JP3504719B2 - Substance having amylase inhibitory activity - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel substance derived from wheat having amylase inhibitory activity and a method for preparing the same,
Also, it relates to an amylase inhibitor, a blood glucose elevation inhibitor, an insulin secretion regulator, an appetite suppressant and a food containing the substance having the amylase inhibitory activity . The substance having a novel amylase inhibitory activity of the present invention is particularly useful for human pancreatic α-
It has extremely high inhibitory activity against amylase,
It is possible to effectively suppress increase in blood glucose level of humans and control insulin secretion. Furthermore, the substance having a novel amylase inhibitory activity of the present invention can maintain a feeling of satiety after meals, so that it becomes possible to easily suppress appetite and effectively prevent overeating and obesity which are likely to cause adult diseases. can do.

[0002]

2. Description of the Related Art In recent years, eating habits have become rich in Japan, and metabolic diseases such as diabetes are rapidly increasing. Excessive nutrient intake indirectly causes metabolic balance disruption by inducing a large amount of insulin secretion, leading to impaired glucose tolerance (hyperglycemia), diabetes, hyperlipidemia, arteriosclerosis, and the like. In particular, in diabetic patients, the insulin action is insufficient and the glucose tolerance is lowered, so that the postprandial blood glucose level is significantly increased, which causes complications such as capillary damage and arteriosclerosis. In order to prevent and treat such diseases, it is said that ingestion of foods or substances whose blood glucose level is difficult to increase is effective, and therefore substances that can suppress or inhibit the ingested starch from decomposing into sugars. Is required. Also, in the present situation of Japan, where food and drink are abundant, it is easy to overeat,
This is a cause of adult diseases such as obesity, high blood pressure, diabetes and heart disease.

From the above point of view, various studies have been conducted on so-called amylase inhibitors having an activity of inhibiting the activity of amylase which decomposes starch into sugars, and wheat also contains amylase inhibitors. Since it was reported that various amylase inhibitors derived from wheat have been studied and developed [for example, JP-A-46-1833 and JP-A-61-1714].
31 publication, Phytochemistry. Vol.20, No.8, pp1781-
1784 (1981), Eur. J. Biochem. 183, 37-40 (1989)].

However, although the above-mentioned conventional wheat-derived amylase inhibitor has some inhibitory activity against amylases of animals other than humans, it has extremely low or almost no inhibitory activity against human pancreatic α-amylase, When administered orally to humans, the expected effect was not seen, especially for cooked starch such as cooked rice,
It has been reported that the effect of inhibiting its decomposition is low.
Therefore, it has a strong inhibitory activity against human pancreatic α-amylase, and when administered orally to humans, a small amount of it can effectively suppress an increase in blood sugar level and insulin secretion in humans, and especially sugar of cooked starch. There is a need for an amylase inhibitor having a high activity that can effectively suppress the decomposition into water.

It has also been reported that the amount of free fatty acids in blood is increased when an agent that suppresses an increase in blood glucose level and insulin secretion is used [Puls and Keup "Dia.
betologia ” 9 , 97-101 (1973)], an increase in the amount of free fatty acids in the blood is generally said to cause hunger and is likely to lead to overeating. It seems that it resulted in offsetting the effect of suppressing insulin secretion, making it difficult to effectively treat and prevent diabetes and the like.

[0006]

Under the above circumstances, the present inventors have conducted various studies on substances having amylase inhibitory activity in wheat. As a result, a liquid containing a substance having an amylase inhibitory activity, which is obtained by extracting raw materials such as wheat and wheat flour with water, or a wheat starch waste liquid (wheat extract) obtained during the production of wheat starch, is conventionally used. Has a novel amylase inhibitory activity with high activity when treated with a specific method different from the method
That substance has been found can be obtained. That is, an extract containing a substance having an amylase inhibitory activity or a wheat starch waste solution obtained during the production of wheat starch is treated with a cation exchanger to adsorb the substance having an amylase inhibitory activity and then treated with an alkaline solution having a specific pH. By treating the cation exchanger to elute the substance having an amylase inhibitory activity and immediately acidifying the eluate containing the substance having an amylase inhibitory activity with an acid, a conventionally known substance having an amylase inhibitory activity derived from flour is obtained. It was found that the eluate contains a substance having a higher amylase inhibitory activity, especially a substance capable of strongly inhibiting the activity of human pancreatic α-amylase.
Then, the present inventors collected the substance having a high amylase inhibitory activity contained in the eluate, measured the molecular weight and determined the primary structure, and found that SEQ ID NO: 1
It was revealed to be a novel protein consisting of two subunits represented by

[0007] Furthermore, further research on the novel substance having an amylase inhibitory activity obtained above revealed that the substance having an amylase inhibitory activity consisting of two subunits represented by SEQ ID NO: 1, particularly SEQ ID NO: 1 A substance having amylase inhibitory activity, which contains a protein consisting of two subunits represented by
Compared with conventional wheat-derived amylase inhibitors, it significantly inhibits human pancreatic α-amylase activity by administration in an extremely small amount, and can significantly suppress increase in blood glucose level and insulin secretion in human, and the amount of free fatty acid in blood. Does not increase,
It was found that a feeling of satiety after a meal can be sustained, and therefore, suppression of an increase in blood glucose level and suppression of insulin secretion can be facilitated at the same time as suppression of appetite, which is extremely effective for treatment and prevention of diabetes and prevention of obesity.

In addition, amylase inhibition obtained from wheat
A protein consisting of two subunits represented by SEQ ID NO: 2 has already been known as an active substance. Although this protein has some inhibitory activity against human salivary amylase, human pancreatic α-amylase However, it has been considered to be ineffective in the intended use of the present invention. Under such circumstances,
The present inventors have conducted various studies on a protein consisting of two subunits represented by SEQ ID NO: 2 which is a known substance. As a result, the protein and sequence consisting of two subunits represented by SEQ ID NO: 1 described above <br/> substance having an amylase inhibitory active substance having an amylase inhibitory activity which contains both proteins composed of two subunits, in particular the total content of both proteins is 20 wt% or more represented by the number 2 Also, contrary to the above-mentioned conventional expectations, it exerts an extremely high inhibitory activity against human pancreatic α-amylase, has an inhibitory effect on blood glucose level elevation and insulin secretion in humans, and the amount of free fatty acid in blood is The present invention has been completed by finding that the feeling of satiety after meal can be maintained without increasing.

Therefore, the present invention provides a protein consisting of two subunits represented by SEQ ID NO: 1 (hereinafter referred to as “0.2
6AI ”), which is a substance having an amylase inhibitory activity (hereinafter, a“ substance having an amylase inhibitory activity ”is referred to as an“ amylase inhibitor ”).

The present invention is a liquid containing an amylase inhibitor obtained by extracting a wheat-derived substance such as wheat, flour or wheat gluten with water, a dilute aqueous salt solution, a buffer, an acid, a dilute alkali or a hydrous alcohol. Is purified to remove impurities, if necessary, and then treated with a cation exchanger to adsorb the amylase inhibitor, and then to pH 9-
After the amylase inhibitor was eluted from the cation exchanger by treatment with the alkaline solution of 13, the pH of the eluate containing the amylase inhibitor was immediately adjusted to acidic, and the amylase inhibitor was recovered from the pH-adjusted liquid. And a method for preparing an amylase inhibitor comprising 0.26 AI.

According to the present invention, the wheat starch waste liquor (wheat extract) obtained during the production of wheat starch is purified if necessary to remove impurities and then treated with a cation exchanger to inhibit amylase. After adsorbing the substance and then treating it with an alkaline solution having a pH of 9 to 13 to elute the amylase inhibitor from the cation exchanger, immediately adjust the pH of the eluate containing the amylase inhibitor to acidic to adjust the pH. A method for preparing an amylase inhibitor consisting of 0.26 AI, characterized in that the amylase inhibitor is recovered from the solution.

