JP4686792B1 - Angiotensin I converting enzyme inhibitor from wheat bran, barley meal, rice bran - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an angiotensin I converting enzyme from wheat bran, barley koji, and rice bran without adding a protease or amylase, without adding a harmful substance to the human body, and economically and efficiently. An object is to provide an active ingredient having an inhibitory activity and a blood pressure lowering function.
The present invention provides at least one powder raw material selected from the group consisting of wheat seed powder containing at least wheat bran, barley seed powder containing at least barley meal, and rice seed powder containing at least rice bran. Added to and mixed with water or buffer, pH of the water or buffer after addition is adjusted to 3.05 to 3.95, and stored at a temperature of 30 to 45 ° C. by the action of endogenous protease for at least 4 hours. An angiotensin I-converting enzyme inhibitor characterized by comprising decomposing a protein; and containing the obtained reaction product as an active ingredient; and an antihypertensive action comprising the angiotensin I-converting enzyme inhibitor The pharmaceutical which has this is provided.
[Selection] Figure 1

Description

  TECHNICAL FIELD The present invention relates to a novel antihypertensive peptide from wheat bran, barley koji, and rice bran and a method for producing the same, and more particularly, a novel polypeptide having an inhibitory activity on angiotensin I converting enzyme involved in increasing blood pressure and also having a hypotensive function. And its manufacturing method.

The processing of food processing waste is a major problem for the food industry. In order to require the reuse of useful waste, the “Law Concerning Promotion of Reuse of Food Recycling Resources (Food Recycling Law)” was enacted (promulgated in June 2000 and enforced in May 2001).
In response to this, each food manufacturer has started production of commodities such as methane gas, liquid fertilizer, cosmetics, food additives, and functional foods from food waste.
Wheat bran and rice bran are by-products of primary processing, with bran producing about 1.3 million tons per year and rice bran producing 800 to 900,000 tons.
Since these are only used for livestock feed, rice oil, mushroom nurseries, etc., and many of them are discarded, there is a need for the development of effective usage.
Recently, the value of whole grain (whole grain) has been reviewed, and the whole grain including bran and rice bran, which was previously the subject of disposal, has attracted attention as food. In the background, there is an expectation for the preventive effect of lifestyle-related diseases by ingesting functional factors contained in germ and seed coat.

One of the typical lifestyle-related diseases is hypertension. Hypertension is known to cause various complications such as cerebral hemorrhage, subarachnoid hemorrhage, cerebral infarction, myocardial infarction, angina pectoris and nephrosclerosis.
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 related to pressurization, angiotensinogen secreted from the liver is converted into angiotensin I by renin produced in the kidney, and further converted into angiotensin II by “angiotensin I converting enzyme”. Angiotensin II contracts blood vessels and increases blood pressure. In the kallikrein-kinin system related to hypotension, bradykinin produced by the action of kallikrein on kininogen relaxes blood vessels and lowers blood pressure. “Angiotensin I converting enzyme” also has an activity of degrading bradykinin.
Therefore, by suppressing or inhibiting the enzyme activity of the “angiotensin I converting enzyme”, it is possible to suppress an increase in blood pressure and further to decrease the blood pressure.

As a substance having an angiotensin I converting enzyme inhibitory activity, it is known that a polypeptide having a specific sequence exhibits an angiotensin I converting enzyme inhibitory activity. It is known that polypeptides having strong inhibitory activity of the enzyme can be separated and purified.
Examples of edible proteins capable of separating and purifying a polypeptide having an angiotensin I converting enzyme inhibitory activity include fish sardines (see, for example, Patent Document 1 and Non-Patent Document 1), and skipjack (for example, Patent Document 2 and Non-Patent Documents). Patent Documents 2 and 3) and tuna (for example, see Non-Patent Document 4), casein that is a milk protein (for example, see Patent Document 3 and Non-Patent Document 5) and lactoglobulin (for example, Patent Document 4 and Non-Patent Document) 6), and cereals such as corn protein (for example, see non-patent document 7), buckwheat whole grain (for example, refer to non-patent document 8), wheat germ (for example, refer to non-patent document 9), soy protein (for example, Non-patent document 10).
Among the peptides separated and purified from the above edible protein, lactopeptide, casein peptide, sardine peptide, and skipjack peptide are registered as foods for specific health use and sold as health foods.

However, the conventional method of adding a protease as described above requires the addition of 1% (w / v) or more of protease, which is a concentration sufficient to degrade edible proteins. In addition, in the case where cereals rich in starch are used as raw materials, addition of amylase (about 1% (w / v)) is also required in order to increase the reaction efficiency.
Since these enzyme agents are extremely expensive, they are a great obstacle in mass production and commercialization.
In addition, when producing from fish meat, there are a large amount of contaminants such as odor components, pigment components, and proteins, so the purification efficiency is poor, and a large amount of purified material is required to remove these contaminants, resulting in rapid deterioration. The manufacturing cost is also bad. In addition, the concentration of the sardine peptide (valyltyrosine (Val-Tyr)) marketed in the food for specified health use is about 1 mg / g protein, which is a purity that cannot be said to be high purity. In addition, sardines are hardly caught in the waters near Japan, and catches of anchovy and urchin eagle are decreasing and prices are rising. Consumption of fishery resources is increasing globally, and fishery resources such as sea bonito and tuna are soaring.
Furthermore, in any of the above methods, it is impossible to produce a polypeptide having a plurality of types of polypeptides having significantly high angiotensin I converting enzyme inhibitory activity.

  It is possible to synthesize a functional polypeptide by artificial synthesis of the polypeptide (see, for example, Non-Patent Document 11), but in addition to the cost of the synthesis process itself, a person can ingest chemicals used in the synthesis process. Since it is necessary to remove and purify to the extent, it is more difficult in terms of production amount and cost than the above methods.

JP 2006-56805 A JP 2001-112470 A WO95 / 28425 publication JP-A-11-98978

Agricultural and Biological Chemistry, 55, 2169-2170. (1991) Bioscience Biotechnology and Biochemistry, 56, 1541-1545. (1992) Bioscience Biotechnology and Biochemistry, 57, 695-697. (1993) Agricultural and Biological Chemistry, 55, 2169-2170. (1991) Agricultural and Biological Chemistry, 51, 1581-1586. (1987) FEBS letters, 402, 99-101. (1997) Agricultural and Biological Chemistry, 55, 1313-1318. (1991) Journal of Peptide Science, 8, 267-274. (2002) Journal of Peptide Science, 5, 289-297. (1999) Soy Protein Research, 6, 73-77. (2003) The Journal of Biological Chemistry, 25, 401-407. (1980)

An object of the present invention is to provide a novel polypeptide having an angiotensin I converting enzyme inhibitory activity.
Next, the present invention produces a polypeptide having an inhibitory activity on angiotensin I converting enzyme from wheat bran, barley koji, and rice koji without adding protease or amylase and without adding substances harmful to the human body. It is intended to provide a possible method.
Furthermore, an object of the present invention is to provide a method capable of economically and efficiently producing a polypeptide having an angiotensin I converting enzyme inhibitory activity from wheat bran, barley meal, and rice bran.

Another object of the present invention is to provide a novel polypeptide having a blood pressure lowering function.
Next, the present invention relates to a method capable of producing a polypeptide having a blood pressure lowering function from wheat bran, barley koji, and rice koji without adding protease or amylase and without adding substances harmful to the human body. It is intended to provide.
Furthermore, an object of the present invention is to provide a method capable of economically and efficiently producing a polypeptide having a blood pressure lowering function from wheat bran, barley meal, and rice bran.

As a result of intensive studies to solve the above-described conventional problems, the present inventor has previously used wheat bran, barley koji, and rice bran powder materials that do not contain a large amount of starch, water or a buffer solution. The mixture is added to and mixed, and the pH of water or buffer after the addition and mixing is adjusted to 3.05 to 3.95, and reacted at a temperature of 30 to 45 ° C. for at least 4 hours, whereby the wheat bran, barley koji, rice bran It was found that a polypeptide having an angiotensin I converting enzyme inhibitory activity was produced by the degradation of stored proteins by the action of endogenous protease in the powder raw material.
Furthermore, the present inventor has found that by the above method, polypeptides having inhibitory activity of seven types of angiotensin I converting enzyme derived from wheat bran, barley koji, and rice bran storage proteins can be obtained. It was found that a tripeptide consisting of the amino acid sequence of Ile-Gln-Pro, which is a polypeptide having angiotensin I converting enzyme inhibitory activity, exists.
Further, the present inventor has found that the polypeptide has a blood pressure lowering function.
The present invention has been completed based on such findings.

That is, the present invention according to claim 1 is at least one powder selected from the group consisting of wheat seed powder containing at least wheat bran, barley seed powder containing at least barley straw, and rice seed powder containing at least rice bran. The raw material is added to and mixed with water or a buffer solution, and the pH of the water or buffer solution after the addition and mixing is set to 3.05 to 3.95, and the endogenous protease is kept at a temperature of 30 to 45 ° C. for at least 4 hours or more. An object of the present invention is to provide an angiotensin I converting enzyme inhibitor characterized by comprising decomposing a storage protein by action; and containing the obtained reaction product as an active ingredient.
The present invention according to claim 2 is characterized in that the wheat seed powder is wheat bran powder not containing a starch storage part, the barley seed powder is barley straw powder not containing a starch storage part, and the rice seeds The angiotensin I converting enzyme inhibitor according to claim 1, wherein the powder is rice bran powder containing no starch reservoir.
This invention which concerns on Claim 3 provides the angiotensin I converting enzyme inhibitor of Claim 1 or 2 which degreases the said powder raw material using hexane before the said addition mixing.
In the present invention according to claim 4, the powder raw material is previously added to and mixed with a cleaning liquid composed of water or a buffer, and the pH of the cleaning liquid after the addition mixing is set to 3.0 to 7.0. The angiotensin I converting enzyme inhibitor according to any one of claims 1 to 3, wherein the supernatant is removed after immersion at a temperature for 5 to 30 minutes.
The present invention according to claim 5 is the angiotensin I converting enzyme inhibitor according to any one of claims 1 to 4, wherein the reaction solution obtained after the reaction is purified by reverse phase chromatography using an ODS column. Is to provide.
According to a sixth aspect of the present invention, in the purification process by reverse phase chromatography using the ODS column according to the fifth aspect, the reaction solution is passed through the ODS column and adsorbed, and then 10 to 25% ethanol or 10 The angiotensin I converting enzyme inhibitor according to claim 5, which is eluted and collected with ~ 25% acetonitrile.
The present invention according to claim 7 provides a medicine having a blood pressure lowering action, comprising the angiotensin I converting enzyme inhibitor according to claims 1 to 6.

According to the present invention, a novel polypeptide having an angiotensin I converting enzyme inhibitory activity is provided.
Next, according to the present invention, a polypeptide having inhibitory activity of angiotensin I converting enzyme from wheat bran, barley koji, and rice koji without adding protease or amylase and without adding substances harmful to the human body. Is provided.
Furthermore, according to the present invention, there is provided a method capable of economically and efficiently producing a plurality of types of polypeptides having an angiotensin I converting enzyme inhibitory activity from wheat bran, barley meal, and rice bran.

In addition, according to the present invention, a novel polypeptide having a blood pressure lowering function is provided.
Next, according to the present invention, a polypeptide having a blood pressure lowering function can be produced from wheat bran, barley koji, and rice koji without adding protease or amylase and without adding substances harmful to the human body. A method is provided.
Furthermore, according to the present invention, there is provided a method capable of economically and efficiently producing a plurality of types of polypeptides having a blood pressure lowering function from wheat bran, barley koji, and rice koji.

In Example 2, it is the figure which each showed the relationship between the pH value of an enzyme reaction liquid, and the inhibition rate of a refinement | purification product, and the relationship between the pH value of an enzyme reaction solution, and the yield of a refinement | purification product. In Example 3, it is the figure which each showed the relationship between the temperature of an enzyme reaction, and the inhibition rate of a crudely purified product, and the relationship between the temperature of an enzyme reaction and the yield of a crudely purified product. In Example 4, it is the figure which showed the relationship between the reaction time of an enzyme reaction, and the inhibition rate of a crudely purified product. In Example 8, it is the figure which showed the relationship between various protease inhibitors and the suppression rate of the inhibitory activity of angiotensin I converting enzyme. In Example 10, it is the figure which showed the peak of the fractions F-1 to F-11 isolate | separated by gel filtration HPLC. In Example 10, it is the figure which showed the peak of each component isolate | separated by the reverse phase HPLC from the fraction F-3. In Example 10, it is the figure which showed the peak of each component isolate | separated from the fraction F-7 by reverse phase HPLC. In Example 15, it is the figure which showed the influence which the single administration of an ODS column process rough refinement | purification has on the blood pressure of a spontaneous hypertensive rat. In Example 16, it is the figure which showed the influence which single administration of Ile-Gln-Pro exerts on the blood pressure of a spontaneous hypertensive rat. In Example 17, it is the figure which showed the influence which the long-term administration of an ODS column process rough refinement | purification has on the blood pressure of a spontaneous hypertensive rat.

