably performed under 0.01 MPa or less or a pressure at which moisture contained in the beans boils. Under such a reduced pressure, the beans expand about 1.01-1.10 times the volume under normal pressure, and the air present inside the beans and the peptide bond hydrolase present on the surface of the beans are in the seed coat. It is easily replaced through the formed damaged part, and the enzyme can be uniformly introduced into the beans. The time of leaving under reduced pressure is preferably selected according to the situation such as the type and size of the beans so as to avoid the deterioration of the quality of the beans, and can be, for example, about 30 seconds to 30 minutes. Release of the reduced pressure holding state can be performed by returning to the normal pressure. The decompression treatment can be performed at room temperature, and can be performed at a low temperature of about 10 ° C. so as not to impair the quality.

  The pressure treatment in the enzyme introduction step uses a pressure-resistant sealed container and a device having a gas supply device, etc., and the beans immersed in the enzyme solution or the beans with the enzyme attached to the surface are placed in the pressure-resistant sealed container. It can be carried out by a method such as supplying a gas until it is under a desired pressure and leaving it for a predetermined time. The pressurization is preferably performed at 50 to 400 MPa. Under such pressure, the beans contract about 0.90 to 0.99 times the volume under normal pressure, and the air present inside the beans and the peptide bond hydrolase present on the surface of the beans are in the seed coat. The enzyme can be uniformly introduced into the beans by being replaced through the damaged portion formed. The time for leaving under pressure is preferably selected according to the situation, such as the type and size of the beans. The pressure treatment can be performed at room temperature, and can be performed at a low temperature of about 10 ° C. so as not to deteriorate the quality. A flexible plastic film retort packaging bag can also be used as a pressure-resistant sealed container, and the packaging bag can be decompressed by suction, heat sealed, and the film can be in close contact with beans to form a pressurized state. is there.

  The enzyme reaction step of degrading peptide bonds by the peptide bond hydrolase introduced inside by the enzyme introduction step can be performed in an immersed state when it is immersed in an enzyme solution and subjected to reduced pressure or pressure treatment, It is preferable to take out from the enzyme solution because the enzyme reaction between the surface and the inside can proceed uniformly and the shape of the beans before the enzyme introduction step can be maintained. Even when the optimum temperature of the peptide bond hydrolase exceeds 30 ° C., the enzyme reaction is performed at 30 ° C. to suppress the deterioration of beans when the enzyme reaction is performed for a long time, for example, more than 4 hours. The following is preferable, and it is more preferable to carry out at a low temperature of 10 ° C. or lower. In addition, the enzyme reaction is performed until the content of the antihypertensive peptide is more than twice the content of the antihypertensive peptide contained in the beans before the enzyme introduction step. In the enzyme reaction, it is preferable to select the treatment time from the kind and size of beans, the suppression of the production of bitter components, and in the case of soybean, for example, about 1 to 24 hours can be mentioned.

  The enzyme deactivation step is a step of suppressing an excessive enzyme reaction in order to stop the enzyme reaction and maintain the shape of the beans before the introduction of the enzyme. Enzyme deactivation is preferably by heating at 65 ° C. or higher, more preferably at 100 ° C. for 1 minute or longer or equivalent heating. When the retort packaging bag is used, it can be distributed at room temperature as it is by heating at 120 ° C. for 5 to 60 minutes.

  The beans for food obtained by the above method retain the shape of the beans before production, have little bitterness, and can be cooked in the same manner as ordinary beans.

  Further, the food of the present invention is obtained by drying or freezing the above-mentioned food beans, and used as a food processing material and applied to various processed foods such as sugar beet, retort food, frozen food, vacuum cooked food, canned food, etc. The foodstuff which can be taken and can take an antihypertensive peptide as a meal can be provided.