Furthermore, the present invention provides a protein having a content of 0.26 AI of 20% by weight or more, or a protein comprising the 0.26 AI and two subunits represented by SEQ ID NO: 2 (hereinafter referred to as “0.19 AI”). Content of 20% by weight or more and the content of 0.26 AI is 0.26 AI.
20% by weight or more based on the total weight of 0.19 AI , an amylase inhibitor, a blood glucose elevation inhibitor, an insulin secretion regulator or an appetite suppressant.
And the present invention provides 0.26 AI 30 to 25 per serving.
Foods adjusted to 00 mg, or 0.26 AI and 0.
The total content of 19AI is 30 ~ 2500m per serving and
The content of 0.26AI is 0.26AI and 0.19AI
Including foods adjusted to 20% by weight or more based on the total weight of

The novel 0.26 AI of the present invention has a molecular weight of approximately 25,000 as determined by gel filtration chromatography using Sephadex G-75, which will be specifically described below. In addition, this protein was modified by Davis' method [Annals New
York Academy of Science, 121, 404-427 (1964)], polyacrylamide gel electrophoresis (hereinafter referred to as “Nati
VE-PAGE ”), mobility 0.2
A single band was seen at position 6. In addition, SDS polyacrylamide gel electrophoresis (SDS-PA) according to the method of Orth et al. [Cereal Chem. Vol. 50, p190-197 (1973)].
In GE), a single band was observed at a molecular weight of 12,500. From this, 0.26 AI has a molecular weight of 1
It was found to be a protein composed of 2,500 two subunits.

Then, the amino acid sequence of the subunit was examined by using a peptide sequencer PSQ-1 (manufactured by Shimadzu Corp.), and it was found that it had a structure in which 124 amino acids represented by SEQ ID NO: 1 were bound. Therefore, 0.26AI is a protein composed of 248 amino acids consisting of two subunits represented by SEQ ID NO: 1. Also, this 0.26 AI is SS
It has a bond and is spherical.

This 0.26 AI has an extremely high inhibitory activity against amylase, particularly human pancreatic α-amylase, and is a subunit represented by SEQ ID NO: 3 which is a conventionally known wheat-derived amylase inhibitor. A protein consisting of two proteins "hereinafter referred to as" 0.53AI ") [Biochem. Biophys.
Acta. 743, 52-57 (1983)] or the protein represented by SEQ ID NO: 4 (hereinafter referred to as "0.28AI") [Phyt described above.
Ochemistry. Vol.20, No.8, 1781-1784 (1981)], it has an α-amylase inhibitory activity that is several to several tens of times higher than that of 0.18 AI. In some cases, it has about 20 to 30% higher α-amylase inhibitory activity.

Further, in the above-mentioned agent of the present invention, 0.2
0.19AI used with 6AI is Sephadex G-
About 25 by gel filtration chromatography using 75,
It has a molecular weight of 000 and, like 0.26 AI,
It has an S-S bond and is spherical. Also, 0.19A
I use the Davis method [Annals New York Academy of Scie
nce, 121, 404-427 (1964)], polyacrylamide gel electrophoresis (hereinafter referred to as “Native-PAGE”) showed a single band at the position of mobility 0.19, and the method of Orth et al. [Cereal]. Chem. Vol.50, p190-
197 (1973)], SDS polyacrylamide gel electrophoresis (SDS-PAGE) shows a single band with a molecular weight of 12,500. Therefore, 0.19AI is a protein composed of two subunits having a molecular weight of 12,500, and the amino acid sequence of the subunit has a structure in which 124 amino acids represented by SEQ ID NO: 2 are bound. It is clear that they are doing [Biochi
m. Biophys. Acta, Vol. 828, 213-221 (1985)].
Then, 0.19 AI is 0.26 AI as described above.
Although its α-amylase inhibitory activity is about 20% lower than that of, it has an extremely high α-amylase inhibitory activity when compared with 0.53AI and 0.28AI.

The 0.26AI of the present invention includes any protein composed of two subunits represented by SEQ ID NO: 1 and having an amylase inhibitory activity, and the production method thereof is not particularly limited. It may be the one according to the above. However, the 0.26 AI of the present invention
The liquid obtained by extracting the wheat-derived substance with water or the like and the wheat starch waste liquid obtained during the production of wheat starch are subjected to the above-mentioned specific treatment using a cation exchanger to ensure the above-mentioned preparation method of the present invention. Can be obtained, and the method will be described below.

(1) First, a wheat-derived substance is extracted with water, a dilute aqueous salt solution, a buffer solution, an acid, a dilute alkali or hydrous alcohol to prepare a liquid containing 0.26 AI. As the wheat-derived substance at that time, any one can be used regardless of its type, production place, harvest time, grain size and the like.
Among them, it is preferable to use wheat flour (including wheat semolina) or wheat gluten in terms of extraction efficiency. In addition, among the various wheat varieties, particularly when durum wheat is used, 0.26 of the present invention is used.
AI can be obtained in high yields, so it is particularly preferred to use durum flour or gluten obtained therefrom as wheat-derived material.

The extraction liquid is preferably water, a dilute salt aqueous solution, a buffer solution, an acid aqueous solution, or a dilute alkaline aqueous solution, but a hydrous alcohol may be used. The dilute salt aqueous solution is preferably an aqueous solution of a neutral salt such as sodium chloride or potassium chloride, and the concentration thereof is preferably 20 mM to 200 mM. Further, the buffer solution has a pH of 4 to 8.5.
A phosphate buffer solution, an acetate buffer solution, a Tris buffer solution and the like adjusted to 1 are preferably used. When an acid aqueous solution is used, it is preferable to use an acidic aqueous solution whose pH is adjusted to about 2 to 5 with an inorganic acid such as hydrochloric acid or phosphoric acid, or an organic acid such as acetic acid. When using a dilute alkaline aqueous solution, it is preferable to use an alkaline aqueous solution adjusted to pH 8 to 10 with ammonia, sodium hydroxide or the like. Further, when using a hydrous alcohol, an alcohol aqueous solution having an alcohol concentration of about 1 to 50% is preferable, and the alcohol at that time is methanol,
Examples thereof include ethanol and isopropyl alcohol. Further, it is preferable to use Tris buffer, acetate buffer or the like as the extraction liquid so that the pH of the liquid is kept constant.

In the extraction treatment, a sufficient amount (usually about 3 to 50 times) of the above extraction liquid is added to the wheat-derived substance, and the extraction treatment is usually performed at a temperature of about 10 to 40 ° C. while stirring. Then, a method of removing the solid content by an arbitrary method such as centrifugation, filtration, or standing to obtain an extract containing 0.26 AI can be adopted.

However, in the present invention, as the liquid containing the amylase inhibitor, the above-mentioned liquid obtained by extracting the wheat-derived substance with water, a dilute aqueous salt solution, a buffer, an acid, a dilute alkali or hydrous alcohol is used. Instead, a wheat starch waste solution obtained during the production of wheat starch, that is, a dough or batter washing waste solution at the time of collecting starch or gluten from wheat flour is used as it is as a solution containing an amylase inhibitor 0.26AI to give the following: (2) The subsequent steps may be carried out, and in that case, the effective utilization of the wheat starch waste liquid, which has been difficult to handle in the past, can be achieved at the same time.

(2) Then, the liquid containing 0.26 AI obtained by the extraction treatment of the above (1) or the wheat starch waste liquid obtained during the production of the above wheat starch is directly treated with a cation exchanger as it is. Alternatively, a purification treatment is applied to remove contaminants, and then a cation exchanger is used. In any case, 0.26AI of the present invention can be obtained, but it is more efficient to obtain 0.26AI of the present invention with high purity by performing purification treatment to remove impurities and then treating with a cation exchanger. This is preferable because it can be obtained well.

When the purification treatment is carried out before the treatment with the cation exchanger, any of the purification methods conventionally used for obtaining an amylase inhibitor from wheat can be adopted, and it is not particularly limited. The methods (1) to (3) can be mentioned, and the method (2) is particularly preferable.

Ammonium sulfate, ethanol, etc. were added to the extract containing 0.26 AI or the wheat starch waste liquid obtained by the extraction treatment of (1) above to obtain 0.26 AI.
A precipitate containing AI is generated, and after the precipitate is collected, it is suspended in a small amount of water and dialyzed using a dialysis membrane, and a phosphate buffer is added to the dialyzed liquid to generate a precipitate. The precipitate obtained was removed, the resulting supernatant was treated with an anion exchanger such as DEAE cellulose, and the passing solution was directly or once dried to give a powder, which was dissolved in a buffer solution to give 0.26 AI. A method of using as a containing solution, or collecting fractions having an amylase inhibitory activity by further subjecting to hydrophobic chromatography and using the fractions as a 0.26 AI containing solution.