Hereinafter, the present invention will be described in detail.
In the present invention, the “polypeptide having angiotensin I converting enzyme inhibitory activity” refers to a polypeptide having a function of inhibiting or suppressing the activity of angiotensin I converting enzyme.
The polypeptide suppresses or inhibits the enzyme activity of “angiotensin I converting enzyme” to suppress the conversion of angiotensin I to angiotensin II that increases blood pressure and the degradation of bradykinin that lowers blood pressure, thereby increasing blood pressure. Is a polypeptide that finally suppresses blood pressure and has a “function of lowering blood pressure”.

The polypeptide having an angiotensin I converting enzyme inhibitory activity and the polypeptide having a blood pressure lowering function in the present invention are a tripeptide and a dipeptide having a specific sequence, specifically, Ile-Gln-Pro, Leu-Gln. -A polypeptide comprising the amino acid sequences of Pro, Leu-Arg-Pro, Ile-Arg-Pro, Val-Tyr, Thr-Phe and Ile-Tyr.
Among the above, the tripeptide consisting of the amino acid sequence of Ile-Gln-Pro is a polypeptide having a novel angiotensin I converting enzyme inhibitory activity, which has not been reported so far, and a polypeptide having a blood pressure lowering function.

  A polypeptide other than the tripeptide consisting of the amino acid sequence of Ile-Gln-Pro, that is, a polypeptide consisting of the amino acid sequence of Leu-Gln-Pro and a polypeptide consisting of the amino acid sequence of Leu-Arg-Pro are derived from maize (for example, , Non-patent document 7), a polypeptide comprising the amino acid sequence of Ile-Arg-Pro is bonito (for example, see non-patent document 3), and a polypeptide comprising the amino acid sequence of Val-Tyr is sardine (for example, non-patent document). A polypeptide comprising the amino acid sequence of Thr-Phe is derived from wheat germ (see, for example, Non-patent Document 9) from soybean (for example, see Non-patent Document 10) and soybean (for example, see Non-patent Document 10). Peptides include sardines (see, for example, Non-Patent Document 1), skipjack ( Eg to non-patent reference 2) and wheat germ (e.g., from a non-Patent Document 9), is a polypeptide that has been reported, respectively.

In the polypeptide having angiotensin I converting enzyme inhibitory activity of the present invention, “having angiotensin I converting enzyme inhibitory activity” means that the IC ₅₀ value is 1.0 (mg / mL) or less, preferably 0.5 (mg / ML) refers to the following.
In the polypeptide having a blood pressure lowering function of the present invention, “having a blood pressure lowering function” means that the IC ₅₀ value is 1.0 (mg / mL) or less, preferably 0.5 (mg / mL) or less. Point to.
The value of IC _{50 in} the present invention is the value of the final concentration (mg / mL) of the sample when the activity of angiotensin I converting enzyme is inhibited by 50%. Show.
In addition, when the value of the IC ₅₀ is higher than 1 mg / mL, it is not preferable because the titer cannot be expected to increase even if the components are purified.

The inhibitory activity of angiotensin I converting enzyme was measured by the Liberman modified method (DW Cushmanand HS Cheung, Biochem. Pharmacol., 20, 1637-1648. (1971)), the method of Yoshimoto et al. 45-49. (2007)), etc., for example, according to the Liberman modified method.
In the Liberman modified method, Hip-His-Leu (HHL) is used as a pseudo-substrate, hippuric acid (N-benzoylglicine) is released from Hip-His-Leu by angiotensin I converting enzyme, and then the released hippuric acid is ethyl acetate. And the amount of liberated hippuric acid is measured by measuring the absorbance at 228 nm. In this reaction, if a sample whose inhibitory activity is to be measured is contained, if the sample has inhibitory activity, the amount of hippuric acid released is reduced and the measured absorbance at 228 nm is also reduced.
In this method, the angiotensin I converting enzyme inhibitory activity of each sample can be determined based on the measured change in absorbance at 228 nm. Specifically, it can be determined using the following equation (1).

In the formula (1), C is the absorbance at 228 nm of the control to which water was added, B is the absorbance at 228 nm of the blank measured by adding water and adding 0.5 M HCl in advance, and S is the absorbance at 228 nm of the sample. SB represents the absorbance at 228 nm of the sample blank measured by adding 0.5 M HCl in advance to the sample.
Here, the final sample concentration when the activity of the angiotensin I converting enzyme of the formula (1) is inhibited by 50% is the IC ₅₀ value.

The “angiotensin I converting enzyme inhibitor” and “blood pressure lowering agent” of the present invention contain a polypeptide having the specific sequence described above as an active ingredient.
Specifically, a novel tripeptide comprising the amino acid sequence of Ile-Gln-Pro as an active ingredient, a novel tripeptide comprising the amino acid sequence of Ile-Gln-Pro, Leu-Gln-Pro, Leu A polypeptide comprising one or more amino acid sequences selected from the group consisting of -Arg-Pro, Ile-Arg-Pro, Val-Tyr, Thr-Phe and Ile-Tyr as an active ingredient; is there.

The angiotensin I converting enzyme inhibitor and antihypertensive agent in the present invention have an IC ₅₀ value of 1.0 (mg / mL) or less, preferably 0.5 (mg / mL) or less.
Incidentally, if the value of the IC ₅₀ of angiotensin I converting enzyme inhibitor and antihypertensive agent is higher than 1 mg / mL, because the increase in the titers was purified it can not be expected, which is not preferable.

Specifically, the dose of the angiotensin I converting enzyme inhibitor and the blood pressure lowering agent in the present invention is an adult (adult adult) when the IC ₅₀ value is a crude product having an IC ₅₀ value of about 40 μg / ml to 0.5 mg / ml. The weight is 0.6 to 7.0 g, preferably 1.3 to 7.0 g per day (with a weight of 60 kg). Here, a dose of less than 0.6 g per day for an adult is not preferable because a sufficient blood pressure lowering function cannot be obtained.
In addition, when Ile-Gln-Pro is administered as a single peptide, an effect is expected at 90 to 900 mg.

The angiotensin I converting enzyme inhibitor and antihypertensive agent in the present invention can be administered either orally or parenterally.
Examples of parenteral administration include intravenous injection and rectal administration.
On the other hand, the forms of the angiotensin I converting enzyme inhibitor and the blood pressure lowering agent in the present invention for oral administration are not particularly limited, but other than powder, crushed granules, granules, and capsule filling forms. It can be used in the form of a solution dispersed in water or ethanol, or in the form of a tablet obtained by mixing with an excipient or the like.

Next, the production method of “polypeptide having inhibitory activity of angiotensin I converting enzyme” of the present invention will be described.
The method for producing an angiotensin I converting enzyme inhibitor in the present invention comprises at least one powder selected from the group consisting of wheat powder containing at least wheat bran, barley powder containing at least barley meal, and rice powder containing at least rice bran. The raw material is added to and mixed with water or a buffer solution, and the pH of the water or the buffer solution after the addition and mixing is set to 3.05 to 3.95, and the reaction is performed at a temperature of 30 to 45 ° C. for at least 4 hours. To do.
In the present invention, powder raw materials containing wheat bran, barley koji, and rice bran are added and mixed in water or a buffer solution, and the pH of the water or buffer solution after the addition and mixing is set in a predetermined range, and immersed for a predetermined time at a predetermined temperature. By doing so, the protein in the raw material is decomposed by the activity of endogenous aspartic protease and serine protease contained in a large amount in the raw material itself to produce a polypeptide having an angiotensin I converting enzyme inhibitory activity.
The polypeptide obtained by the production method is also a “polypeptide having a blood pressure lowering function”.

Here, wheat bran, barley meal, and rice bran are tissues, excluding wheat, barley, and rice seed starch storage, in detail, embryo, germ, hypocotyl, scutellum, cotyledon, root progenitor, seed coat , Refers to tissues such as the peel and glue layer.
The raw material that can be used in the present invention includes at least one powder selected from the group consisting of wheat powder containing at least wheat bran, barley powder containing at least barley straw, and rice powder containing at least rice bran. If it is. Therefore, in the present invention, wheat, barley, and rice seed whole powders can be used, but preferably wheat bran, barley koji, and rice bran that do not include a starch storage part are used.

  Moreover, in this invention, it can be used regardless of the generation | occurrence | production stage of the said wheat, barley, and the seed of rice. That is, those immature seeds, ungerminated seeds, germinated seeds and the like can be used. In particular, germinated seeds are suitable for use as a raw material of the present invention because they consume stored starch in the course of embryo development and the proportion of starch storage is low. For example, in the case of a germinated seed (malt) of barley, it is preferable to use a seed from 0 to 3 days after germination. In the case of germinated seeds of wheat, it is preferable to use the seeds of 0 to 3 days after germination.

When wheat bran, barley meal, and rice bran are used as the raw material of the present invention, there is an advantage that the tissue can be decomposed without adding amylase because these raw materials do not contain a starch storage part. . Furthermore, wheat bran, barley straw, and rice bran are conventionally objects of disposal and have high economic efficiency in terms of cost when mass production is assumed. It is also desirable in that it can reduce waste and reduce the environmental burden.
In addition to the wheat bran, barley meal, and rice bran, corn germ, buckwheat, rye, durum wheat, and the like can be used as the raw material used in the present invention.

As wheat bran used in the present invention, large bran, small bran and powder can be used, but it is desirable to use large bran and small bran.
In addition, examples of the type of wheat used to prepare wheat bran include Fusayaka, Norin 61, Nambu Wheat, Kitanokaori, Haruyaka, and the like. Specifically, Fusayaka, Norin 61 is used. Is desirable.
Next, barley koji used in the present invention refers to a portion of 20 to 40 koji of the weight of brown barley. Examples of barley used for adjusting barley straw include Nijo barley (Sky Golden, Nishino Chikara, Takaho Golden, Large HP19), Rokujo Barley (Intermediate Mother Farm 2, 9551, Mantenboshi). Specifically, it is desirable to use Sky Golden and Intermediate Mother Farm 2, 9551.
In addition, rice bran is a general term for brown rice, which is obtained by removing rice husk from rice, and is a generic name for germ, outer endosperm, and paste layer. As these, red cocoon, middle cocoon, and white cocoon can be used. Examples of the types of rice used to obtain the rice bran used in the present invention include Hinohikari, Koshihikari, Haiminori, Mame-mochi, Haruyo, LGC software, etc., specifically, Hinohikari, Koshihikari, Haiminori, Shunyo It is desirable to use LGC software.

When germinating barley seeds, that is, malt is used as a raw material used in the present invention, malt can be prepared by a usual method. For example, barley seeds, specifically Nijo barley seeds, are immersed in water at 15 to 20 ° C. for about 4 days to germinate. After germination, after drying at 35 to 45 ° C. for 10 to 12 hours, the temperature is raised to 55 to 65 ° C. in about 15 minutes and dried at that temperature for about 2 to 4 hours. Thereafter, the temperature is further raised to 75 to 85 ° C. in about 15 minutes, dried at that temperature for about 4 to 5 hours, and used as dried malt.
Moreover, when using the germinated seed of wheat as a raw material used in the present invention, it is prepared by immersing in water at 15 to 40 ° C. for about 3 days for germination, and then drying at 40 to 50 ° C. overnight after germination. Can do.

In the method for producing the polypeptide of the present invention, the raw material is used in powder form. The pulverization of the raw material in the present invention refers to crushing the raw material into powder, sand, and particles, and refers to those crushed to an average particle diameter of about 1 mm or less, preferably about 0.84 mm or less.
The raw material can be pulverized by any method known in the art, such as a Buehler test mill, a Brabender test mill, a solid rice mill, a small rice mill, a grinding rice mill, a brush rice mill, and a centrifugal mill. A crusher, a stone mortar or the like can be used. In addition, when the raw material which is not pulverized is used, since the efficiency of enzymatic decomposition and extraction efficiency in the following steps decrease, it is not preferable.

When preparing wheat bran, a Buehler test mill, a Brabender test mill, or the like can be used. In the pulverization (milling) step, about 20% of the total weight of the wheat seeds can be milled and recovered to obtain large bran. Specifically, the large bran is shaped like a thin skin that is fragmented, and has a large size of 400 μm or more, and the long side has a non-uniform grain size of about several millimeters. This large bran may be used as it is, but it is preferable to re-grind to a particle size of 100 to 400 μm using a screen [500 μm] with a centrifugal mill.
In the powdering (milling) step, about 10% of the total weight of wheat seeds can be milled and recovered from the remaining seeds after milling the large bean as small bran. . Furthermore, in the pulverization (milling) step, about 10% of the total weight of wheat seeds can be milled and recovered from the remaining seeds after milling the small bran as powder. it can. Specifically, the small bran is pulverized into a powder having a particle size of 120 to 500 μm and the powder is 20 to 100 μm.
Moreover, when preparing the whole wheat flour, a centrifugal mill, a pulverizer, a stone mill, or the like can be used. For example, when wheat seeds are crushed with a pulverizer and whole wheat flour is prepared, a whole wheat flour of 560 to 840 μm is obtained.
On the other hand, when pulverized by a centrifugal mill, whole grains having a particle size of 100 to 400 μm are obtained.