Hereinafter, although the Example for food beans of this invention is demonstrated in detail by an Example, the technical scope of this invention is not limited to these Examples. Moreover, in the following examples, in order to produce more ACE inhibitory active peptides, a heating step at 100 ° C., a freezing step, and a drying step are combined in the seed coat damage step, but at 100 ° C. for 1 minute or longer. It has been confirmed that a peptide having an ACE inhibitory activity is produced even when the seed coat damage process of only heating is carried out, and an amount of ACE that causes an antihypertensive action by increasing the amount of intake per day of the soybean is increased. It is possible to take active peptides.
[Example 1]
The dried soybeans were soaked in water for 16 hours or longer and absorbed until the soybean mass became about 2.2 times and stabilized. The absorbed soybean was heated in steam or hot water at a rate of 4 ° C. per minute, and heated for 1 hour after reaching 100 ° C. Immediately after the heating was completed, cold water was added for cooling, frozen in a freezer at −30 ° C., freeze-dried for 3 hours, and stored again frozen. Frozen soybeans were immersed in a proteolytic enzyme solution (Protease N “Amano” G, Amano Enzyme) adjusted to 2.0% by mass using a phosphate buffer (pH 7.0) at 50 ° C. Thawed for minutes. While immersed in the enzyme solution, the pressure was reduced to 0.01 MPa, and the reduced pressure state was maintained for 5 minutes. The soybean which had been subjected to the decompression operation was taken out from the enzyme solution, subjected to an enzyme reaction at 50 ° C. for 4 hours and then steamed for 10 minutes to deactivate the enzyme to obtain food-grade soybean. The obtained soybean for food was frozen at −30 ° C. and then dried for 48 hours at a temperature of vacuum of 10 Pa or less and 30 ° C. heating. The mass after drying was 35% with respect to the mass before drying. The dried soybeans were pulverized using an electric mill to obtain soybean powder, vacuum-packed, and stored in a −30 ° C. freezer.

[ACE inhibitory activity]
A 20-fold amount of purified water was added to the produced soybean flour, and the mixture was stirred and extracted for 1 hour to obtain a sample solution for measuring ACE inhibitory activity. 50 mM potassium phosphate buffer-300 mM NaCl (pH 8.5) 200 μl, 50 mM substrate solution (Hippuril-Histidyl-Leucine, manufactured by Nacalai Tesque) 25 μl, sample solution 5 μl, 37.5 mU / ml angiotensin converting enzyme solution (ACE, Sigma) 20 μl) was mixed and reacted at 37 ° C. for 1 hour, and then 250 μl of 1N hydrochloric acid was added and mixed to stop the enzyme reaction. To this solution was added 1.5 ml of ethyl acetate and the mixture was stirred well and allowed to stand. After extracting 1 ml of ethyl acetate and evaporating to dryness, purified water was added and the absorbance (As) at 233 nm was measured. Absorbance (Ac) in which distilled water was added to the substrate solution and enzyme solution instead of the sample solution, Absorbance (Ab) added to the substrate solution in which the sample solution and enzyme solution were changed to 30 μm distilled water, distilled water instead of the enzyme solution Absorbance (Asb) obtained by adding 20 μl to the sample solution and the substrate solution was measured, and the ACE inhibition rate (%) was determined.
ACE inhibition rate (%) = ((Ac−Ab) − (As−Asb)) / (Ac−Ab) × 100
[Component Analysis 1]
To the produced soybean powder, 10 times the amount of purified water was added and stirred for 1 hour to extract the protein degradation product in water, followed by centrifugation at 6000 rpm, 4 ° C. for 15 minutes to obtain a supernatant. Hexane was added to the supernatant and stirred vigorously, and after centrifuging at 6000 rpm, 4 ° C. for 15 minutes, the aqueous layer was extracted and hexane was added again several times to remove oils and fats. The aqueous layer was extracted, and a protein degradation product was obtained by drying under reduced pressure. The protein degradation product was dissolved in purified water to 5 mg / 1 ml, and a sample solution was prepared by 0.45 μm cellulose acetate filter treatment. 100 μl of the sample solution was fractionated by high performance liquid chromatography (HPLC). COSMOSIL 5C18-MS-II (250 mm × 4.6 mm ID, manufactured by Nacalai Tesque) was used as the column, and (A solution) as the mobile phase: acetonitrile / water = 10/90 (v / v), (Liquid B): Using acetonitrile / water = 70/30 (v / v), the concentration of liquid B in liquid A was 0% to 0 to 1 minute, 0 to 50% to 1 to 45 minutes, 45 to 50 minutes. The flow rate was 0.5 ml / min and the detection wavelength was 220 nm while applying a concentration gradient of 50 to 100%, 50 to 55 minutes, 100%, and 55 to 60 minutes, 100 to 0%. Fractionation was performed for each peak, and ACE inhibitory activity was measured. Strong activity was observed in the fraction with an elution time of 15.0 minutes.