The extract containing 0.26 AI or the wheat starch waste liquor obtained by the above-mentioned extraction treatment (1) is heated to, for example, 70 to 90 ° C. to remove heat-labile proteins and the like which remain in the liquid. A method in which impurities are denatured and solidified, separated and removed, and the resulting liquid is used as a 0.26AI-containing liquid.

The liquid obtained by the above method is further subjected to an ultrafiltration membrane (preferably having a molecular weight cutoff of 20000 or less).
Or a method using gel filtration chromatography to remove excess salts and other low-molecular contaminants, concentrating if necessary, and using the solution as a 0.26 AI-containing solution.

A liquid obtained by adjusting the pH of the extract containing 0.26 AI obtained by the extraction treatment of the above (1) to about 3 and then adjusting the pH to neutral again to separate and remove the generated precipitate. Is used as a 0.26 AI-containing solution.

(3) 0.26 A obtained in (1) above
The extract containing I or the wheat starch waste liquor is directly treated with a cation exchanger, or is subjected to the purification treatment as described in (2) above and then treated with a cation exchanger. As the exchanger, a cation exchanger such as a polymer cation exchange resin, silicic acid or aluminum silicate can be used. Specifically, CM-Toyopearl (trade name; manufactured by Tosoh Corporation), Diaion HPK-
It is preferable to use a polymer cation exchange resin such as 55 (trade name; manufactured by Mitsubishi Kasei Co., Ltd.). The treatment with the cation exchanger includes 0.26 AI in the cation exchanger packed in the column even in the batch system in which the cation exchanger is added to a liquid containing 0.26 AI and stirred to perform cation exchange. It may be carried out by any of the continuous methods of passing the liquid used, but the continuous method is preferred. When treating with a cation exchanger, it is preferable to preliminarily equilibrate the cation exchanger with a buffer solution having a pH of 3 to 5, and then to treat a solution containing an amylase inhibitor adjusted to pH 3 to 5, As a result, 0.26 AI in the liquid is adsorbed on the cation exchanger.

(4) Next, if necessary, the cation exchanger is treated with a neutral solution containing sodium chloride or the like to elute and remove impurities from the cation exchanger, and then the pH is adjusted to 9 to 10.
The cation exchanger is treated with an alkaline solution of 13 or an alkaline buffer solution of pH 9 to 10 to elute 0.26 AI of the present invention adsorbed on the cation exchanger. As the alkaline solution in this case, the kind of the alkaline compound is not particularly limited as long as the pH is 9 to 13, and the alkaline compound such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and ammonium hydroxide. Aqueous solutions of can be used, with aqueous solutions of sodium hydroxide being preferred. When using an aqueous solution of sodium hydroxide, adjust the concentration of sodium hydroxide to 0.05 to 1.0.
N is desirable. In the case of an alkaline buffer solution, it is desirable that the concentration is 20 mM to 1M. If the pH of the alkaline solution is lower than 9, the pH of the cation exchanger will be 0.
26AI cannot be smoothly eluted. On the other hand, when the pH of the alkaline solution is higher than 13, 0.26AI is denatured at the time of elution and the desired 0.26AI cannot be obtained.

(5) Then, an acid is immediately added to the 0.26AI-containing solution eluted from the cation exchanger to obtain p of the solution.
Adjusting H to 2-5, 0.26A contained in the extract
Prevent I denaturation. In this case, the pH adjustment of the alkaline eluate with an acid is performed by temporarily accumulating the alkaline eluate from the cation exchanger in a container or the like and adding the acid thereto.
The acid may be continuously added to the container at the same time as the alkaline container liquid flows into the container, or the acid solution may be put in the container in advance and the alkaline solution may be allowed to flow thereinto. In this case, it is necessary to adjust the pH of the alkaline solution containing 0.26 AI eluted from the cation exchanger to 2 to 5 with acid as soon as possible. As the acid for adjusting the pH of the alkaline eluate, inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid, and organic acids such as acetic acid can be used, but hydrochloric acid is preferred.

(6) Finally, p obtained in (5) above
A solution containing 0.26 AI in which H is adjusted to 2 to 5 is subjected to dialysis and other purification treatments if necessary, and then dried by any method such as freeze drying, reduced pressure drying, spray drying, and ball drying. By doing so, the desired 0.26 AI can be obtained. Then, in the case of the above method, in general, an amylase inhibitor containing 0.26 AI at a high concentration of 20% by weight or more can be obtained. This 0.2
6AI has an extremely high inhibitory activity against human pancreatic α-amylase and has a high function as an insulin secretion regulator, and suppresses digestion of cooked starch such as cooked rice with a small amount of use, that is, It is possible to effectively suppress the decomposition of heat-treated starch into sugar, and it has a function as a high blood sugar level elevation inhibitor, and also has an appetite suppressing function.

Generally, a protein having a physiological function such as an enzyme is easily denatured under a strong alkaline condition of pH 9 or higher,
Therefore, even in the conventional technique for preparing an amylase inhibitor from a wheat-derived substance, an alkaline solution having a pH of 9 or higher was not used. On the other hand, in the present invention, an alkaline solution having a pH of 9 to 13 which has not been conventionally used is used, and the cation exchanger is treated with the alkaline solution to obtain 0.53 AI or 0.28 which is a conventional wheat-derived amylase inhibitor.
It is possible to obtain the novel 0.26AI of the present invention having a remarkably high amylase inhibitory activity as compared with AI and the like, and it is possible to obtain such a treatment operation of the present invention and the excellent properties of the novel 0.26AI obtained thereby. Is totally unexpected.

Further, 0.19AI used together with 0.26AI in the present invention is a known protein before the application of the present invention, and its preparation method is not particularly limited, and it may be prepared by any method. However, the amylase inhibitor containing 0.19 AI at a high concentration of 20% by weight or more can be smoothly obtained.

[0035] A containing not less than 20% by weight of 0.19 AI
Preparation Example of Millase Inhibiting Substance : (a) Wheat-derived substance is extracted with water, acid (pH about 2 to 6), alkali (pH about 8 to 10) or hydrous alcohol to obtain an extract containing 0.19 AI. After that, (b) a polysaccharide that forms an insoluble complex with 0.19AI is added to the extract to form an insoluble complex of 0.19AI and the polysaccharide, and the complex is separated from the liquid. And (c) separating and removing the polysaccharide from the fractionated complex to fractionate a liquid containing 0.19 AI, and (d) using the cation exchanger for the fractionated liquid containing 0.19 AI. 0.19 AI is recovered from the fraction not adsorbed by the cation exchanger to obtain an amylase inhibitor having a content of 0.19 AI of 20% or more.

More specifically, the above step (a) is usually carried out by adding about 3 to 50 times the extraction liquid to the wheat-derived substance, and usually about 1
It is preferable to carry out at 0 to 40 ° C., and the 0.19AI-containing extract obtained in step (a) is preferably subjected to a purification treatment to obtain impure soluble proteins, soluble saccharides, inorganic salts, soluble pigments, etc. contained therein. The impurities are removed and the mixture is subjected to the step (b).

In step (b), 0.1 from step (a)
An extract containing 9AI is water-soluble and has a cation exchange function such as sodium alginate, CMC (sodium carboxymethyl cellulose), κ-carrageenan, ν-carrageenan, and λ-carrageenan capable of forming an insoluble complex with 0.19AI. Water-soluble polysaccharides such as polysaccharides, pectin, xanthan gum, gellan gum, etc., preferably sodium alginate, are generally added at a ratio of 50 to 600 ppm, preferably stirred under conditions of pH 1 to 6 and 1 to 30 ° C. An insoluble complex of .19AI and a water-soluble polysaccharide is formed, and the complex is separated from the liquid by filtration, centrifugation, or any other suitable method.