When barley koji is prepared, a solid kneader, a test kneader (parest), or the like can be used. In the powdering (grinding) step, about 40% of the total weight of the barley seeds is ground and recovered, and barley straw can be obtained. Specifically, barley straw having a particle size of 150 to 350 μm can be obtained. Moreover, when preparing the whole grain powder of a barley seed, a centrifugal mill, a grinder, etc. can be used. For example, when barley seeds are crushed with a pulverizer and barley whole grains are prepared, 560-840 μm whole grains are obtained.
On the other hand, when pulverized by a centrifugal mill, whole grains having a particle size of 100 to 400 μm are obtained.

When preparing rice bran, a small rice mill, a grinding rice mill, a brush rice mill or the like can be used. In the pulverization (grinding) step, about 10% of the total weight of the brown rice seed is ground and recovered, and rice bran can be obtained. Specifically, rice bran having a particle size of 150 to 350 μm can be obtained. Moreover, when preparing the whole grain flour of brown rice, a centrifugal mill, a grinder, etc. can be used. For example, when brown rice is crushed with a grinder and whole grain flour of brown rice is prepared, a whole grain flour of 560 to 840 μm is obtained.
On the other hand, when pulverized by a centrifugal mill, whole grains having a particle size of 100 to 400 μm are obtained.

  When preparing the malt powder, a centrifugal mill, a pulverizer, a stone mill or the like can be used.

  In the present invention, when a raw material containing a large amount of lipid, for example, wheat bran, rice bran, barley koji or the like is used, it is desirable to perform a degreasing treatment after the powdering step. When a raw material containing a large amount of lipid is used, the lipid tends to suppress the activity of the endogenous protease in the enzyme reaction step in which a polypeptide is produced by the following endogenous protease.

  The solvent that can be used for the degreasing treatment may be any non-polar solvent that is volatile, does not deactivate the protease, and is harmless to the human body. Examples thereof include hexane and acetone. Preferably, hexane is used.

In this degreasing treatment step, first, 5 to 40 parts by mass of the nonpolar solvent is added to the total mass of the powder raw material, and 10 to 40 ° C., preferably about 10 to 30 ° C., more preferably 20 The mixture is stirred for 0.5 to 4 hours, preferably about 1 hour at room temperature of about 0 ° C., and the lipid is eluted from the raw material.
Subsequently, the nonpolar solvent from which the lipid was eluted is separated and removed by filtration using a buchner funnel, filter paper, Miracloth, centrifugal filter, etc., preferably by suction filtration using a buchna funnel, and the resulting residue Let stand at room temperature of about 10-25 ° C. for 6-18 hours to dry. The drying treatment can also be performed by methods such as ventilation drying, nitrogen stream drying, and vacuum drying.
It is desirable that the non-polar solvent is completely volatilized in the defatted powder raw material obtained after drying.

Furthermore, in the present invention, it is desirable to remove soluble proteins and pigment components, which are contaminants contained in the powder raw material, by washing before performing the enzyme reaction step, which is the next step.
In this raw material cleaning treatment step, first, 10 to 50 parts by mass of water or buffer solution is added and mixed with respect to the total mass of the powder raw material, and then the pH value of the water or buffer solution after addition and mixing is set to 3. It is 0 to 7.0, preferably 3.2 to 6.9, and the temperature is about 0 to 28 ° C., preferably about 4 to 28 ° C., and is immersed for about 5 to 30 minutes, preferably about 10 minutes. Elute protein and dye components.
By passing through this step, for example, when wheat bran or barley koji is used as a raw material, an acidic or neutral solution other than gliadin (a kind of prolamin type protein) containing a peptide sequence having an angiotensin I converting enzyme inhibitory activity And soluble protein and dye components can be eluted and removed.
In addition, "immersion" in this process refers to standing, shaking and stirring after mixing a raw material and water or a buffer solution. Preferably, the washing efficiency can be further increased by shaking at 100 to 200 rpm.

In this step, if the pH value of the water or buffer after addition and mixing is lower than 3.0, the enzyme is denatured, which is not preferable. Moreover, when the pH value of the water or buffer solution after addition mixing is higher than 7.0, gliadin dissolves, which is not preferable.
Moreover, when the temperature of the water or buffer solution after addition mixing is lower than 0 degreeC, since the solubility of soluble protein falls, it is unpreferable. Moreover, when the temperature after addition mixing is higher than 30 degreeC, since an enzyme reaction advances and the degradation rate of gliadin increases, it is not preferable.
Furthermore, when the immersion time after addition and mixing is longer than 30 minutes, the amount of degradation of gliadin increases, which is not preferable. When the soaking time after addition and mixing is shorter than 5 minutes, the soluble protein is not sufficiently dissolved, which is not preferable.

As the cleaning liquid used in this step, any water or aqueous solution can be used as long as the pH after adding and mixing the raw materials is adjusted to the pH within the predetermined range. For example, soft water, deionized water Water, distilled water, or sodium phosphate aqueous solution, sodium citrate aqueous solution, sodium chloride aqueous solution, sodium bicarbonate aqueous solution, sodium sulfate aqueous solution, etc., preferably prepared using deionized water, distilled water, sodium citrate aqueous solution An aqueous solution can be used.
The washing solution is preferably a buffer solution, for example, citrate-sodium phosphate buffer solution, citrate-potassium phosphate buffer solution, sodium phosphate buffer solution, potassium phosphate buffer solution, Tris-HCl buffer. Solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, lactic acid-sodium lactate buffer solution, phosphate buffer, MES buffer, MOPS buffer, formic acid-potassium phosphate buffer, formic acid-phosphate Sodium buffer can be used. In particular, the use of a citrate-potassium phosphate buffer, a citrate-sodium phosphate buffer, a sodium phosphate buffer, and a potassium phosphate buffer in terms of ease of pH adjustment, buffer capacity, and economy Is preferred.
The concentration of the buffer solution is preferably 5 to 50 mM when citric acid-sodium phosphate or citrate-potassium phosphate is used, for example.

In addition, the bactericidal effect which prevents the microbial contamination in the enzyme reaction process which is the next process can be acquired by making the said washing | cleaning liquid contain the substance which has a bactericidal effect, and performing a washing | cleaning sterilization.
Examples of substances that can impart a bactericidal effect to the cleaning solution without impairing the cleaning treatment effect of this step include sodium hypochlorite (antiformin), hydrogen peroxide, sodium sulfite, and sodium hyposulfite. Specifically, it is preferable to use sodium hypochlorite.
Sodium hypochlorite is a substance generally used as a food disinfectant for vegetables, fruits, drinking water, production equipment and utensils. Since sodium hypochlorite is decomposed by heating after use and is easily removed by washing with water, the labeling is exempted as a processing aid. Currently, prohibited food is limited to black sesame.

When sodium hypochlorite is used as a substance that imparts a bactericidal effect to the cleaning liquid, it is desirable to contain an effective chlorine concentration of 100 to 600 ppm as the concentration of sodium hypochlorite contained in the cleaning liquid. If it is less than 100 ppm, a bactericidal effect cannot be expected. If it exceeds 600 ppm, it will cause oxidation of the sample, and there is a concern that the reaction yield will decrease.
Even when this cleaning treatment step is performed with sodium hypochlorite contained in the cleaning solution, suitable temperature conditions and time conditions are the same as the predetermined range, but the temperature of the cleaning treatment is 60. Since sodium hypochlorite decomposes at a high temperature of ℃ or higher, it is not particularly preferred for the bactericidal effect. Further, if the time for the washing treatment is less than 5 minutes, sterilization is insufficient, which is not preferable.

After eluting the protein and dye component soluble in the washing solution, the protein and dye soluble in an acidic or neutral solution are centrifuged at 2,000 to 10,000 × g for 10 to 20 minutes. The powder raw material from which the components have been eluted can be separated and recovered as a precipitate.
In addition, you may wash | clean precipitation by performing the process which adds water again to the obtained precipitation, and removes water by centrifugation once, Preferably twice.

  In the present invention, the cleaning process may be performed before the degreasing process.

In the present invention, after adding and mixing the powder raw material obtained above (including all powder raw materials subjected to pretreatment such as a degreasing treatment step and a raw material washing treatment step, the same applies hereinafter) to water or a buffer solution, addition mixing The pH of the subsequent water or buffer solution is set within a predetermined range and reacted at a predetermined temperature for a predetermined time, so that the activity of endogenous aspartic protease and serine protease contained in a large amount in the raw material itself can be increased in the raw material. To produce a polypeptide having an angiotensin I converting enzyme inhibitory activity.
In the enzyme reaction step, unlike the conventional technique, the enzyme reaction can be performed without adding any protease.

In this step (the present enzyme reaction step), first, the powder raw material obtained above is immersed in an enzyme reaction solution of 5 to 20 parts by mass, preferably about 10 parts by mass with respect to the mass of the raw material. Add and mix.
The enzyme reaction solution used in this step is water or a buffer solution. The raw material is added to and mixed with the water or buffer solution, and the pH value of the water or buffer solution after addition and mixing is set to 3.05 to 3.95, It is preferably 3.15 to 3.87, more preferably 3.15 to 3.77, and most preferably 3.15 to 3.26.
In addition, when the pH value of the water or the buffer solution after addition and mixing is lower than 3.05 or higher than 3.95, the inhibition rate is 65% when the maximum value of the inhibitory activity is 100 (inhibition rate). 43.9%), which is not preferable.

As the enzyme reaction solution, any aqueous solution can be used as long as the pH after adding and mixing the powder raw material is adjusted to the pH within the predetermined range. For example, phosphoric acid, citric acid, hydrochloric acid, An aqueous solution prepared using acetic acid, sulfuric acid, lactic acid, formic acid, etc., preferably phosphoric acid, citric acid, hydrochloric acid, acetic acid, sulfuric acid, formic acid can be used.
In addition, the enzyme reaction solution is preferably a buffer solution, such as citrate-potassium phosphate buffer solution, citrate-sodium phosphate buffer solution, citrate-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, Lactic acid-sodium lactate buffer solution, phosphate buffer solution, MES buffer solution, MOPS buffer solution, formic acid-potassium phosphate buffer solution, formic acid-sodium phosphate buffer solution and the like can be used. In particular, it is preferable to use a citrate-sodium phosphate buffer or a citrate-potassium phosphate buffer in terms of ease of pH adjustment and buffer capacity.
The concentration of the buffer used for the enzyme reaction solution is, for example, 0.02 to 0.15M, preferably 0.05 to 0.1M when citric acid-sodium phosphate or citrate-potassium phosphate is used. It is desirable to be.
The enzyme reaction solution may contain salts as long as the enzyme reaction in this step is not suppressed. Specifically, it is desirable that the concentration of sodium salt is 100 mM or less, preferably 50 mM or less.
The enzyme reaction solution can be prepared and used before adding and mixing the raw materials, but prepared by adding the pH adjusting component and the component having a buffering action directly to the water added and mixed with the raw materials. You can also

In this enzyme reaction step, a bactericidal effect for preventing microbial contamination in this step can be obtained by including a substance having a bactericidal effect in the enzyme reaction solution.
Examples of substances that can impart a bactericidal effect to the cleaning solution without impairing the effect of the enzyme reaction treatment in this step include sodium hypochlorite (antiformin), hydrogen peroxide, sodium sulfite, and sodium hyposulfite. Specifically, it is preferable to use sodium hypochlorite.
When sodium hypochlorite is used as a substance that imparts a bactericidal effect to the enzyme reaction solution, the concentration of sodium hypochlorite contained in the enzyme reaction solution may include an effective chlorine concentration of 100 to 600 ppm. desirable. If it is less than 100 ppm, a bactericidal effect cannot be expected. If it exceeds 600 ppm, it will cause oxidation of the sample, and there is a concern that the reaction yield will decrease.
In addition, when a substance having a bactericidal effect is added to the washing liquid in the washing treatment step, it is sufficient to prevent microbial contamination of the enzyme reaction liquid without adding a substance having a bactericidal effect to the enzyme reaction liquid in this step. Sterilizing effect can be obtained.

In this enzyme reaction step, the enzyme reaction of the endogenous protease is desirably performed at a temperature of 30 to 45 ° C, preferably 35 to 40 ° C, more preferably 37 ° C to 40 ° C.
In addition, when the temperature of an enzyme reaction is higher than 45 degreeC, since inhibitory activity comes to fall below 65% of the maximum value, it is unpreferable. Further, even when the temperature of the enzyme reaction is lower than 30 ° C., it is not preferable because the inhibitory activity becomes 65% lower than the maximum value.
In this enzyme reaction step, the enzyme reaction solution in which the raw materials are added and mixed and immersed is shaken or allowed to stand for at least 4 hours, preferably 8 hours, more preferably 12 hours, thereby allowing angiotensin I converting enzyme to stand. A polypeptide having an inhibitory activity can be produced.
In addition, Preferably, reaction efficiency can be improved more by shaking at 50-160 rpm.
The reaction solution after the enzyme reaction is immersed in boiling water for about 5 minutes to deactivate the enzyme, cooled to about 4 ° C. to room temperature, preferably about 4 ° C., and then 2,000 to 10,000 × g. The “enzyme reaction treatment liquid” can be separated from the residue and recovered by centrifugation for 10 to 20 minutes.