  The fractionation operation was repeated for this fraction, and the obtained fraction was concentrated under reduced pressure, and dissolved in purified water to give a sample of 40 cycle fraction / 1 ml to prepare a sample. Fractionation was performed again by HPLC under different conditions. COSMOSIL 5C18-AR-II (250 mm × 4.6 mm ID, manufactured by Nacalai Tesque) was used as a column, and (A solution) as a mobile phase: acetonitrile / water / trifluoroacetic acid = 5/95/0. 0.1 (v / v), (Liquid B): acetonitrile / water / trifluoroacetic acid = 35/65 / 0.1 (v / v), and the concentration of liquid B in liquid A was 0 until 0 to 45 minutes. It was carried out at a flow rate of 0.5 ml / min and a detection wavelength of 220 nm while applying a concentration gradient of -100%, 100% up to 45-50 minutes, and 100-0% up to 50-55 minutes. When fractionation was performed for each peak and ACE inhibitory activity was measured, strong activity was observed in the fraction with an elution time of 46.1 minutes. The chromatogram is shown in FIG.

  Fractionation of this fraction was repeated again, and the obtained fraction was concentrated under reduced pressure and dissolved in purified water to give a sample of 10 cycle fraction / 100 μl to prepare a sample. This sample solution was analyzed by a high performance liquid chromatograph / tandem quadrupole mass spectrometer (LC / MS / MS, Quattro micro API (manufactured by Waters)). COSMOSIL 5C18-AR-300 (250 mm × 4.6 mm ID, manufactured by Nacalai Tesque) was used as a column, and (A solution) as a mobile phase: acetonitrile / water / trifluoroacetic acid = 5/95/0. 0.1 (v / v), (Liquid B): acetonitrile / water / trifluoroacetic acid = 35/65 / 0.1 (v / v), and the concentration of liquid B in liquid A is 0 to 0 to 30 minutes. While applying a concentration gradient of -30%, 30-100% for 30-40 minutes, 100% for 40-60 minutes, 100-0% for 60-61 minutes, flow rate 0.5 ml / min, detection wavelength 220 nm, MS Was performed by setting Ion Mode: Electrospray +, Scan Type: MS Scan, and MS Range: 100-1000. The chromatogram is shown in FIG.

  Daugher Ion Scan analysis was performed on the peak observed around 28 minutes. The analysis result is shown in FIG. From the Leu-Tyr pattern shown in FIG. 2 (a) and the Ile-Tyr pattern shown in FIG. 2 (b), it is inferred that the sample having the pattern of FIG. 2 (c) is Ile-Tyr or Leu-Tyr. It was done. Analysis for Ile-Tyr and Leu-Tyr under the same conditions as in LC / MS / MS analysis revealed that this component was Ile-Tyr from the elution time and MS / MS pattern. The analysis results are shown in FIG. (A) is a chromatogram of a mixture of Ile-Tyr and Leu-Tyr, (b) is an MS chromatogram of a mixture of Ile-Tyr and Leu-Tyr, (c) is a chromatogram of a sample solution, (d ) Shows the MS chromatogram of the sample solution.

[Component Analysis 2]
To the produced soybean powder, 10 times the amount of purified water was added and stirred for 1 hour to extract the protein degradation product in water, followed by centrifugation at 6000 rpm, 4 ° C. for 15 minutes to obtain a supernatant. The sample solution which removed the fats and oils and was treated with the 0.45 μm cellulose acetate filter was analyzed by LC / MS / MS, and the amount of Ile-Tyr in the soybean ground was measured. COSMOSIL 5C18-AR-300 (250 mm × 4.6 mm ID, manufactured by Nacalai Tesque) was used as a column, and (A solution) as a mobile phase: acetonitrile / water / trifluoroacetic acid = 5/95/0. 0.1 (v / v), (Liquid B): acetonitrile / water / trifluoroacetic acid = 35/65 / 0.1 (v / v), and the concentration of liquid B in liquid A is 0 to 0 to 30 minutes. While applying a concentration gradient of -30%, 30-100% for 30-40 minutes, 100% for 40-60 minutes, 100-0% for 60-61 minutes, flow rate 0.5 ml / min, detection wavelength 220 nm, MS Is determined by setting Ion Mode: Electrospray +, Scan Type: MRM, Parent Ion: m / z = 295.2, Daughter Ion: m / z = 182.2. It was. A calibration curve was prepared using a standard solution of Ile-Tyr of 0.010 to 0.100 mg / ml.

  The amount of Ile-Tyr in the sample solution was 0.031 mg / ml. Therefore, the amounts of Ile-Tyr in the soybean powder and the steamed soybean after the enzyme reaction were 0.31 mg / soybean powder 1 g and 0.11 mg / soybean 1 g, respectively.