Then, in the step (c), the insoluble complex of 0.19AI and the polysaccharide obtained by fractionation in the step (b) is treated with water, ammonia, a weakly alkaline aqueous solution such as ammonium hydrogen carbonate, calcium, potassium. It is preferably about 30 to 70 when placed in a solution consisting of an aqueous solution of salts containing no
After heating to a temperature of ℃ to dissociate into 0.19AI and polysaccharides, metal ions such as potassium ions, calcium ions, and magnesium ions are added to this solution to gelate the polysaccharides to form solid insoluble matter. In addition, 0.19AI is dissolved and left in the solution as it is, and the gelled polysaccharide is separated and removed from the solution by an appropriate method to recover a solution containing 0.19AI.

Then, the solution containing 0.19 AI after the polysaccharides are separated and removed in the step (d) is treated with a cation exchanger to obtain the target 0.
An amylase inhibitor containing 19 AI in a proportion of 20% by weight or more is obtained. In this step (d), the solution containing 0.19 AI obtained in the above step (c) may be directly treated with the cation exchanger in the step (d) or may be subjected to another purification step as necessary. It may be subjected to a cation exchanger treatment after being treated with a cation exchanger, and it is generally preferable that the treatment with a cation exchanger after undergoing another purification step has a higher amylase inhibitory activity and other undesirable substances such as trypsin inhibitor. 0.19 AI with a lower substance content can be obtained, which is desirable.

In the case where another purification step is carried out before the cation exchanger treatment in the step (d), the solution containing 0.19AI obtained in the step (c) is heated to, for example, 70 to 90 ° C. After heat-unstable proteins and other impurities that remain in the solution are denatured and solidified and separated and removed, the remaining solution is further processed through an ultrafiltration membrane or gel filtration chromatography to remove excess salts and other impurities. It is desirable to remove low-molecular contaminants and perform concentration. In that case, as the ultrafiltration membrane, it is preferable to use a polyacrylonitrile-based, polyolefin-based, polysulfine-based, polyimide-based or cellulose-based material having a molecular weight cutoff of 20,000 or less.

When treating a solution containing 0.19 AI with a cation exchanger, a cation exchanger such as a polymer cation exchange resin, silicic acid or aluminum silicate can be used, and DIAION HPK-55. Polymer type cation exchange resins such as (trade name; manufactured by Mitsubishi Kasei Co., Ltd.) are preferable. The treatment with the cation exchanger may be carried out by either a batch method in which the cation exchanger is added to the liquid and stirred for cation exchange, or a continuous method in which the liquid is passed through the cation exchanger packed in the column. Good, but continuous is preferred. The treatment with a cation exchanger is preferably carried out by adjusting the pH of the solution containing 0.19AI to 6 to 9, so that 0.19AI in the solution is not passed through the column or the cation exchanger. It is collected as an adsorption fraction (in the case of batch type). By this cation exchanger treatment, the harmful trypsin inhibitor is adsorbed to the cation exchanger, and the solution containing 0.19AI without the trypsin inhibitor at a high concentration can be efficiently recovered. After being subjected to sterilization or sterilization treatment (for example, heating, alcohol sterilization, sterilization filtration, etc.) or concentration treatment as necessary, it is dried to obtain an amylase inhibitor containing 0.19 AI at a high concentration of 20% or more. A powder can be obtained. In addition,
A method for preparing an amylase inhibitor containing 0.19 AI in an amount of 20% or more is specifically described in Japanese Patent Application No. 4-127970 (JP-A-5-301898) filed by the present applicant.

In the present invention, the above-mentioned 0.26A
The content of I is 20% by weight or more, or 0.26
The total content of AI and 0.19AI is 且 at 20 wt% or more
The content of 0.26AI is 0.26AI and 0.19AI
20% by weight or more based on the total weight of the above is used as an amylase inhibitor, a blood sugar level increase inhibitor, an insulin secretion inhibitor or an appetite suppressant . In that case , 0.
26AI and 0.19AI may be highly purified products or roughly purified products, and may be used in an appropriate combination of both.

0.26 A in amylase inhibitor
When the content of I or the total content of 0.26 AI and 0.19 AI is less than 20% by weight, the amylase inhibitory action is small even if a large amount of such amylase inhibitor is administered, and the blood sugar level is suppressed. The action and insulin secretion regulating action are reduced, and moreover, it cannot be effectively used as an appetite suppressant. On the other hand, the upper limit of the content of 0.26 AI or the total content of 0.26 AI and 0.19 AI is not limited, and the higher the content of these proteins in the amylase inhibitor, the higher the amylase inhibitory effect and The effect of suppressing increase in value, the effect of regulating insulin secretion and the effect of suppressing appetite are also increased. However, the higher the purity, the lower the productivity. From the viewpoint of productivity and economy, the upper limit of the content of 0.26 AI in the amylase inhibitor or the total content of 0.26 AI and 0.19 AI is set. Is preferably about 80% by weight.

The amylase inhibitor used in the present invention is, in addition to 0.26 AI and 0.19 AI, other proteins, peptides and other proteins derived from wheat if the amount is less than 80% by weight. Other substances (eg starch, dietary fiber, vitamins, minerals, etc.),
In general, amylase inhibitors used in the present invention include
7 of its total weight, including 26AI and 0.19AI
It is preferred that 0% by weight or more consists of protein.

The amylase inhibitor containing 0.26 AI or the amylase inhibitor containing 0.26 AI and 0.19 AI may be used as it is or in the form of a liquid, granule, tablet using a known carrier or auxiliary agent. And the like, and can be used as an amylase inhibitor, a blood sugar level elevation inhibitor, an insulin secretion regulator and / or an appetite suppressant. Furthermore, it can be used by adding it to foods, especially carbohydrate-based foods such as bread and cookies containing a lot of starch, teas,
It can be used by adding it to spreads such as soups, sprinkles, butter and jam. The amount of amylase inhibitor containing 0.26AI or 0.26AI and 0.19AI to humans and the amount to be added to foods depends on whether they are administered to diabetic patients or healthy subjects, or Depending on the condition and symptoms of the recipient, it can be appropriately adjusted depending on the type of food to which it is added and its intake amount, but in the case of the agent of the present invention, a small amount is high, It is possible to achieve an amylase inhibitory effect, a blood sugar level increase suppressing effect, an insulin secretion regulating effect and a satiety sensation sustaining effect. Especially when added to foods,
The content of 0.26A is adjusted to 30 to 2500 mg per serving, or the total content of 0.26 AI and 0.19 AI is 30 to 2500 mg per serving and 0.2
Total weight of 6AI content of 0.26AI and 0.19AI
The amount is adjusted to 20% by weight or more based on the amount .
It is preferable to contain 26 AI or 0.26 AI and 0.19 AI.

For example, the content of 0.26 AI, or the total content of 0.26 AI and 0.19 AI is 30% by weight.
When the blood sugar level elevation inhibitor of the present invention is administered to a healthy person, the blood glucose level elevation suppression effect is observed even at a single dose of 100 mg, and 500 to 2,500 mg per dose
When administered three times a day, the rise in blood sugar level can be suppressed very well. Further, in a diabetic patient, a lower dose (generally about 300 to 1,500 mg per day) can effectively suppress an increase in blood glucose level. Furthermore,
0.26 AI content, or 0.26 AI and 0.19
When an appetite suppressant having a total AI content of 30% by weight is administered to a person at a rate of 1,000 to 5,000 mg per meal with a person, the amount of free fatty acid in the blood does not increase and hunger is suppressed. The appetite can be suppressed without being distressed.

Therefore, the agent of the present invention is 0.26A
The content of I is 20% by weight or more, or 0.26
The total content of AI and 0.19AI is 且 at 20 wt% or more
The content of 0.26AI is 0.26AI and 0.19AI
20% by weight or more based on the total weight of the above is formulated so as to be contained in an amount of 100 to 5,000 mg, preferably 500 to 2,500 mg per dose per administration. Desirably, for example, one capsule, tablet,
Per pill or pellet, per packet of powder, granules, or 100 to 5,000 mg contained in one liquid, and if they are administered one at a time, there will be no excess or deficiency. And it can be taken easily.