Next, in the present invention, the reaction solution (enzyme reaction treatment solution) obtained through the above steps is a coexisting protein, low molecular polysaccharide (oligosaccharide), polar lipid by reverse phase chromatography using an ODS column. The pigment component and ions can be removed and roughly purified. Ions coexisting in the enzyme reaction treatment liquid include sodium ions, potassium ions, sodium ions, magnesium ions, iron ions, and the like. Since these ions affect the measurement of the inhibitory activity of angiotensin I converting enzyme, it is not preferable to mix these ions as impurities.
Prior to purification by reverse phase chromatography using an ODS column, the pH of the enzyme reaction treatment solution is adjusted to 5.0 to 7.0, preferably about 7.0, and neutralized. Is desirable. For the neutralization treatment, an aqueous ammonia solution, a sodium hydroxide solution, potassium hydroxide, or the like can be used. Preferably, an aqueous ammonia solution of about 1 to 10 N can be used.
When the neutralization treatment is performed, centrifugation is performed at 5,000 to 10,000 × g for 20 to 30 minutes, and further, filtration, preferably suction filtration using a Buchna funnel is performed, and the residue is completely removed. It is desirable to do.

In addition, before purification by reverse-phase chromatography using an ODS column, the enzyme reaction treatment solution is passed through an activated carbon packed column, and the liquid that has passed through the column is collected to adsorb impurities such as pigment components to the activated carbon. Can be removed. It is not preferable that the dye component is adsorbed non-specifically on the resin for purification and mixed as a contaminant to reduce the separation ability.
In the activated carbon column treatment, pulverized, powdered, or columnar activated carbon such as coal-based, coconut shell-based, and sawdust-based for liquid layer adsorption can be used. Specifically, spherical activated carbons manufactured by Nacalai Tesque and Wako Pure Chemicals, Kuraray Coal (manufactured by Kuraray), and Samplif (manufactured by Santesque) can be used.

As the ODS column that can be used in the reverse phase chromatography step using the ODS column in the present invention, LiChroprep RP-18 (manufactured by Merck), YMC Gel ODS-AQ (manufactured by YMC) SepPak (manufactured by Millipore), YMC Dispo SPE (made by YMC) etc. can be used. Specifically, LiChroprep RP-18 (manufactured by Merck) or the like can be used.
A column of any scale filled with ODS resin can be created and scaled up as necessary. In addition, a plurality of sets of columns can be prepared and the purification operation can be performed simultaneously.

In the reverse phase chromatography step using the ODS column in the present invention, first, ethanol and then water are passed through to activate the ODS resin. Thereafter, the enzyme reaction treatment liquid (hereinafter including the case of the neutralized enzyme reaction treatment liquid and the enzyme reaction treatment liquid treated with the activated carbon column in this step) is applied to the ODS column and adsorbed. By this treatment, polypeptides, proteins, polar lipids, pigment components, and the like contained in the applied enzyme reaction treatment solution are adsorbed to the ODS resin within a range of the ODS resin adsorption allowable amount.
In this step, the enzyme reaction treatment solution can be applied in an amount corresponding to the volume of ODS resin packed in the column. Specifically, when a column packed with 100 to 200 mL of ODS resin is used, 2,000 to 4,000 mL of the enzyme reaction treatment solution can be applied.

Next, water is allowed to flow and the adsorption column is washed to remove non-adsorbed impurities that have not been adsorbed to the ODS resin. The water used here can be any water such as distilled water, tap water, deionized water or ultrapure water, but it is preferable to use distilled water.
After washing away non-adsorbed impurities, it can be recovered by elution in an elution solvent having a volume of 3 to 10 times, preferably about 5 times the volume of the ODS resin used. Examples of the elution solvent that can be used in this step include ethanol, acetonitrile, and methanol.
In this step, the extraction solvent is used in the form of an aqueous solution. Specifically, when ethanol is used, the aqueous solution contains about 5 to 30%, preferably about 10 to 25%, and most preferably about 10% ethanol. It is desirable that In addition, it is not preferable that the ethanol content is eluted with an aqueous solution of less than 5% or an aqueous solution of more than 30% because the inhibitory component does not elute or inactive components are mixed.
When acetonitrile is used in this step, about 5 to 30% and about 65 to 99.5%, preferably about 10 to 20% and about 70 to 99%, more preferably about 10 to 20%, Most preferably, it is an aqueous solution containing about 10% acetonitrile. In the high polar fraction having a low acetonitrile content and the low polar fraction having a high acetonitrile content, the high polar fraction has a higher inhibitory activity of angiotensin I converting enzyme, and the polypeptide having the inhibitory activity High content.
In addition, when eluted with an aqueous solution having an acetonitrile content of less than 5% or an aqueous solution of greater than 30% and less than 65%, the inhibitory component does not elute or inactive components are mixed, which is not preferable.

  In this step, when eluted with 5-30% ethanol and when eluted with 5-30% acetonitrile, the inhibitory activity of angiotensin I converting enzyme and the content of the polypeptide having the inhibitory activity are Although both are comparable and can be used for the eluate in this step, it is particularly desirable to use ethanol for reasons of safety to the human body and waste liquid treatment costs.

Through the above steps, the eluate containing a polypeptide having an angiotensin I converting enzyme inhibitory activity is recovered as an “ODS column-treated crude purified solution”.
The crude purified solution treated with ODS column is made dry powder of “crude purified product containing polypeptide having inhibitory activity of angiotensin I converting enzyme” by evaporating and drying ethanol, acetonitrile and methanol as solvents. Can do.
In addition, although this drying process can be obtained by methods, such as natural drying, freeze drying, spray drying, nitrogen stream drying, and reduced pressure drying, specifically, it is desirable to evaporate a solvent by freeze drying or reduced pressure drying. .

In addition, as a specific example up to the above step, 100 g of wheat bran was immersed in 1.0 L of the enzyme reaction solution to perform a protease reaction, and then the obtained enzyme reaction treatment solution was adsorbed on an ODS column. Show the case.
From the eluate eluted with 10% ethanol from the ODS column, 1.5 to 3.0 g and a crude purification containing a polypeptide having an inhibitory activity of angiotensin I converting enzyme having an IC ₅₀ value of 65 to 90 μg / mL A dry powder of the product is obtained. The crude product is white and soluble in water.
The eluate eluted from the ODS column with 25% ethanol contains 3.0 to 5.0 g of angiotensin I converting enzyme having an inhibitory activity of angiotensin I converting enzyme having an IC ₅₀ value of 90 to 110 μg / mL. A crude dry powder is obtained. The crude product is pale yellow and soluble in water.
In addition, after eluting the said ODS column with 10% ethanol, it can also elute with 25% ethanol. In that case, from the obtained eluate, a crude dry powder containing a polypeptide having an angiotensin I converting enzyme inhibitory activity of 1.2 to 1.8 g and an IC ₅₀ value of 280 to 360 μg / mL is obtained. can get. The crude product is pale yellow and soluble in water.

  The dry powder of the roughly purified product obtained in the above step is a purified product or an isolated and purified product having an increased content of a polypeptide having an angiotensin I converting enzyme inhibitory activity obtained through the following steps. Like the polypeptide, it can be used as a material for foods, beverages, and preparations. The above process is composed only of processes that can be mass-produced. In addition, it is not necessary to add an expensive protease, which is one of the technical features of the present invention. Has an effect.

In the present invention, a polypeptide having an angiotensin I converting enzyme inhibitory activity is further obtained by purifying the crude ODS column-treated crude solution or the solution obtained by dissolving the crude product in water by anion exchange chromatography. The content rate can be increased.
The column used for anion exchange chromatography that can be used in the present invention is a column packed with a strongly basic anion exchanger, such as AG MP-1 (manufactured by BIO-RAD), Amberlite IRA400J (manufactured by Organo). A column packed with a general anion exchange resin such as Amberjet 4002 (manufactured by Organo) can be used. Specifically, AG MP-1, Amberlite IRA400J, etc. can be used.
A column of any scale filled with an anion exchange carrier can be prepared and scaled up as necessary. In addition, a plurality of sets of columns can be prepared and the purification operation can be performed simultaneously.

In the anion exchange chromatography step in the present invention, first, an aqueous ammonia solution of 0.5 to 5N, preferably about 1N, is passed through and washed with about 5 times the amount of water of the resin to obtain an anion exchange resin. To the ammonia type. The ODS column-treated crude purified solution or a solution obtained by dissolving the crude purified product in water has a pH of 7.0 to 12.0, preferably 9.0 with an aqueous ammonia solution of 1 to 10 N, preferably about 1 N. It is desirable to adjust to the extent.
The ODS column-treated crude purified solution or the solution obtained by dissolving the crude purified product in water after adjusting the pH to the predetermined value with an aqueous ammonia solution is equilibrated with the aqueous ammonia solution, and then is subjected to about 5 times the amount of water passed through. It can be applied to a column packed with an ion exchange resin.

In this step, the ODS column-treated crude purified solution or a solution obtained by dissolving the crude purified product in water can be applied in an amount corresponding to the capacity of the anion exchange column. Specifically, when a column having a diameter of 20 to 40 mm and a length of 10 to 20 cm is used, 100 to 1000 mL of the crude purified solution can be applied.
Next, 0.05M ammonia is fed, and the anion exchange column is washed to collect the non-adsorbed fraction that has not been adsorbed on the anion exchange carrier. In this step, the polypeptide having an angiotensin I converting enzyme inhibitory activity is recovered as an anion exchange column non-adsorbed fraction. As conditions for liquid feeding, it is desirable that the flow rate be 2 to 10 mL / min, within several hours.
In this step, acidic proteins, acidic peptides and the like can be adsorbed and removed from the anion exchange carrier.

The “anion exchange column treatment solution” obtained in the above step can separate and purify a fraction containing a polypeptide having an angiotensin I converting enzyme inhibitory activity by gel filtration HPLC.
Superdex 75HR (manufactured by Amersham), Superdex Peptide HR (manufactured by Amersham), and Bio-Gel P-6 (manufactured by BIO-RAD) can be used as a column used for gel filtration HPLC that can be used in the present invention. For example, Superdex 75HR (manufactured by Amersham) can be used. Moreover, as an apparatus for performing gel filtration HPLC, LC-10AD system (made by Shimadzu) etc. can be used.
The column can be of any scale. In addition, a plurality of sets of columns can be prepared and the purification operation can be performed simultaneously.
In this gel filtration HPLC step, the column is first equilibrated with 20 to 40%, preferably about 30% acetonitrile containing 0.05 to 1.0%, preferably about 0.1% trifluoroacetic acid. It is desirable to keep it.
Further, instead of trifluoroacetic acid, 0.5 to 2.0%, preferably about 1.0% of acetic acid or formic acid can be used.

In this step, the anion exchange column treatment liquid can be applied in an amount corresponding to the capacity of the column used for gel filtration HPLC. Specifically, when a column having a diameter of 10 to 15 mm and a length of 30 to 40 cm is used, 0.5 to 1.5 mL can be applied.
Next, a 30% acetonitrile aqueous solution is passed under conditions of a flow rate of 1.0 to 2.0 mL / min for 30 to 40 minutes, and the absorbance, specifically, the absorbance peak at 220 nm is monitored and used for gel filtration HPLC. The components eluted from the column according to the molecular weight are fractionated and collected for each peak.
A fraction containing a polypeptide having an angiotensin I converting enzyme inhibitory activity can be obtained from the fraction collected. Specifically, a fraction containing the third (F-3) and seventh (F-7) peaks can be obtained as a fraction containing a polypeptide having angiotensin I converting enzyme inhibitory activity.

The “gel filtration HPLC fraction” obtained through the above steps is made into a dry powder of “a crudely purified product containing a polypeptide having an inhibitory activity of angiotensin I converting enzyme” by evaporating the solvent and drying. be able to.
In addition, although this drying process can be obtained by methods, such as natural drying, freeze drying, spray drying, nitrogen stream drying, and reduced pressure drying, specifically, it is desirable to evaporate a solvent by freeze drying or reduced pressure drying. .
The dry powder of the roughly purified product obtained in the above step can be used as a material for foods, beverages, and preparations in the same manner as the roughly purified product and the isolated and purified polypeptide obtained in the following steps. .

Further, as a specific example up to the above step, 100 g of wheat bran was immersed in 1.0 L of the enzyme reaction solution and subjected to protease reaction, and then the ODS column treated crude eluted from the ODS column with 10% ethanol. When the purified solution is used, a dry powder of a purified product containing a polypeptide having an angiotensin I converting enzyme inhibitory activity of 150 to 200 mg and IC ₅₀ value of 40 to ₅₀ μg / mL is obtained from fraction F-3. It is done. Further, from the fraction F-7, a dry powder of a purified product containing a polypeptide having an angiotensin I converting enzyme inhibitory activity of 70 to 100 mg and an IC ₅₀ value of 70 to 80 μg / mL is obtained.

Furthermore, in the present invention, the “gel filtration HPLC fraction” containing the polypeptide having the inhibitory activity of the angiotensin I converting enzyme obtained in the above step is converted into a reverse phase using a reverse phase system column such as butyl, phenyl, ODS. By treating with HPLC, it can be isolated and purified as a single polypeptide having the inhibitory activity.
As a reverse phase column that can be used in this step, a Jupiter 5 μC ₄ 300A column (Phenomenex), Cosmosil 5Ph-AR-300 (Nacalai Tesque), μRPC C18 ST (Amersham) can be used. For example, a Jupiter 5μ C ₄ 300A column (Phenomenex) can be used. Moreover, LC-10AD etc. can be used as an apparatus for performing reverse phase HPLC.
The column can be of any scale. In addition, a plurality of sets of columns can be prepared and the purification operation can be performed simultaneously. In the reverse-phase HPLC step using a reverse-phase column, which is this step, it is desirable to equilibrate the column first with a 30% acetonitrile aqueous solution and 0.1% trifluoroacetic acid.