[Animal test]
Seven weeks old male spontaneously hypertensive rats (SHR / Izm, Shimizu experimental material) were preliminarily kept for one week, blood pressure was measured for one week, 12 were divided into 3 groups of 4 animals, and 9 weeks old The following feeds were administered for 45 days from During the test period, the food was freely consumed, but the amount of food consumed was almost the same between the groups.
Group 1 (Comparative Example 1): Feed intake group containing no soy flour Group 2 soy flour mixed feed (Comparative Example 2): Daily intake of soy flour without enzyme reaction is 5 g / kg body weight Group 3 enzyme-treated soy flour mixed feed added so as to be: 2 times from the start of the feed intake group test added so that the daily intake of the prepared soy flour was 5 g / kg body weight The systolic blood pressure was measured using a non-invasive sphygmomanometer Softron BP-98A at a frequency of / week. The results are shown in FIG. Statistical processing was performed by Dunnet's multiple comparison test.

  From the results, it is clear that soybean powder containing Ile-Tyr has an action of significantly suppressing an increase in blood pressure.

  The food beans of the present invention have ACE inhibitory activity, and the peptide effectively inhibits ACE when ingested in a small amount and is free from side effects, and can be easily taken orally by hypertensives in daily life. In addition, by proposing a method for producing a composition containing the peptide, excellent in flavor, high in safety, and easy to ingest as a food, it can greatly contribute to the improvement of the quality of life of hypertensives. Become.

[Example 2]
As the proteolytic enzyme used in the proteolytic enzyme solution, in place of protease N “Amano” G, the proteolytic enzyme and buffer shown in Table 1 were used, and the enzyme reaction temperature was changed and the same as in Example 1. Food soy was prepared and ACE inhibitory activity was determined. The results are shown in Table 1.

Claims (11)

  It contains a peptide containing Ile-Tyr produced by the action of a peptide bond hydrolase introduced inside, and the total content of peptides composed of 2 to 20 amino acids is before the peptide bond hydrolase introduction. A food legume characterized by being at least twice the content of a peptide and retaining the shape prior to introduction of a peptide bond hydrolase.   2. The food legume according to claim 1, wherein the total content of the peptide composed of 2 to 20 amino acids is 0.001 mg to 100 mg in 100 g of the food legume.   The food legume according to claim 1 or 2, comprising any one or more selected from sodium chloride, amino acid sodium salt, phosphatidic acid, lysophosphatidic acid, and exopeptidase.   4. The food beans according to claim 1, wherein the food beans are soybeans or water-absorbed soybeans.   It is a manufacturing method of the beans for foodstuffs in any one of Claim 1 to 4, Comprising: The seed coat damage process which damages the seed coat of beans, The enzyme contact process which makes a peptide bond hydrolase contact the beans which damaged the seed coat, pressure reduction Alternatively, the content of peptides composed of 2 to 20 amino acids is more than doubled by the enzyme introduction step of introducing peptide bond hydrolase into the interior by pressure treatment, and the peptide bond hydrolase introduced inside. A method for producing edible beans, comprising: an enzyme reaction step for degrading peptide bonds; and an enzyme deactivation step for deactivating peptide bond hydrolases by heating.   6. The method for producing food beans according to claim 5, wherein the seed coat damaging step includes a step of heat treatment at 100 ° C. or more for 1 minute or more.   The method for producing legumes for food according to claim 5 or 6, wherein the seed coat damage step comprises a surface drying treatment for 10 minutes to 3 hours under a reduced pressure of 100 Pa or less.   The method for producing beans for food according to any one of claims 5 to 7, wherein the seed coat damage step comprises a freezing and thawing step for freezing and thawing the beans.   The method for producing food beans according to any one of claims 5 to 8, wherein the enzyme introduction step is performed at 0.01 MPa or less or 50 MPa or more.   In the enzyme contact step, together with the peptide bond hydrolase, it is brought into contact with any one or more selected from salt, sodium salt of amino acid, phosphatidic acid, lysophosphatidic acid, and exopeptidase. 10. One or more selected from sodium chloride, amino acid sodium salt, phosphatidic acid, lysophosphatidic acid, and exopeptidase are introduced into the inside together with the binding hydrolase. The manufacturing method of the beans for foodstuffs in any one.   A food product obtained by drying or freezing the beans for food according to any one of claims 1 to 4.

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