[0047]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Also,
In the following examples, molecular weight determination by gel filtration chromatography using Sephadex G-75, 0.26 AI
Determination of amino acid sequence, total protein content, 0.26
Determination of AI or 0.19AI content, amylase inhibitors (0.26AI, 0.19AI, 0.53AI, 0.
The measurement of the inhibitory activity of 28AI) on human pancreatic α-amylase, the measurement of blood glucose level, the measurement of insulin amount, and the evaluation of the satiety persistence were carried out as follows. Further, the obtained 0.26-AI Native-PAGE was prepared by the method of Davis [Annals New York Academy of Scienc] as described above.
e, 121, 404-427 (1964)]. Furthermore, S
DS-PAGE is the method of Orth et al. [Cereal Chem. Vol.
0, 190-197 (1973)].

Determination of molecular weight by gel filtration chromatography using Sephadex G-75: 0.26 A obtained
For I, 4 ml of buffer (20 mM Tris, 200 mM
NaCl, pH 8.0) was added to prepare a sample solution, and the solution was applied to a Sephadex G-75 gel filtration column (1.6 cm × 90 cm) that had been equilibrated with a buffer solution in advance, and 0.5 ml /
Gel filtration chromatography was performed at a flow rate of min to measure the molecular weight.

Determination of amino acid sequence of 0.26AI : The obtained 0.26AI was pyridylethylated and then hydrolyzed using V8 protease and lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.). The hydrolyzed sample was subjected to HPLC to obtain the hydrolyzed peptide fragment. The obtained peptide plugment was used as a peptide sequencer P
Using SQ-1 (manufactured by Shimadzu Corporation), analysis was performed from the N-terminal of the peptide to determine the total primary structure. The C-terminus was digested with carboxypeptidase and the released amino acid was analyzed and determined.

Measurement of total protein content : Keltec Auto 1
It was measured by the Kjeldahl method using a 030 type machine (manufactured by Tecator). The nitrogen / protein conversion factor was 5.70.

Measurement of 0.26 AI or 0.19 AI content
Assay : Dissolve the sample in 0.1% trifluoroacetic acid aqueous solution,
Subjected to high performance liquid chromatography under the conditions shown in Table 1 below, 0.26 AI or 0.
The peak area of 19 AI was measured. On the other hand, 0.26 AI
Alternatively, 0.19AI standard (purity 100%) is subjected to high performance liquid chromatography under the same conditions to measure the peak area of 0.26AI or 0.19AI in the chromatogram, The .26AI or 0.19AI content was calculated.

[0052]

[Equation 1] 0.26 AI or 0.19 AI content in the sample
(%) = (Sa / St) × 100 where Sa = peak area of 0.26 AI or 0.19 AI of sample St = peak area of 0.26 AI or 0.19 AI of standard

[0053]

[Table 1] Chromatographic conditions Column : Packing material: CAPCELL PAK C18 SG120A (particle size 5 μm) (manufactured by Shiseido Co., Ltd.) Size: 4.6 mmφ × 250 mm Temperature: 50 ° C. Flow rate : 1 ml / min Detection : Absorbance at 280 nm Mobile phase : Liquid A; 0.1% trifluoroacetic acid aqueous solution B solution; high-pressure linear gradient end elution time (minutes) A solution (%) B solution consisting of 80% acetonitrile and 0.1% trifluoroacetic acid aqueous solution with the following time / concentration gradient (%) 0 65 35 3 62 38 38 15 62 38 38 16 0 100 20 0 100

Of human pancreatic α-amylase inhibitory activity
Measurement : 50 mM aqueous solution of sample and human pancreatic α-amylase
20 mM piperazine-N, N'-bis (2-ethanesulfonic acid) buffer solution (pH 6.9) containing NaCl, 5 mM CaCl ₂ and 0.02% ovalbumin, and added at 37 ° C.
After standing at room temperature for 30 minutes, 1.5% soluble starch solution (pH
0.5 ml of 6.9) was mixed. Add this solution to 37 ° C for 10
After allowing the reaction to proceed for 2 minutes, 2.5 ml of the quenching solution (0.08M HCl and 0.4M acetic acid) was added. 0.2 ml was sampled from the reaction solution, 2.5 ml of iodine solution (0.05% KI and 0.005% iodine) was added thereto, and the absorbance at 660 nm was measured. In the measurement, the amount of amylase that reduces the absorbance by 80% when the sample solution was not used was used, and the amount of amylase inhibitor that inhibits the amylase activity by 50% was expressed as 1 unit (U).

Measurement of blood glucose level : Blood was collected from the forearm vein of the subject, and the blood glucose level was immediately measured by the glucose oxidase method. For measurement by the glucose oxidase method,
Glucose-B-Test Wako [Wako Pure Chemical Industries, Ltd.
Manufactured] was used.

Measurement of insulin amount : Blood was collected from a forearm vein of a subject, serum was immediately obtained by centrifugation, and the amount of insulin in the serum was measured by enzyme immunoassay. For the measurement by the enzyme immunoassay, Grazyme Insulin-EIA test [manufactured by Wako Pure Chemical Industries, Ltd.] was used.

Evaluation of persistence of satiety : The subject was asked to select one of the seven grades shown in Table 2 below, which seemed appropriate, and the average value was taken.

[0058]

[Table 2]

Example 1 2 g of water was added to 500 g of durum flour and stirred at room temperature for 3 hours. After that, the precipitate was removed by centrifugation, and the resulting supernatant was adjusted to pH 3 with hydrochloric acid and left as it was for 1 hour. Then, an aqueous solution of sodium hydroxide was added to the supernatant to adjust the pH to 6
The mixture was adjusted to 1, and left for 1 hour, and then centrifuged to remove the precipitate. Ammonium sulfate was added to the obtained supernatant liquid while stirring so as to be 45% saturated, and after standing for 2 hours, this liquid was centrifuged to collect a precipitate.

To the precipitate collected above, 50 ml of water was added to suspend the precipitate, and desalting was performed overnight using a dialysis membrane (manufactured by Visking). A phosphate buffer (pH 7.6) was added to the desalted solution so that the concentration was 20 mM, and the mixture was stirred sufficiently and then centrifuged to remove the precipitate. The obtained supernatant is applied to an anion exchange resin column (manufactured by Tosoh Corporation; “DEAE-Toyopearl column”) that has been equilibrated with 20 mM phosphate buffer in advance, and the column is thoroughly washed with the same buffer. The fractions that passed through were collected. After dialysis of the collected fractions against water, 20 mM containing 200 mM NaCl
Again dialyzed against Tris buffer. After dialysis, use a mini module (Asahi Kasei) to concentrate to 4 ml,
The gel filtration column (manufactured by Pharmacia; "Sephacryl S-200") pre-equilibrated with the same buffer was applied to collect fractions having amylase inhibitory activity. The collected fractions were dialyzed against water, and then the acetate buffer (pH 4.0) was added to 2 times.
The mixture was added to a concentration of 0 mM, equilibrated with the same acetate buffer in advance, and applied to a cation exchange resin column (manufactured by Tosoh Corporation; “CM-Toyopearl column”).

Then, an acetate buffer containing 100 mM sodium chloride was passed through a cation exchange resin column to elute impurities, and then a 0.1 N sodium hydroxide aqueous solution was passed through the column to elute 0.26 AI from the column. It was Hydrochloric acid was immediately added to the eluate to adjust the pH of the liquid to 3.0. This solution was dialyzed against water and then freeze-dried to obtain 60 mg of a white product mainly composed of 0.26 AI.

Sephad as described above using the product obtained above
When the molecular weight was measured by gel filtration chromatography using ex G-75, it had a molecular weight of about 25,000. Further, when subjected to SDS-PAGE as described above, a single band was provided at a position corresponding to a molecular weight of 12,500. Therefore, it was revealed from these that the above product was composed of two subunits having a molecular weight of 12,500. When the amino acid sequence of the subunit was examined by the method described above, it had a structure in which 124 amino acids represented by SEQ ID NO: 1 were bound.

The total protein content and 0.26AI content in the above product were measured by the above method and were 95% and 91%, respectively. In addition to 0.26 AI, the product contained 4% of contaminating proteins and 5% of water. The human pancreatic α-amylase inhibitory activity of the product obtained in this Example 1 was measured by the above-mentioned method and was as shown in Table 3 below.