In this step, the eluate of the gel filtration HPLC fraction can be applied in an amount corresponding to the capacity of the column used for reverse phase HPLC. Specifically, when a column having a diameter of 4.6 to 10 mm and a length of 15 to 20 cm is used, 0.05 to 0.20 mL can be applied.
Next, by passing the solvent used for equilibration and washing the column used for reverse-phase HPLC, the non-adsorbed fraction that had not been adsorbed on the column was washed, and then the flow rate was 1.0 to Acetonitrile-water is passed under a linear gradient condition at 2.0 mL / min for 30 to 50 minutes.
The eluate from the column can be isolated and purified as a single polypeptide by monitoring the absorbance, specifically the absorbance peak at 220 nm, and fractionating and collecting each peak.

If the fraction obtained after the above steps is not sufficiently isolated and purified, reverse-phase HPLC using Jupiter 5μ C18, 300A (manufactured by Phenomenex) is performed again, so that the polypeptide alone Can be isolated and purified. Specifically, it is desirable to perform this step when isolating and purifying a single polypeptide from the F-3 fraction.
In addition, the procedure of gel filtration HPLC of this process can be performed similarly to the reverse phase HPLC of a previous process except using Jupiter 5 (micro | micron | mu) C18, 300A (made by Phenomenex).

The isolated and purified molecular fraction obtained in the above step is subjected to molecular weight measurement and analysis with an N-terminal amino acid analyzer to select a molecular fraction that is a polypeptide and determine the amino acid sequence of the polypeptide. Can be done by.
In this step, the molecular weight is measured by MALDI-TOF MS (Applied Biosystems, USA), ESI-TOF MS (Mariner Biospectrometry, PerSeptive Biosystems, USA), LC MS (MAGIC 2002, Microc Bios, USA). Specifically, it can be performed by MALDI-TOF MS (Voyager-DE STR, manufactured by Applied Biosystems, USA).
In this step, the amino acid sequence can be determined by an amino acid sequencer (PPSQ-10, Shimadzu) or an amino acid sequencer (Procise 494 cLC, manufactured by Applied Biosystems, USA). Specifically, the amino acid sequencer (PPSQ −10, Shimadzu).

By the above analysis, a polypeptide consisting of a specific amino acid sequence having an angiotensin I converting enzyme inhibitory activity can be identified from the molecular fraction isolated and purified by the reverse phase HPLC step.
In the present invention, specifically, from the fraction F-3, Ile-Gln-Pro, Leu-Gln-Pro, Leu-Arg-Pro, Ile-Arg-Pro, from the fraction F-7, Val-Tyr , Thr-Phe and Ile-Tyr amino acid sequences can be isolated and purified as a polypeptide having an angiotensin I converting enzyme inhibitory activity.
Among these, the tripeptide consisting of the amino acid sequence of Ile-Gln-Pro is a polypeptide having a novel angiotensin I converting enzyme inhibitory activity that has not been reported so far.
As described above, the polypeptide “having an inhibitory activity of angiotensin I converting enzyme” obtained in the above step is also a polypeptide “having a blood pressure lowering function”.

The dried powder of the polypeptide obtained in the above step can be used as a material for foods, beverages, and preparations as in the case of the roughly purified product and purified product.
Specifically, it can be used for foods and beverages such as soft drinks, powdered soft drinks, carbonated drinks, tablet confectionery, biscuits, snacks, cereals, cup noodles, powder soups, and jelly.
As a preparation, it can be used in the form of powder, pulverized granules, granules, capsules, solutions dispersed in water or ethanol, tablets obtained by mixing with excipients, etc. .

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the scope of the present invention is not limited at all by these Examples.

Production Example 1 (Production of wheat bran, rice bran, barley bran powder, etc.)
Wheat seeds (blossoms) were milled with a Bühler test mill (Bühler Inc., Switzerland) to prepare a whole grain powder, which was separated into large bran, small bran, powder and 60% flour. In the present invention, large bran is mainly composed of the outer skin such as pericarp and seed coat, and small bran is composed mainly of embryos including cotyledons, root progenitors, hypocotyls, scutellum and the like, and a paste layer. The flour includes a paste layer and a portion of endosperm. The 60% powder is a pulverized product made of endosperm excluding large bran, small bran and powder.
The large bran accounted for approximately 20% of the total flour amount, the small bran accounted for approximately 10% of the total flour amount, and the powder flour accounted for approximately 10% of the total flour amount. In this example, a combination of large and small bran was designated as “wheat bran”.
Wheat germination seeds were prepared by immersing wheat seeds (fukasayaka) in water at 20 ° C. and incubating them for 3 days while allowing them to germinate. This was dried overnight at 40 ° C., and then ground in a centrifugal mill (Retsch, Mitamura Riken Kogyo Co., Ltd.) to prepare whole wheat germinated seed flour.
In addition, immature wheat seeds were prepared by harvesting, threshing 4 weeks after heading and drying at 40 ° C. overnight. Then, the wheat immature seed whole grain powder was prepared by grind | pulverizing with a centrifugal mill (Retsch, Mitamura Riken Kogyo).
For rice bran, rice (Hinohikari) was prepared with a small rice mill (manufactured by Zojirushi). Whole grains were prepared with a centrifugal mill (Retsch, manufactured by Mitamura Riken Kogyo).
For barley koji, barley seeds (intermediate mother farm 2, 9,551, Mantenboshi, which is six-row barley) were scoured with a parest small mill (manufactured by Kett), and grinding powder of approximately 40% of the seed weight was used as barley koji. Whole grains were prepared with a centrifugal mill (Retsch, manufactured by Mitamura Riken Kogyo).
The malt was prepared from Nijo barley by a normal manufacturing method. That is, Nijo barley is soaked in water at 15-20 ° C for 4 days, germinated, dried at 41 ° C for 12 hours, heated to 61 ° C in 15 minutes, dried at 61 ° C for 2 hours 45 minutes, and then in 15 minutes. The temperature was raised to 83 ° C. and dried at 83 ° C. for 4 hours and 45 minutes. Then, the whole wheat flour was prepared by grinding with a centrifugal mill (Retsch, Mitamura Riken Kogyo).

Example 1 (Examination of elution conditions of crude product from ODS column)
As a buffer solution that is an enzyme reaction solution, 0.1 M citrate-sodium phosphate buffer (pH 3.0) is used, and 0.1 g of wheat bran (prepared by the method described in Production Example 1) in 4 mL thereof. Was immersed and kept warm at 40 ° C. for 12 hours with shaking. The pH of the enzyme reaction solution after adding and mixing the raw materials in the buffer solution and being immersed was 3.2.
Thereafter, the mixture was centrifuged at 8000 × g for 20 minutes at a low temperature of 4 ° C., and the supernatant was collected to obtain an enzyme reaction treatment solution.
In order to remove pigment components and coexisting ions in the enzyme reaction treatment solution, a crudely purified product purified by the technique shown below was subjected to an inhibition test.
An enzyme reaction treatment solution (4 ml) was passed through ODS resin (0.5 g) (SepPak (manufactured by Millipore)) activated with ethanol and subsequently with water to adsorb an inhibitory component of angiotensin I converting enzyme. The resin was washed with 5 mL of water, and 5 mL of 10%, 25%, 50%, and 95% ethanol aqueous solutions were sequentially passed through to recover each eluate. The eluate was lyophilized after removing ethanol with a centrifugal evaporator (CVE-3100 (manufactured by Tokyo Rika Kikai Co., Ltd.)) to obtain a crude product. Table 1 shows the relationship between the ethanol concentration used for elution, the inhibitory activity (inhibition rate (%)), and the yield (mg) of the crude product.
In addition, the inhibition rate (%) of angiotensin I converting enzyme in Table 1 indicates the inhibition rate when the concentration of the sample (crudely purified product) was adjusted to 167 μg / mL in the measurement method shown below.

The inhibition rate of angiotensin I converting enzyme was measured according to the modified Liberman method. Hip-His-Leu was used as a pseudo-substrate, hippuric acid (N-benzoylglicine) released from Hip-His-Leu with angiotensin I converting enzyme was extracted with ethyl acetate, and the inhibitory activity was evaluated from the change in absorbance at 228 nm.
Angiotensin I converting enzyme was dissolved in 0.2 M borate buffer (pH 8.3) at a concentration of 25 mU / mL. Hip-His-Leu was dissolved in 0.2 M borate buffer (pH 8.3) containing 1 M NaCl at a concentration of 12.5 mM.
The hippuric acid release reaction was performed by adding a solution (100 μL) containing the angiotensin I converting enzyme to a sample (50 μL) for measuring inhibitory activity, incubating at 37 ° C. for 5 minutes, and then containing a solution (100 μL) containing the Hip-His-Leu. ) Was added and incubated at 37 ° C. for 60 minutes. After the reaction, 0.5M HCl (250 μL) was added to terminate the reaction.
Thereafter, 1.5 mL of ethyl acetate was added to the solution after the hippuric acid release reaction, and the mixture was centrifuged at 2000 × g for 10 minutes, and 500 μL of the upper layer was collected and evaporated to dryness. To this, 3 mL of 1M NaCl solution was added and the absorbance at 228 nm was measured. In addition, the angiotensin I converting enzyme inhibition rate (%) was performed using the above-described formula (1).

  As Table 1 shows, all inhibitory components were eluted at ethanol concentrations of 10% and 25%. Moreover, it was shown that about 60% is recovered when it is eluted at an ethanol concentration of 10%.

  Next, in the said process, it carried out similarly except having used acetonitrile as an elution solvent instead of ethanol. The results are shown in Table 2.

As shown in Table 2, the inhibitory component was divided into a high polar fraction eluting at a low concentration of up to 20% and a low polar fraction eluting at 70% or higher. The low polar fraction had a lower yield and lower inhibitory activity than the high polar fraction. In addition, compared with the fraction eluted with ethanol up to 20%, the highly polar fraction of acetonitrile had the same level of recovery and inhibitory activity. Therefore, it was judged that ethanol or acetonitrile could be used for the preparation of the crude product.
Note that ethanol has advantages over acetonitrile in terms of safety to the human body and waste liquid treatment costs.
Thereafter, in this example, the fraction eluted with 10% ethanol was used as a crude purified fraction, and the dried product was used as a crude purified product.

Example 2 (Examination of optimum pH conditions in enzyme reaction)
As a buffer solution that is an enzyme reaction solution, 0.1 M citrate-sodium phosphate buffer solution (each pH shown in Table 3) is used, and 0.2 g of wheat bran (described in Production Example 1) is added to each 4 mL. (Prepared by the above method) was added to and mixed with the buffer solution, and the mixture was kept warm by shaking at 40 ° C. for 12 hours.
Table 3 shows the pH of the buffer solution after addition and mixing, that is, the pH of each enzyme reaction solution.
Thereafter, ODS column chromatography was performed in the same manner as in Example 1 to prepare a crude product obtained by drying 10% ethanol crude purified fraction.
The relationship between the pH value and the inhibitory activity (inhibition rate (%)) of the crude product and the relationship between the pH value and the yield (mg) of the crude product are shown in FIG. 1 and Table 3, respectively.
In addition, the inhibition rate (%) of the angiotensin I converting enzyme in FIG. 1 and Table 3 is the inhibition rate when the concentration of the sample (crudely purified product) is adjusted to 167 μg / mL as in the measurement method shown in Example 1. Indicates.

As shown in FIG. 1 and Table 3, the inhibition rate (%) of the angiotensin I converting enzyme was highest when the pH value of the enzyme reaction solution after mixing the raw materials was 3.15, 3.26, and the inhibition rate was 67. % Activity. Then, when the pH is 3.77, the inhibition rate is 62.1%, when the pH is 3.77, the inhibition rate is 62.1%, and when the pH is 3.05, the inhibition rate is 49.3%. When the pH was 3.95, the inhibition rate was 45.7%.
Moreover, the yield of the crude product was highest at 7.1 mg at pH 3.77, and a yield exceeding 5 mg was obtained at pH 2.75 to 4.64. It was shown that a sufficient yield was obtained when the reaction was carried out at the indicated pH.
As a result, the pH range suitable for the formation reaction of the inhibitory component of angiotensin I converting enzyme is pH 3.05 to 3.95, preferably pH 3.15 to 3.87, and more preferably pH 3.15 to 3.77. Most preferably, it was determined to be 3.15 to 3.26.

Example 3 (Examination of optimum temperature conditions in enzyme reaction)
Except that the enzyme reaction was carried out at each temperature shown in FIG. 2, the enzyme reaction was carried out in the same manner as in Example 1 (shaking at pH 3.2 for 12 hours), and then the same method as in Example 1. ODS column chromatography was performed to prepare a crude product obtained by drying 10% ethanol crude purified fraction.
FIG. 2 shows the relationship between the temperature and the inhibitory activity (inhibition rate (%)) of the crude product, and the relationship between the temperature and the yield (mg) of the crude product.
In addition, the inhibition rate (%) of the angiotensin I converting enzyme in FIG. 2 shows the inhibition rate when the concentration of the sample (crudely purified product) is adjusted to 167 μg / mL as in the measurement method shown in Example 1.

As shown in FIG. 2, the inhibition rate (%) of angiotensin I converting enzyme was highest at 40 ° C., followed by 35 ° C., 30 ° C., and 45 ° C. in this order. The yield of the crude product was highest at 35 ° C., followed by 40, 30 and 45 ° C. in this order. As a result, the temperature range suitable for the production reaction of the inhibitory component was determined to be 30 to 45 ° C, more preferably 35 to 40 ° C, from the viewpoint of both the inhibition rate and the yield.

Example 4 (Examination of optimal reaction time in enzyme reaction)
The enzyme reaction (temperature) was carried out in the same manner as in Example 1 except that the enzyme reaction was carried out at each pH shown in FIG. 3 and the reaction time was 4, 8, 12, 16, 20, and 24 hours. After shaking at 40 ° C., ODS column chromatography was performed in the same manner as in Example 1 to prepare a crude product obtained by drying a 10% ethanol crude product fraction.
The relationship between the reaction time and the inhibitory activity (inhibition rate (%)) of the crude product is shown in FIG.
In addition, the inhibition rate (%) of the angiotensin I converting enzyme in FIG. 3 shows the inhibition rate when the concentration of the sample (crudely purified product) is adjusted to 167 μg / mL as in the measurement method shown in Example 1.

As shown in FIG. 3, the inhibition rate (%) of angiotensin I converting enzyme increased until 12 hours after the start of the reaction, and remained at a substantially constant level until 24 hours. Except when the enzyme reaction was performed at pH 2.7, a significant increase was observed by 4 hours after the start of the reaction. As a result, it was judged that the reaction time was 4 hours or longer, preferably 12 hours or longer.

Example 5 (Effect of degreasing treatment with hexane)
Wheat bran contains a large amount of lipids, which can affect proteolytic reactions. Therefore, 0.2 g of bran was soaked in 5.0 mL of hexane of each temperature condition shown in Table 4 for 1 hour, and then suction filtered with a Buchna funnel (manufactured by ASONE), dried at 20 ° C. overnight, and defatted wheat bran. Was prepared.
To this whole defatted wheat bran, 4.0 mL of a citrate-sodium phosphate buffer (pH 3.0) was added, and an enzyme reaction (shaking at a temperature of 40 ° C. for 12 hours) was carried out in the same manner as in Example 1. The pH of each enzyme reaction solution after adding and mixing the raw materials to the buffer solution was 3.2.
Thereafter, ODS column chromatography was performed in the same manner as in Example 1 to prepare a crude product obtained by drying 10% ethanol crude purified fraction.
Table 4 shows the relationship between the temperature during immersion in hexane, the inhibitory activity (inhibition rate (%)) of the crude product, and the yield (mg) of the crude product.
In addition, the inhibition rate (%) of angiotensin I converting enzyme in Table 4 indicates the inhibition rate when the concentration of the sample (crudely purified product) was adjusted to 167 μg / mL as in the measurement method shown in Example 1. Moreover, the numerical value in parentheses in Table 4 shows the rate of increase (%) of the inhibition rate and the rate of increase (%) of the yield with respect to the case where an untreated raw material not subjected to degreasing treatment with hexane is used.

As shown in Table 4, it was shown that the wheat bran was degreased with hexane to increase the inhibition rate (%) and the yield of the angiotensin I converting enzyme in the crude product. Among these, when the degreasing treatment was performed at 10 to 40 ° C., the inhibition rate and the increase rate of the yield were high, and among these, the increase rate of the degreasing treatment group at 10 to 30 ° C. was high. In particular, the rate of increase in degreasing treatment at 20 ° C. was the highest, the rate of increase in inhibition effect was 13.1%, and the yield increased 27.6%.

Example 6 (Examination of pH adjusting reagent in enzyme reaction)
In Examples 1 to 5 described above, 0.1 M citrate-sodium phosphate buffer was used for the enzyme reaction. Therefore, in this example, it was examined whether other pH adjusting reagents are applicable. Select 0.5N hydrochloric acid, 5N acetic acid, 0.5N formic acid, 0.5N phosphoric acid, 0.5N sulfuric acid, citric acid powder as a reagent, and add 4 g of water to 0.1 g of small bran (China No. 155). After the addition, the pH was adjusted to 3.2 with each reagent.
Moreover, what used 50 mM citrate-potassium phosphate buffer (pH 3.0) was used as control. The pH of the enzyme reaction solution after adding and mixing the raw materials in the buffer solution and being immersed was 3.2.
Next, an enzyme reaction (shaking at a temperature of 40 ° C. for 12 hours) was carried out in the same manner as in Example 1, and then ODS column chromatography was carried out in the same manner as in Example 1 to obtain a 10% ethanol crude purified fraction. A dried crude product was prepared.
In addition, the measurement of the inhibition rate (%) of an angiotensin I converting enzyme shows the inhibition rate when the density | concentration of a sample (crude refinement | purification product) is adjusted to 167 microgram / mL similarly to the measuring method shown in Example 1. FIG. The results are shown in Table 2.

As a result, compared to the sample using the citrate-phosphate buffer, no significant difference was observed in the inhibition rate and yield. Therefore, it was judged that any of these pH adjusting reagents can be used for the production of an inhibitory component of angiotensin I converting enzyme.

Example 7 (Examination of influence of salt concentration on enzyme reaction)
In order to examine the influence of the salt concentration in the production reaction of the inhibitory component of angiotensin I converting enzyme, the production reaction was performed using a buffer solution containing NaCl.
After adjusting 4.0 mL of 0.1 M citrate-sodium phosphate buffer (pH 3.0) to which 0.2 g of bran was added so as to contain sodium chloride at 50 mM, 100 mM, 200 mM, and 400 mM, Examples In the same manner as in No. 1, an enzyme reaction (shaking at 40 ° C. for 12 hours) was performed. The pH of each enzyme reaction solution after adding and mixing the raw materials to the buffer solution was 3.2.
Thereafter, ODS column chromatography was performed in the same manner as in Example 1 to prepare a crude product obtained by drying 10% ethanol crude purified fraction.
The inhibition rate (%) of angiotensin I converting enzyme was measured in the same manner as the measurement method shown in Example 1.

As a result, there was no effect at 50 mM or less, but the inhibition rates of 100, 200, and 400 mM decreased to 80%, 50%, and 40% of the control (no sodium chloride added), respectively. Therefore, it was judged that the reaction for producing an inhibitory component of angiotensin I converting enzyme is preferably carried out at a salt concentration of 100 mM or less.

Example 8 (Properties of wheat bran protease)
In order to investigate the properties of proteases that produce angiotensin I converting enzyme inhibitory peptides from wheat bran protein, the effect on enzyme reaction when various protease inhibitors (protease inhibitors) were added was examined.
0.1 M citrate-sodium phosphate buffer (pH 3.0) added to contain 1 mM EDTA, 0.1 M citrate-sodium phosphate buffer (pH 3.0) added to contain 1 mM PMSF ), 0.1 M citric acid-sodium phosphate buffer (pH 3.0) added to contain 20 μM Pepstatin A, 0.1 M citric acid-phosphoric acid added to contain 4.4 μM E-64 After adjusting each of the sodium buffer solutions (pH 3.0), enzymatic reaction (shaking at a temperature of 40 ° C. for 12 hours) was performed in the same manner as in Example 1. The pH of each enzyme reaction solution after adding and mixing the raw materials to the buffer solution was 3.2.
Thereafter, ODS column chromatography was performed in the same manner as in Example 1 to prepare a crude product obtained by drying 10% ethanol crude purified fraction.
Here, EDTA is a metalloprotease, PMSF is a serine protease, Pepstatin A is an aspartic protease, and E-64 is an inhibitor of cysteine protease. The added concentration of the proteinase inhibitor is a concentration usually used as an inhibitor.
The inhibition rate (%) of angiotensin I converting enzyme was measured in the same manner as the measurement method shown in Example 1.
FIG. 4 shows the relationship between various protease inhibitors and the inhibition rate of the inhibitory activity of angiotensin I converting enzyme. The inhibition rate (%) of the inhibitory activity of angiotensin I converting enzyme in FIG. 4 indicates that the higher the value, the more difficult it is to produce a polypeptide having the inhibitory activity of angiotensin I converting enzyme from the raw material protein.

As shown in FIG. 4, about 15% inhibition was observed in PMSF and 100% in Pepstatin A, respectively, with respect to the enzyme reaction that produced the angiotensin I converting enzyme inhibitory peptide. Therefore, it has been clarified that aspartic protease plays a major role in the production of angiotensin I converting enzyme-inhibiting peptide, and serine protease is also involved.
Aspartic protease is an acidic protease typified by pepsin, and the optimum pH is on the acidic side.
As shown in FIG. 1 and Table 3, aspartic protease is mainly used for the production of angiotensin I converting enzyme inhibitory peptide because the optimum pH of the enzyme reaction for generating the angiotensin I converting enzyme inhibitory peptide in the present invention is acidic. It is supplemented that it is working properly. On the other hand, it is known that seed germination increases cysteine protease activity and degrades gluten (Journal of Agricultural and Food Chemistry, 48, 6271-6279. (2000)). It was shown from the results of this example that is not involved in the production of angiotensin I converting enzyme inhibitory peptide.

Example 9 (Angiotensin I converting enzyme inhibitory effect of crudely purified product from wheat, barley and rice seeds)
Crude products were produced using the tissues of wheat, barley and rice seeds produced by the method described in Production Example 1 as raw materials, and the angiotensin I converting enzyme inhibitory effect was examined.
From wheat seeds (fukusayaka), whole grains, large bran, small bran, bran, powder, 60% flour, whole grains of germinated seeds, and whole grains of immature seeds 4 weeks after heading were used. In addition, the combination of large and small bran was used as bran. From the barley seeds, intermediate mother farm 2 cocoons, 6551 barley, mantenboshi cocoons, Nijo barley sky golden cocoons, sky golden whole grains, and sky golden malt whole grains were used. From rice seeds, Hinohikari rice bran was used.
The above raw material powder (0.2 g) was added to 4.0 mL of 0.1 M citrate-sodium phosphate buffer (pH 3.0), and the enzyme reaction (at a temperature of 40 ° C. at 12 ° C. was performed in the same manner as in Example 1). Shake time). The pH of each enzyme reaction solution after adding and mixing the raw materials to the buffer solution was 3.2.
Thereafter, ODS column chromatography was performed in the same manner as in Example 1 to prepare a crude product obtained by drying 10% ethanol crude purified fraction.
Table 6 shows the inhibitory activity IC ₅₀ (mg / mL) of angiotensin I converting enzyme of each crude product.
In Table 6, the value of the inhibitory activity IC ₅₀ (mg / mL) of angiotensin I converting enzyme is the final concentration value of the sample (crude product) when the inhibition rate of the activity of angiotensin I converting enzyme is 50%. Show. The inhibition rate (%) was measured in the same manner as the measurement method shown in Example 1.

As Table 6 shows, all samples showed angiotensin I converting enzyme inhibitory activity except 60% wheat seed flour. Among them, wheat bran, large bran, and small bran showed strong inhibitory activity.

Example 10 (Purification of inhibitory peptide of angiotensin I converting enzyme)
A component (peptide) exhibiting angiotensin I converting enzyme inhibitory activity was purified from wheat bran. Purification was performed in the order of the steps shown in Table 7, and the yield and inhibitory activity in each step were measured. Inhibitory activity was measured in the same manner as in Example 9.

First, 100 g of wheat bran is suspended in 500 mL of 0.1 M citrate-sodium phosphate buffer (pH 3.0) and shaken at the same enzyme reaction conditions as in Example 1 (temperature 40 ° C. for 12 hours). ). Note that the pH of the enzyme reaction solution after adding and mixing the raw materials to the buffer solution was 3.2.
Then, it centrifuged at 8000 * g for 20 minutes, and collect | recovered supernatant (enzyme reaction processing liquid). The yield of the enzyme reaction treated product obtained when this supernatant was dried was 20.25 g, and the IC ₅₀ value was 1040 μg / mL (1.04 mg / mL).
Next, this enzyme-treated reaction solution was added to a column (5 × 25 cm) YMC Gel ODS-AQ (manufactured by YMC) filled with ODS resin, and after column washing with 1000 mL of water, 500 mL of 10%, The adsorbed components were eluted by passing 25%, 50%, and 95% aqueous ethanol solutions. In this step, from the 10% aqueous ethanol fraction, 2100 mg of the crude purified product having an IC ₅₀ value of 86 μg / mL (0.086 mg / mL), which is significantly stronger in inhibitory activity than the other aqueous ethanol fractions. was gotten.
Ethanol was removed from the 10% ethanol aqueous fraction using an evaporator, adjusted to pH 9.0 with 1N ammonia aqueous solution, and anion exchange resin (3 × 20 cm) Amberlite AG MP equilibrated with 1N ammonia aqueous solution and water. -1 (manufactured by BIO-RAD), and the non-adsorbed fraction was collected. From this fraction, 1200 mg of a dried product having an IC ₅₀ value of 45 μg / mL was obtained.