Reference Example 1 Gluten was added to the product obtained in Example 1 above to give a 0.26 AI content of 15%.
Was prepared and its human pancreatic α-amylase inhibitory activity was measured. The results are shown in Table 3 below.

Reference Example 2 (1) Add 800 liters of wheat flour to 110 liters of water,
Kneaded to form a dough. The dough was washed with 7,600 liters of water to recover 410 kg of gluten and 505 kg of wheat starch. At that time, 6,200
Since 1 liter of waste liquid was generated, this waste liquid (water extraction liquid)
Hydrochloric acid was added to the mixture to adjust the pH to 3, the mixture was allowed to stand for 30 minutes, and then the pH was adjusted to 6.5 with ammonia, insoluble matters were precipitated. The precipitate was removed and 5,200 liters of the supernatant was recovered.

(2) Sodium alginate was added to the above-obtained supernatant in an amount such that its concentration would be 300 ppm, the pH was adjusted to 4.2, and the mixture was stirred for 30 minutes. As a result, a water-insoluble matter was produced, and this water-insoluble matter was recovered using a DeLaval centrifuge. The recovered material was dispersed in 10 times the amount of water, 4.7 kg of calcium chloride was added, and the mixture was stirred well, the pH was adjusted to 8.5 with ammonia, and the mixture was left standing for 1 hour. Then, using a de Laval centrifuge, the solids are separated and removed, and the supernatant liquid 60
0 liter was collected.

(3) The supernatant recovered above was neutralized with hydrochloric acid, the neutralized solution was heated at 80 ° C. for 30 minutes, and the produced insoluble matter was separated by a de Laval centrifuge to obtain the supernatant. The supernatant was collected and the supernatant was used as an ultrafiltration membrane [Nitto Denko Corporation
(Manufactured by NTU-3250 CIR (20,000 fractionation)], and the excess calcium salt was desalted to obtain a concentrated liquid.

(4) 140 L of the concentrate obtained above was adjusted to pH 7.5 with ammonia, and the cation exchange resin [DIAION HPK-55; manufactured by Mitsubishi Kasei Co.] 28
Column filled with liter (length 900 mm, inner diameter 20
0 mm) at a flow rate of 1 liter / min, and a fraction eluted without being adsorbed on the cation exchange resin was collected. The above-mentioned eluate fraction was sterilized and filtered using a ceramic filter and then freeze-dried to obtain 1,400 g of a dry powder product. (5) When the total protein content and the content of 0.19 AI of the dry powder product obtained in (4) above were measured,
The human pancreatic α-amylase inhibitory activities were as shown in Table 3 below, which were 84% and 21%, respectively, and contained 0.19AI at a high concentration.

Reference Example 3 The concentrated liquid obtained in the step (3) of Reference Example 2 was dried to give a powder, and the total protein content and the content of 0.19 AI in the obtained powder were measured. % And 15%, and the human pancreatic α-amylase inhibitory activity was as shown in Table 3 below.

Example 2 To 50 mg of the white product obtained in Example 1, 20 mM acetate buffer (pH 4.0) was added, and the CM-Sepharose column (Pharmacia) was equilibrated with the same buffer in advance. (Made by the company). After thoroughly washing the column with the same buffer, a linear gradient of 0 to 300 mM sodium chloride was applied to separate and elute 0.26 AI, which was the target substance. The obtained separated solution was thoroughly dialyzed against water and then freeze-dried to obtain 35 mg of a white powder. The total protein content, the 0.26 AI content and the amylase inhibitory activity of the obtained powder were measured by the above-mentioned methods and were as shown in Table 3 below.

Reference Example 4 120 ml of the acetic acid buffer solution (pH 4.0) was added to 10 g of the dried product obtained in Reference Example 2 and the mixture was stirred for 30 minutes, and acetic acid was added to adjust the pH to 4.5. This solution was previously equilibrated with the same buffer solution "C
20 m from "M-Toyopearl column" (manufactured by Tosoh Corporation)
After thoroughly washing with M acetate buffer (pH 4.0), 350 mM
Acetate buffer containing sodium chloride (20 mM, pH
In 4.0), 0.19 AI, which is the target substance, was separated and eluted. The obtained eluate was further washed thoroughly by applying it to a "Butyl-Toyopearl column" (manufactured by Tosoh Corporation) equilibrated with a 20 mM acetate buffer (pH 4.0) containing 350 mM sodium chloride in advance. Then, a 20 mM acetate buffer (pH 4.
0.19AI, which is the target substance, was separated and eluted in 0), and the resulting solution was thoroughly dialyzed against water and freeze-dried.
5 g of white powder was obtained. The total protein content of the obtained powder,
The 0.19AI content and amylase inhibitory activity were measured by the methods described above, and were as shown in Table 3 below.

Example 3 To 6.2 tons of the washing waste liquid of dough or batter discharged when collecting starch and gluten from wheat flour was added hydrochloric acid to adjust the pH to 3.0, and
After standing for a minute, the pH was adjusted to 6.5 with ammonia, and insoluble matter was precipitated. This precipitate was removed and 5.2
Tons of supernatant was collected. This supernatant has a concentration of 30
Sodium alginate was added in an amount such that the concentration became 0 ppm, and then hydrochloric acid was added to adjust the pH to 4.2 and the mixture was stirred for 30 minutes. The resulting precipitate is collected by centrifugation (65k
g) and 650 liters of water were added and dispersed, 4.7 kg of calcium chloride was added and well stirred. Then, the pH of the dispersion liquid was adjusted to 8.5 with ammonia, the mixture was allowed to stand for 1 hour, and then the precipitate was removed by centrifugation to collect 600 liters of the supernatant liquid.

Sodium phosphate was added to the recovered supernatant in an amount such that its concentration was 0.1%, the pH was adjusted to 6.5 with ammonia, and then heat treatment was performed at a temperature of 80 ° C. for 15 minutes. After removing the precipitate due to heat denaturation by centrifugation, desalting and concentration were performed using an ultrafiltration membrane (molecular weight 20,000; “NTU-3250CIR” manufactured by Nitto Denko), freeze-dried and dried powder 1 .4 kg was obtained. To 500 mg of this dry powder, 5 ml of 20 mM buffer solution (pH 4.0) containing 100 mM sodium chloride was added and dissolved, and the insoluble matter was removed by centrifugation, and the resulting solution was separated by Sephadex.
Gel filtration chromatography was performed on G-100. The eluate of the active peak portion was collected, placed in a dialysis tube and dialyzed against 20 mM acetate buffer (pH 4.0) at 4 ° C. overnight. The solution after the dialysis was passed through a cation exchange resin column (CM-Topal) which had been equilibrated with the same buffer in advance to adsorb the target substance.

20 mM acetate buffer (pH 4.0) containing 300 mM sodium chloride was passed through the cation exchange resin column to elute impurities, and then the target substance was added with 0.05N sodium hydroxide solution (pH 12.2). Was eluted.
Hydrochloric acid was immediately added to the eluate to adjust the pH to 3.0, desalted sufficiently using a dialysis tube, and then freeze-dried to obtain a white powdery product. The protein content and 0.26 AI content of the obtained product were measured by the above method,
94% and 88%, respectively. In addition, the human pancreatic α-amylase inhibitory activity of this product was measured by the above-mentioned method and was as shown in Table 3 below.

Example 4 To 100 g of flour was added 1 liter of a 1% sodium chloride aqueous solution, and the mixture was stirred at room temperature for 2 hours. The precipitate was removed by centrifugation, the resulting supernatant was separated, ammonium sulfate was added to 50% saturation, and the mixture was stirred at room temperature for 1 hour and then left still for 1 hour. The precipitate was collected by centrifugation, dispersed in 200 ml of distilled water, and dialyzed against deionized water overnight. After adjusting the pH to 4.0 by adding hydrochloric acid to the dialyzed solution, the remaining precipitate was removed by centrifugation to collect the supernatant. The obtained supernatant was passed through a cation exchange resin column (CM-Sepharose column) that had been equilibrated with an acetate buffer (20 mM, pH 4.0) in advance to adsorb the target substance onto the ion exchange resin. . After thoroughly washing with the same buffer, impurities were eluted with 20 mM acetate buffer (pH 4.0) containing 400 mM sodium chloride.