Subsequently, the fraction showing the inhibitory activity was separated and purified by performing gel filtration HPLC on the solution of the non-adsorbed fraction of the anion exchange resin. First, the solution of the non-adsorbed fraction of the anion exchange resin was added to a Superdex 75 HR column (1 × 30 cm) (Amersham) equilibrated with 30% acetonitrile containing 0.1% trifluoroacetic acid. The flow was at a flow rate of min and monitored at 220 nm. As an apparatus, LC-10AD HPLC system (made by Shimadzu) was used. Eleven peaks from F-1 to F-11 were collected based on the absorbance peak. The results are shown in FIG. Each peak was concentrated by an evaporator, freeze-dried, and the inhibitory activity of angiotensin I converting enzyme was measured. Among these, purification was advanced with respect to F-3 (IC ₅₀ : 44 μg / mL) and F-7 (IC ₅₀ : 76 μg / mL) showing strong inhibitory activity.

The active ingredient was separated and purified by performing reverse-phase HPLC using a C4 column on the fraction solution of F-3 and F-7 that showed strong inhibitory activity. F-3 and F-7 fraction solutions are added to a C4 column (1 × 25 cm) (Jupiter 5μ C4, 300A, Phenomenex) and run at a flow rate of 2 mL / min, containing 0.1% trifluoroacetic acid Water-acetonitrile was passed under a linear gradient condition to separate the active ingredient. The active ingredient obtained from the F-3 fraction was further purified with an ODS column (1 × 25 cm) (Jupiter 5 μC18, 300A, manufactured by Phenomenex). The elution pattern from the F-3 fraction solution is shown in FIG. 6, and the elution pattern from the F-7 fraction solution is shown in FIG.
Each peak was concentrated with an evaporator, freeze-dried, and the inhibitory activity of angiotensin I converting enzyme was measured. Among these, 4 components showing strong inhibitory activity were isolated from the F-3 fraction, and 3 components were isolated from the F-7 fraction.

The structure of the isolated inhibitory component was analyzed for molecular weight by MALDI-TOF MS ((Applied Biosystems, USA), and analyzed for amino acid sequence by amino acid sequencer (PPSQ-10, Shimadzu), and the results are shown in Table 8.
As a result, the inhibitory component of angiotensin I converting enzyme isolated from the F-3 fraction was Leu-Gln-Pro (Leucylglutamylproline; LQP), Ile-Gln-Pro (Isoleucilglutamylproline; IQP), It was revealed that it was a polypeptide consisting of amino acid sequences of Leu-Arg-Pro (Leucylarginylproline; LRP) and Ile-Arg-Pro (Isoleucilarginylproline; IRP). Inhibitory components of angiotensin I converting enzyme isolated from the F-7 fraction are Val-Tyr (valyltyrosine; VY), Thr-Phe (threonylphenylalanine; TF), Ile-Tyr (isoleucine tyrosine). ; IY) was revealed to be a polypeptide consisting of the amino acid sequence.
Among these, the tripeptide (isoleucylglutamylproline; IQP) consisting of the amino acid sequence of Ile-Gln-Pro is a polypeptide having a novel angiotensin I converting enzyme inhibitory activity that has not been reported so far.
These are both α- or β-gliadin partial sequences, suggesting that the degradation product of gliadin contained in bran is inhibited by an endogenous protease, thereby inhibiting the activity of angiotensin I converting enzyme. It was done.

Inhibitory peptides isolated and purified from wheat bran according to this example include an inhibitory activity (IC) of angiotensin I converting enzyme equivalent to or higher than Val-Tyr (valyltyrosine) approved as a food for specified health use. ₅₀ )) and was shown to contain 7 polypeptides. This result is a remarkable effect that cannot be achieved from the method using other materials (sardines, bonito, tuna, casein, lactoglobulin, corn, wheat gluten, etc.) with the addition of a foreign enzyme as in the prior art. .

Example 11 (preparation of large amount of crude product)
To 1 kg of wheat bran, 5 L of hexane was added and stirred at 20 ° C. for 1 hour to degrease the wheat bran. Then, it filtered, wash | cleaning with 2 L hexane using a Buchner funnel (product made from ASONE). The defatted bran was dried overnight at room temperature.
10 L of water was added to the dried bran and adjusted to pH 3.0 with 2N HCl. While stirring at 40 ° C., the enzyme reaction with endogenous protease was performed for 12 hours.
The enzyme reaction treatment solution was cooled to 4 ° C. and adjusted to pH 7.0 with 10N ammonia. Then, it centrifuged at 8000 * g for 20 minutes, supernatant was collect | recovered, and it filtered with the Buchner funnel. Next, the filtrate was passed through a column (5 × 30 cm) filled with activated carbon (coconut shell system, granular (manufactured by Nacalai Tesque)) to remove the pigment component.
The obtained filtrate was passed through a column (5 × 40 cm) packed with ODS resin (LiChroprep RP-18 (manufactured by Merck)), and the column was washed with 3 L of water.
The inhibitory component of angiotensin I converting enzyme was eluted with 3 L of 10% ethanol aqueous solution, 3 L of 25% ethanol aqueous solution, concentrated with an evaporator, and lyophilized.

From the 10% ethanol elution fraction, 24.68 g (IC ₅₀ : 52 μg / mL) of crude product I was obtained, and from the 25% ethanol elution fraction, 16.91 g (IC ₅₀ : 193 μg / mL) of crude product II was obtained. It was. These can be used in foods as a crude product having an angiotensin I converting enzyme inhibitory activity. In addition, as described above, a product that is further purified with an anion exchange resin and has enhanced inhibitory activity can be used as well.

The crude product I is white, and the crude product II is light yellow, and both are soluble in water. As a functional food material having a blood pressure lowering effect, it can be added to processed foods. For example, solids can be used as tablet-type tablet confections, powdered soups, etc., and dissolved products can be used as beverages such as tea, juice, soft drinks, jelly, and the like. Further, a product whose inhibitory activity is further enhanced by anion exchange chromatography can be similarly applied.

Example 12 (Effect of wheat bran washing treatment before enzyme reaction)
From the above experimental results, it was found that a peptide having an angiotensin I converting enzyme inhibitory activity was a sequence contained in gliadin. Wheat gliadin is a kind of simple protein of prolamin type, soluble in 60-90% ethanol, but insoluble in water, acidic solution, and neutral solution. In the enzyme treatment reaction, soluble proteins other than gliadin dissolve and coexist as contaminants. Thus, if soluble proteins can be removed before the reaction while retaining enzyme activity, it is expected that the production of a crude product can be simplified.
Therefore, as a result of trial and error, 4 mL of each cleaning solution shown in Table 9 was added to 0.2 g of Kobusuma (Chinese No. 155), and the mixture was shaken at room temperature (26 ° C.) for 10 minutes. The supernatant was removed by centrifugation at 2000 rpm to obtain a washed bran which was a precipitate. 4 mL of water was added thereto, and the mixture was shaken for 10 minutes. Similarly, the supernatant was removed after centrifugation. This operation was repeated once more.
4.0 ml of citrate-sodium phosphate buffer (pH 3.0) was added to the whole amount of the washed bran, and an enzyme reaction (shaking at a temperature of 40 ° C. for 12 hours) was performed in the same manner as in Example 1. The pH of each enzyme reaction solution after adding and mixing the raw materials to the buffer solution was 3.2.
Thereafter, ODS column chromatography was performed in the same manner as in Example 1 to prepare a crude product obtained by drying 10% ethanol crude purified fraction. The results are shown in Table 9.
In Table 9, the value of the inhibitory activity IC ₅₀ (μg / mL) of angiotensin I converting enzyme is the final concentration value of the sample (crude product) when the inhibition rate of the activity of angiotensin I converting enzyme is 50%. Show. The inhibition rate (%) was measured in the same manner as the measurement method shown in Example 1.

As Table 9 shows, the pH after suspension of water added to wheat bran was about 6.7. Therefore, the crude purified product prepared after washing with weakly acidic water (sample 1), acidic buffer (sample 2) and neutral buffer (sample 3) was not subjected to washing treatment (sample 6). As compared with the above, it was revealed that the inhibitory activity (IC ₅₀ ) of angiotensin I converting enzyme was remarkably strong, and it was shown that a high-purity crude product was obtained.
In contrast, the inhibitory activity of the crude product produced by washing with an alkaline buffer (samples 4 and 5) was small compared to the control (sample 6), and part of the protease was eluted or inactivated. Indicated.
In terms of yield, the product prepared after washing with weakly acidic water (sample 1), acidic buffer (sample 2) and neutral buffer (sample 3) was 42 of the control (sample 6). ~ 58%. Considering the increase in IC ₅₀ value due to washing, it was at a level that compensated for the decrease in the yield of the crude product. The crude product produced after washing with an alkaline buffer (samples 4 and 5) was 34 to 37%, indicating that a part of gliadin was eluted.
Moreover, while the color tone of the reaction product was light brown in the control (sample 6), each washed product (samples 1 to 5) was white, indicating that the pigment component was removed.

From this result, it is clear that unnecessary contaminating proteins and pigment components can be removed by washing the bran under acidic to neutral conditions, and the content of an inhibitory component of angiotensin I converting enzyme can be increased. It was.

Example 13 (Effect of sodium hypochlorite on the inhibitory activity of angiotensin I converting enzyme in washing treatment)
Since the enzyme reaction treatment of wheat bran takes 4 hours or more, there is concern about microbial contamination during the reaction. Therefore, the bran was washed with an aqueous solution containing sodium hypochlorite generally used in food processing, and the effect of sterilization treatment was examined.

The “cleaning and sterilization treatment” was performed in the same manner as the “cleaning treatment” in Example 12 except that an aqueous solution containing sodium hypochlorite was used.
First, sodium hypochlorite (effective chlorine concentration 6% (manufactured by Wako Pure Chemical Industries, Ltd.)) diluted to effective chlorine concentration 100ppm (sample 8) and 600ppm (sample 9) to 0.2g small bran of No. 155 in China 4 mL of the aqueous solution containing the solution was added and stirred for 10 minutes at room temperature (26 ° C.), and then the supernatant was removed by centrifugation. To the obtained washed and sterilized precipitate, 4 mL of water was added, stirred at room temperature (26 ° C.) for 10 minutes, and then the supernatant was removed by centrifugation. Further, this operation was repeated once more to remove sodium hypochlorite. In addition, in the said process, what was wash-processed only with water, without using sodium hypochlorite aqueous solution was set as the unsterilized control (sample 7).
Next, 4.0 mL of citrate-sodium phosphate buffer (pH 3.0) was added to the washed and sterilized bran precipitate, and the enzyme reaction (shaking at 40 ° C. for 12 hours) was performed in the same manner as in Example 1. Went. The pH of each enzyme reaction solution after adding and mixing the raw materials to the buffer solution was 3.2.
Thereafter, rough purification was performed using SepPak (manufactured by Millipore) to prepare a crude product. The results are shown in Table 10.
In Table 10, the value of the inhibitory activity IC ₅₀ (μg / mL) of angiotensin I converting enzyme is the final concentration value of the sample (crude product) when the inhibition rate of the activity of angiotensin I converting enzyme is 50%. Show. The inhibition rate (%) was measured in the same manner as the measurement method shown in Example 1.

As shown in Table 10, in the cleaning process, when the small bran was “cleaned and sterilized” using an aqueous sodium hypochlorite solution diluted to an effective chlorine concentration of 100 to 600 ppm (samples 8 and 9). It was revealed that there was no difference in the inhibitory activity (IC ₅₀ ) of the angiotensin I-converting enzyme of the roughly purified product produced compared to the control (sample 7: unsterilized) that was washed with water alone. .
As a result, it became clear that the washing and sterilization treatment using hypochlorous acid diluted to an effective chlorine concentration of 100 to 600 ppm can prevent the growth of microorganisms in the enzyme reaction solution without affecting the enzyme reaction. .

Example 14 (Influence of sodium hypochlorite on the inhibitory activity of angiotensin I converting enzyme in enzyme reaction treatment)
For the same purpose as in Example 13, in order to prevent microbial contamination during the enzyme reaction, the influence of the enzyme reaction solution containing sodium hypochlorite on the inhibitory activity of angiotensin I converting enzyme was examined.
First, 0.2 mL of Chinese No. 155 small bran, 4 mL each of 50 mM citrate-sodium phosphate (pH 3.0) containing sodium hypochlorite with an effective chlorine concentration of 100 ppm (sample 11) or 600 ppm (sample 12) And an enzyme reaction was carried out at 40 ° C. for 12 hours in the same manner as in Example 1. In the above reaction, a control (sample 10) was obtained by performing an enzyme reaction treatment only with 50 mM citric acid-sodium phosphate (pH 3.0) without using a sodium hypochlorite aqueous solution. The pH of each enzyme reaction solution after adding and mixing the raw materials to the buffer solution was 3.2.
Thereafter, rough purification was performed using SepPak (manufactured by Millipore) to prepare a crude product. The results are shown in Table 11.
In Table 11, the value of the inhibitory activity IC ₅₀ (μg / mL) of angiotensin I converting enzyme is the final concentration value of the sample (crude product) when the inhibition rate of the activity of angiotensin I converting enzyme is 50%. Show. The inhibition rate (%) was measured in the same manner as the measurement method shown in Example 1.