Then, the target substance was eluted with a 0.05N sodium hydroxide aqueous solution (pH 12.2), hydrochloric acid was added to the eluate to adjust the pH to 3.0, and then 50 mM acetate buffer containing 100 mM sodium chloride was added. It was sufficiently dialyzed against (pH 4.0). The dialyzed solution was concentrated with an ultrafiltration membrane (molecular weight 20,000) and then subjected to gel filtration chromatography (20 mM acetate buffer; p with Sephadex G-75).
H4.0, 100 mM sodium chloride) for further purification. After purification, the product was thoroughly dialyzed against deionized water using a dialysis tube and then freeze-dried to obtain a white powdery product. Protein content of the product obtained and 0.2
When the 6AI content was measured by the above method, it was 9
6% and 93%. In addition, the human pancreatic α-amylase inhibitory activity of this product was measured by the above-mentioned method and was as shown in Table 3 below.

Comparative Examples 1 and 2 For comparison, the human pancreatic α-amylase inhibitory activity was measured in the same manner for the known amylase inhibitor 0.53AI and the known amylase inhibitor 0.28AI. The results are shown in Table 3 below.

[0078]

[Table 3] Total protein content Amylase inhibitor Protein amylase inhibitory activity Product (%) Species content (U / mg) (%) Example 1 95 0.26AI 91 23,300 Example 2 100 0.26AI 100 26,100 Example 3 94 0.26AI 88 22,030 Example 4 96 0.26AI 93 23,500 Reference Example 1 99 0.26AI 15 1,350 Reference Example 2 84 0.19AI 21 3,550 Reference Example 3 60 0.19AI 15 2,160 Reference Example 4 100 0.19AI 100 20,300 Comparative Example 1 100 0.53AI 100 3,940 Comparative Example 2 100 0.28AI 100 840

From the results of Table 3 above, 0.26 of the present invention was obtained.
AI has an extremely high amylase inhibitory activity, which is several times to several tens of times higher than that of conventional amylase inhibitors (0.53 AI and 0.28 AI), and 0.19.
It can be seen that the amylase inhibitory activity is about 30% higher than that of AI.

<< Example 5 >> 0.2 obtained in Example 2
6AI and 0.19AI obtained in Reference Example 4, and wheat gluten as the balance, 0.26AI and 0.
A wheat protein containing 19AI in the amount shown in Table 4 below was prepared based on the total weight of the wheat protein, and its amylase inhibitory activity was measured by the above-mentioned method.

[0081]

[Table 4] 0.26AI content 0.19AI content Total content of 0.26A1 and 0.19AI Amylase inhibitory activity (wt%) (wt%) (wt%) (U / mg) 20 5 25 6,300 15 10 25 5,600 5 20 25 5,100 35 5 40 9,800 20 20 40 8,900 10 30 40 8,400

From the results of Table 4 above, the total content of 0.26 AI and 0.19 AI is 20% by weight or more and
If the content of 0.26 AI is 0.26 AI and 0.19 AI
It can be seen that the protein of 20% by weight or more with respect to the total weight has a high amylase inhibitory activity and can be effectively used as an amylase inhibitor.

Example 6 Healthy Boy 2 Without Diabetes
Fasting one female and one female for 10 hours, then ingesting 300 g of cooked rice and 200 ml of unsweetened black tea, collecting blood every 30 minutes after eating to measure the blood sugar level and insulin level, and maintaining a feeling of fullness The sex was evaluated. The test was conducted 5 times in total for each person at intervals of 1 week. The first of the 5 tests was the product of Example 1, the second was the product of Reference Example 1 and the third was the product of Example 5 (0.26 AI content 20 wt. % Wheat protein having an AI content of 0.19% by weight), and the fourth time, 350 mg each of the product of Reference Example 3 was put into black tea and ingested. In addition, in the fifth time, only rice and black tea were ingested without adding the product (control). The results are shown in Table 5 below. The results in Table 5 are the average values of 3 persons.

[0084]

[Table 5] 1st time 2nd time 3rd time 4th time 5th time Product type Example 1 Reference Example 1 Example 5 Reference Example 3 None (0.26AI) (0.26AI) (0.26AI + 0.19AI) (0.19AI) ) (Reference) (91%) (15%) (20% + 20%) (15%) Blood sugar level (mg / dl) : Before meal 101 103 101 99 102 30 minutes after meal 128 158 141 167 162 60 minutes 150 187 155 178 183 90 minutes 145 151 148 140 148 120 minutes 116 130 123 127 124 Insulin amount (μU / ml ) : Before meal 9 12 8 10 8 After meal 30 minutes 14 27 15 25 23 60 minutes 26 38 30 35 32 90 minutes 22 30 24 36 33 120 minutes 20 33 21 33 30 Full stomach Before meal 1.9 1.7 2.0 2.1 2.1 30 minutes after meal 5.3 4.9 5.0 4.9 4.8 60 minutes 4.3 4.0 4.4 4.0 4.1 90 minutes 4.9 3.3 4.8 3.7 3.5 120 minutes 4.1 3.1 3.7 3.2 3.0

From the results in Table 5 above, 0.26 AI is 2
The product of Example 1 containing 0% by weight or more, and 0.2
When the total content of 6AI and 0.19AI is 20% by weight or more
And the content of 0.26AI is 0.26AI and 0.19A
When the product of Example 5 is used in an amount of 20% by weight or more based on the total weight of I, it is effective in suppressing an increase in blood sugar level in blood and suppressing insulin secretion, and can maintain a feeling of satiety. I understand.

[0086]

INDUSTRIAL APPLICABILITY The novel 0.26AI of the present invention has a high amylase inhibitory activity, and the content of 0.26AI is 20% by weight or more , or the sum of 0.26AI and 0.19AI. The content is 20% by weight or more and 0.
26AI content is the sum of 0.26AI and 0.19AI
The amylase inhibitor, the blood glucose elevation suppressor, the insulin regulator and / or the appetite suppressor of the present invention, which is 20% by weight or more based on the weight , has a high inhibitory activity against human pancreatic α-amylase and has a high inhibitory activity on humans. It is extremely effective, has a high effect on suppressing the rise in blood sugar level and regulating insulin secretion,
You can maintain a feeling of fullness and easily suppress appetite,
It is extremely effective in the prevention and treatment of hyperglycemia, diabetes, hyperlipidemia, arteriosclerosis, obesity, etc. Especially 0.26 AI
The agent of the present invention containing is effective for suppressing the digestion of cooked starch such as cooked rice, inhibits the activity of amylase, suppresses the decomposition of starch into sugar, and raises the blood glucose level. Can be suppressed. The above-mentioned various excellent effects of the agent of the present invention can be extremely easily achieved by oral administration. Furthermore, the above-mentioned agent of the present invention can exert the various excellent effects described above without causing side effects.