As shown in Table 11, even when an enzyme reaction solution containing sodium hypochlorite having an effective chlorine concentration of 100 to 600 ppm is used in the enzyme reaction treatment (samples 11 and 12), the enzyme treatment is performed only with the enzyme reaction solution. It was revealed that there was no difference in the inhibitory activity (IC ₅₀ ) of the angiotensin I-converting enzyme of the roughly purified product produced compared to the control (sample 10: not containing sodium hypochlorite).
As a result, it has been clarified that sodium hypochlorite added to contain an effective chlorine concentration of 100 to 600 ppm can prevent the growth of microorganisms in the enzyme reaction solution without affecting the enzyme reaction. .
Residual sodium hypochlorite can be completely removed after the reaction by heating at 60 ° C. or higher or by a rough purification operation by SepPak.

Example 15 (Effect of ODS column-treated crude product on blood pressure in spontaneously hypertensive rats)
A 10% ethanol-eluted crude product (ODS column-treated crude product) after adsorbing wheat bran on the ODS column was “single dosed” to spontaneously hypertensive rats, and the effect on blood pressure was examined.
First, 100 g of wheat bran is suspended in 500 mL of 0.1 M citrate-sodium phosphate buffer (pH 3.0) and shaken at the same enzyme reaction conditions as in Example 1 (temperature 40 ° C. for 12 hours). ). Note that the pH of the enzyme reaction solution after adding and mixing the raw materials to the buffer solution was 3.2.
Then, it centrifuged at 8000 * g for 20 minutes, and collect | recovered supernatant (enzyme reaction processing liquid). Next, the enzyme-treated reaction solution was added to a column (5 × 25 cm) YMC Gel ODS-AQ (manufactured by YMC) filled with ODS resin, washed with 1000 mL of water, and then 500 mL of 10% ethanol aqueous solution. The adsorbed components were eluted by passing through the solution.
From this 10% aqueous ethanol fraction, 2100 mg of a crude product having an IC ₅₀ value of 86 μg / mL (0.086 mg / mL) was obtained. This was used as the “ODS column-treated crude product” in the following tests.

For animal experiments, 11-week-old male rats (SHR / NCrlCrlj) were obtained from Charles River Japan and used. Rats were purified by Oriental Yeast Co. under conditions of room temperature 21 ± 3 ° C., relative humidity 50 ± 20%, irradiation time 12 hours / day (7: 0 to 19:00), and number of arousals 10-15 times / hour. The solid feed AIN-93M was given, and the drinking water was preliminarily conditioned in an environment where tap water can be freely consumed.
After acclimatization, those having a blood pressure at systolic of 180 mmHg or more at 16 weeks of age are selected, and the following 4 groups (6 in each group), so that blood pressure and body weight (340-370 g) are averaged in each group, that is, The “ODS column-treated crude product” is divided into a group administered with 10 mg / kg body weight, a group administered with 50 mg / kg body weight, a group administered with 200 mg / kg body weight, and a group not administered (group administered with 0 mg / kg: control group). The crudely purified product was administered.
Administration of the crude product was carried out by gavage administration of each test solution dissolved in distilled water so that the prescribed amount defined in each group could be administered, at 10-12 am. The same amount of distilled water was administered to the control group. The administration was “only once” (single administration).
For each group, before administration, 2, 4, 6 and 8 hours after administration, and after pre-incubation (38 ° C.) for 15 minutes before measurement, a non-invasive blood pressure heart rate measuring device (Softron Co., Ltd.) BP-98A) was used to measure tail vein pressure.
The statistical difference between the values before and after the administration was tested by Student's paired t-test. The test between the control group and each administration group was performed by Student's t-test. All values were expressed as mean ± standard error, and the significance level was 5% for both tests. The results are shown in FIG.
In addition, in each measured value in FIG. 8, when the significance probability P before administration is smaller than 0.05, it is marked with “*”, and when smaller than 0.01, it is marked with “**”. Moreover, in the measured value of each time, it described with "#" when the significance probability P with control was smaller than 0.05, and marked with "##" when smaller than 0.01. In the following FIGS. 9 and 10, these symbols have the same meaning.

As a result, in the group administered with “ODS column-treated crude product” 10 mg / kg body weight, blood pressure was significantly reduced from 214.4 ± 13.2 mmHg before administration to 195.9 ± 12.1 mmHg 2 hours after administration ( Antihypertensive value -18.4 mmHg; P <0.05 compared to before administration. Thereafter, although the blood pressure slightly increased, a significant antihypertensive effect was observed up to 6 hours after administration.
Similarly, in the groups administered with 50 mg / kg body weight and 200 mg / kg body weight, a significant decrease in blood pressure was observed 2 hours after administration (P <0.05), and the blood pressure decreased thereafter, and 8 hours after administration. A significant antihypertensive effect was also observed. The antihypertensive effect of the “ODS column-treated crude product” was dose-dependent in the range of 10-200 mg / kg dose.
In contrast, the blood pressure before administration in the control group was 215.8 ± 11.0 mmHg, but no significant reduction in blood pressure was observed 2-8 hours after administration in the control group.

Example 16 (Effect of Ile-Gln-Pro on Blood Pressure of Spontaneously Hypertensive Rats)
Ile-Gln-Pro was “single dose” in spontaneously hypertensive rats and the effect on blood pressure was examined.
First, a novel ACE inhibitory peptide Ile-Gln-Pro (isoleucil glutamylproline; IQP) isolated from bran was synthesized by requesting a synthetic peptide with a purity of 95% or more from Peptide Research Institute. This was used for the following tests.
For animal experiments, an 11-week-old male rat (SHR / NCrlCrlj) was obtained from Charles River, Japan, and preliminarily reared and acclimated in the same environment as in Example 15.
After acclimatization, those at 13 weeks of age and having a systolic blood pressure of 180 mmHg or more were selected, and the following 3 groups (6 in each group), that is, blood pressure and body weight (300-320 g) were averaged in each group, , “Ile-Gln-Pro” was divided into a group administered with 1.5 mg / kg body weight, a group administered with 15 mg / kg body weight, and a group not administered (group administered with 0 mg / kg body weight: control group). Pro ”was administered.
Administration of Ile-Gln-Pro was carried out by gavage administration of each test solution dissolved in distilled water at 10-12 am with a sonde so that the predetermined amount defined in each group could be administered. The same amount of distilled water was administered to the control group. In addition, administration was performed only once (single administration).
For each group, before administration, 2, 4, 6 and 8 hours after administration, and after pre-incubation (38 ° C.) for 15 minutes before measurement, a non-invasive blood pressure heart rate measuring device (Softron Co., Ltd.) BP-98A) was used to measure tail vein pressure.
Further, the test for statistical difference between the values before and after the administration, and the test between the control group and each administration group were carried out in the same manner as in Example 15. The results are shown in FIG.

As a result, in the groups administered with “Ile-Gln-Pro” at 1.5 mg / kg body weight and 15 mg / kg body weight, the blood pressure showed a dose-dependent decrease, and 194.4 ± 13.3 mmHg and 194. Blood pressure decreased significantly from 5 ± 16.2 mmHg to 181.6 ± 6.7 mmHg and 172.2 ± 10.0 mmHg (hypertensive values −12.8 mmHg and −22.3 mmHg; P <0.05) 4 hours after administration did.
Further, 8 hours after the administration, the blood pressure dropped to 181.4 ± 6.2 mmHg and 166.9 ± 10.1 mmHg (hypertensive value—13.0 mmHg and −27.6 mmHg; P <0.05), compared with the control group. Significant hypotension was observed in each case (vs. blood pressure 191.1 ± 4.1 mmHg 8 hours after administration in the control group; P <0.05).
On the other hand, the blood pressure before administration of the control group was 193.3 ± 13.2 mmHg, and there was no change in blood pressure after the start of the test.

Example 17 (Long-term administration test of ODS column-treated crude product to spontaneously hypertensive rats)
A 10% ethanol-eluted crude product (ODS column-treated crude product) after adsorption of wheat bran ODS column was “long-term administered” to spontaneously hypertensive rats, and the effect on blood pressure was examined.
As the “ODS column-treated crude product”, the product obtained in Example 15 was used in the following test.
For animal experiments, an 11-week-old male rat (SHR / NCrlCrlj) was obtained from Charles River, Japan, and preliminarily reared and acclimated in the same environment as in Example 15.
After acclimatization, those having a blood pressure at systolic of 180 mmHg or more at 14 weeks of age were selected, and the following 3 groups (6 in each group), so that blood pressure and body weight (300-330 g) were averaged in each group, that is, , A group that administers water containing 0.02% of “ODS column-treated crude product”, a group that administers water that contains 0.1%, a group that administers water that does not contain (administer water that contains 0%) Group: control group), and the crudely purified product was administered.
For the administration of the crude purified product, each test solution dissolved in distilled water so as to have the predetermined concentration defined in each group was given as drinking water and allowed to “freely” for a long period of time. Distilled water was given to the control group.
Each group was pre-incubated (38 ° C.) for 15 minutes before measurement at 10-12 am before administration and 5, 10, 15, 20, and 25 days after the start of administration. Tail vein pressure was measured using a blood pressure heart rate measurement device (Softron, BP-98A).
Further, the test for statistical difference between the values before and after the administration, and the test between the control group and each administration group were carried out in the same manner as in Example 15. The results are shown in FIG.

As a result, in the group administered with water containing 0.02%, blood pressure was significantly reduced 10 days after the start of administration compared to the control (blood pressure 200.5 ± 5.4 mmHg after 10 days in the control group, administration) 198.6 ± 4.8 mmHg before, 193.4 ± 5.2 mmHg 10 days after administration; P <0.05). Further, the effect was continued until 15 days after administration by continuous administration, and a significant antihypertensive value of −11.1 mmHg as compared with the control group (P <0 against blood pressure 204.2 ± 5.2 mmHg 15 days after administration in the control group). .05). Thereafter, although the antihypertensive action decreased, the antihypertensive effect was significantly maintained up to 25 days after administration compared to the control (relative to the control group; P <0.05).
In the group administered with water containing 0.1%, blood pressure was significantly reduced 5 days after the start of administration compared to the control and before administration (blood pressure 201 ± 6.7 mmHg after 5 days in the control group, administration) Before 197.1 ± 4.9 mmHg, 5 days after administration 185.0 ± 5.2 mmHg; P <0.01). Thereafter, the antihypertensive effect was enhanced until 10 days later (blood pressure 200.5 ± 5.4 mmHg after 10 days in the control group, 197.1 ± 4.9 mmHg before administration, 183.0 ± 5.6 mmHg after 10 days after administration; P < 0.01). Although the antihypertensive effect decreased after 15 days, it maintained a significant difference from before administration until 20 days (before administration after 20 days; P <0.05 vs. control group; P <0.01). ). Thereafter, the antihypertensive effect was significantly maintained until 25 days after administration compared to the control (P <0.01 with respect to the control group).

ADVANTAGE OF THE INVENTION According to this invention, the polypeptide which has the inhibitory activity of the angiotensin I converting enzyme useful as a pharmaceutical, a functional food, etc. effectively using the wheat bran, barley bran, and rice bran which were the object of discard conventionally is provided. .
Therefore, the present invention can be effectively used in the food field, the pharmaceutical field and the like.

Claims (7)

  At least one powder raw material selected from the group consisting of wheat seed powder containing at least wheat bran, barley seed powder containing at least barley bran, and rice seed powder containing at least rice bran; added to water or a buffer solution and mixed And the pH of the water or buffer after addition and mixing is set to 3.05 to 3.95, and the storage protein is decomposed by the action of endogenous protease at a temperature of 30 to 45 ° C. for at least 4 hours; An angiotensin I converting enzyme inhibitor, comprising the obtained reaction product as an active ingredient.   The wheat seed powder is a wheat bran powder that does not contain a starch storage part, the barley seed powder is a barley meal that does not contain a starch storage part, and the rice seed powder does not contain a starch storage part The angiotensin I converting enzyme inhibitor according to claim 1, which is a powder.   The angiotensin I converting enzyme inhibitor according to claim 1 or 2, wherein the powder raw material is degreased using hexane before the addition and mixing.   As the powder raw material, it is added and mixed in advance with a cleaning solution comprising water or a buffer solution, and after the addition and mixing, the pH of the cleaning solution is set to 3.0 to 7.0, and after immersion at a temperature of 0 to 28 ° C. for 5 to 30 minutes. The angiotensin I converting enzyme inhibitor according to any one of claims 1 to 3, wherein the supernatant is used.   The angiotensin I converting enzyme inhibitor in any one of Claims 1-4 which refine | purifies the obtained reaction liquid by the reverse phase chromatography using an ODS column after the said reaction.   6. In the purification process by reverse phase chromatography using the ODS column according to claim 5, the reaction solution is passed through the ODS column and adsorbed, and then eluted and recovered with 10 to 25% ethanol or 10 to 25% acetonitrile. The angiotensin I converting enzyme inhibitor according to claim 5.   A medicament having an antihypertensive action, comprising the angiotensin I converting enzyme inhibitor according to claim 1.

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