[0087]

[Sequence list]

SEQ ID NO: 1 Sequence length: 124 Sequence type: Amino acid Topology: linear Sequence Type: Protein Array Ser Gly Pro Trp Met Cys Tyr Pro Gly Tyr Ala Phe Lys Val Pro Ala  1 5 10 15 Leu Pro Gly Cys Arg Pro Val Leu Lys Leu Gln Cys Asn Gly Ser Gln              20 25 30 Val Pro Glu Ala Val Leu Arg Asp Cys Cys Gln Gln Leu Ala Asp Ile          35 40 45 Ser Glu Trp Cys Arg Cys Gly Ala Leu Tyr Ser Met Leu Asp Ser Met      50 55 60 Tyr Lys Glu His Gly Val Gln Glu Gly Gln Ala Gly Thr Gly Ala Phe  65 70 75 80 Pro Ser Cys Arg Arg Glu Val Val Lys Leu Thr Ala Ala Ser Ile Thr                  85 90 95 Ala Val Cys Lys Leu Pro Ile Val Ile Asp Ala Ser Gly Asp Gly Ala             100 105 110 Tyr Val Cys Lys Gly Val Ala Ala Tyr Pro Asp Ala         115 120

SEQ ID NO: 2 Sequence length: 124 Sequence type: Amino acid Topology: linear Sequence Type: Protein Array Ser Gly Pro Trp Met Cys Tyr Pro Gly Gln Ala Phe Gln Val Pro Ala  1 5 10 15 Leu Pro Ala Cys Arg Pro Leu Leu Arg Leu Gln Cys Asn Gly Ser Gln              20 25 30 Val Pro Glu Ala Val Leu Arg Asp Cys Cys Gln Gln Leu Ala His Ile          35 40 45 Ser Glu Trp Cys Arg Cys Gly Ala Leu Tyr Ser Met Leu Asp Ser Met      50 55 60 Tyr Lys Glu His Gly Ala Gln Glu Gly Gln Ala Gly Thr Gly Ala Phe  65 70 75 80 Pro Arg Cys Arg Arg Glu Val Val Lys Leu Thr Ala Ala Ser Ile Thr                  85 90 95 Ala Val Cys Arg Leu Pro Ile Val Val Asp Ala Ser Gly Asp Gly Ala             100 105 110 Tyr Val Cys Lys Asp Val Ala Ala Tyr Pro Asp Ala         115 120

SEQ ID NO: 3 Sequence length: 124 Sequence type: Amino acid Topology: linear Sequence Type: Protein Array Ser Gly Pro Trp Met Cys Tyr Pro Gly Gln Ala Phe Gln Val Pro Ala  1 5 10 15 Leu Pro Gly Cys Arg Pro Leu Leu Lys Leu Gln Cys Asn Gly Ser Gln              20 25 30 Val Pro Glu Ala Val Leu Arg Asp Cys Cys Gln Gln Leu Ala Asp Ile          35 40 45 Ser Glu Trp Pro Arg Cys Gly Ala Leu Tyr Ser Met Leu Asp Ser Met      50 55 60 Tyr Lys Glu His Gly Val Ser Glu Gly Gln Ala Gly Thr Gly Ala Phe  65 70 75 80 Pro Ser Cys Arg Arg Glu Val Val Lys Leu Thr Ala Ala Ser Ile Thr                  85 90 95 Ala Val Cys Arg Leu Pro Ile Val Val Asp Ala Ser Gly Asp Gly Ala             100 105 110 Tyr Val Cys Lys Asp Val Ala Ala Tyr Pro Asp Ala         115 120

SEQ ID NO: 4 Sequence length: 123 Sequence type: Amino acid Topology: linear Sequence Type: Protein Array Ser Gly Pro Trp Ser Trp Cys Asn Pro Ala Thr Gly Tyr Lys Val Ser  1 5 10 15 Ala Leu Thr Gly Cys Arg Ala Met Val Lys Leu Gln Cys Val Gly Ser              20 25 30 Gln Val Pro Glu Ala Val Leu Arg Asp Cys Cys Gln Gln Leu Ala Asp          35 40 45 Ile Asn Asn Glu Trp Cys Arg Cys Gly Asp Leu Ser Ser Met Leu Arg      50 55 60 Ala Val Tyr Gln Glu Leu Gly Val Arg Glu Gly Lys Glu Val Leu Pro  65 70 75 80 Gly Cys Arg Lys Glu Val Met Lys Leu Thr Ala Ala Ser Val Pro Glu                  85 90 95 Val Cys Lys Val Pro Ile Pro Asn Pro Ser Gly Asp Arg Ala Gly Val             100 105 110 Cys Tyr Gly Asp Trp Cys Ala Tyr Pro Asp Val         115 120

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI A61P 3/04 A61P 3/06 3/06 3/08 3/08 C07K 1/18 C07K 1/18 C12N 9/99 C12N 9 / 99 A61K 37/64 (72) Inventor Satoshi Morimoto 5-3-1, Tsurugaoka, Oi-cho, Iruma-gun, Saitama Nisshin Seifun Co., Ltd. Food Research Laboratory (72) Ryuji Murayama Inventor, Ryuji Murayama 40 Mitani, Yamazaki-cho, Shishiwa-gun, Hyogo 1 (72) Inventor Hiroshi Matsubara 10-16, Tokiwadai, Toyono-cho, Toyono-gun, Osaka Prefecture (56) References JP-A-2-157296 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C07K 14/415 A23J 1/12 A23J 3/18 SwissProt / PIR / GeneS eq

Claims (7)

(57) [Claims] 1. An amylase inhibitor, which is a protein consisting of two subunits represented by SEQ ID NO: 1.
A substance with activity . 2. A liquid containing a substance having an amylase inhibitory activity, which is obtained by extracting a wheat-derived substance such as wheat, flour or wheat gluten with water, a dilute aqueous salt solution, a buffer, an acid, a dilute alkali or a hydrous alcohol. If necessary, after purification treatment to remove impurities, treatment with a cation exchanger to adsorb the substance having amylase inhibitory activity , and then p
After the substance having amylase inhibitory activity was eluted from the cation exchanger by treating with an alkaline solution of H9 to 13, the pH of the eluate containing the substance having amylase inhibitory activity was immediately adjusted to acidic to adjust the pH. process for the preparation of substances having an amylase inhibitory activity according to claim 1, characterized in that the liquid collecting a substance having an amylase inhibitory activity from. 3. A wheat starch waste liquor obtained during the production of wheat starch is purified, if necessary, to remove impurities, and then treated with a cation exchanger to adsorb a substance having an amylase inhibitory activity. after elution things <br/> quality having an amylase inhibitory activity from the cation exchanger is treated with an alkaline solution of pH 9-13, pH of the eluate containing <br/> material immediately with an amylase inhibitory activity It was adjusted to be acidic, organic amylase inhibitory activity according to claim 1, characterized in that recovering the substance having an amylase inhibitory activity from pH adjusted solution
For preparing a substance to be used. 4. The content of the protein consisting of 2 subunits represented by SEQ ID NO: 1 is 20% by weight or more, or the protein consisting of 2 subunits represented by SEQ ID NO: 1 and SEQ ID NO: 2 in the total content of protein composed of two subunits expressed is and at least 20% by weight SEQ ID NO: 1
The content of the protein consisting of two subunits represented by
Protein consisting of two subunits represented by SEQ ID NO: 1
And a protein consisting of two subunits represented by SEQ ID NO: 2
20% by weight or more of the total quality of the amylase inhibitor. 5. The content of the protein consisting of 2 subunits represented by SEQ ID NO: 1 is 20% by weight or more, or the protein consisting of 2 subunits represented by SEQ ID NO: 1 and SEQ ID NO: 2 in the total content of protein composed of two subunits expressed is and at least 20% by weight SEQ ID NO: 1
The content of the protein consisting of two subunits represented by
Protein consisting of two subunits represented by SEQ ID NO: 1
And a protein consisting of two subunits represented by SEQ ID NO: 2
20% by weight or more based on the total weight of quality, a blood sugar level elevation inhibitor or an insulin secretion regulator. 6. The content of the protein consisting of 2 subunits represented by SEQ ID NO: 1 is 20% by weight or more, or the protein consisting of 2 subunits represented by SEQ ID NO: 1 and SEQ ID NO: 2 in the total content of protein composed of two subunits expressed is and at least 20% by weight SEQ ID NO: 1
The content of the protein consisting of two subunits represented by
Protein consisting of two subunits represented by SEQ ID NO: 1
And a protein consisting of two subunits represented by SEQ ID NO: 2
An appetite suppressant characterized by being 20% by weight or more based on the total weight of quality . 7. A food containing 2 subunits of SEQ ID NO: 1 adjusted to 30 to 2500 mg per serving, or a protein consisting of 2 subunits of SEQ ID NO: 1 and SEQ ID NO: The total content of proteins consisting of two subunits represented by 2 is 30 to 2 per serving.
Subunit 2 which is 500 mg and is represented by SEQ ID NO: 1
The protein content consisting of individual sub-sequences represented by SEQ ID NO: 1
A protein consisting of two units and the sequence number 2
20 for the total weight of the protein consisting of 2 subunits
Foods adjusted to more than weight% .