JP2002027979A - Method for producing alpha-glucosidase inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing an α-glucosidase inhibitor which can easily be taken out, does not have a smell and exhibits a strong inhibiting activity. SOLUTION: This method for producing an α-glucosidase inhibitor is characterized by subjecting steamed or boiled beans belonging to the genus Vigna to a solid culture using a mold under a condition having a salt content of <=5 wt.% and then extracting the culture product with >=50 deg.C water.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an α-glucosidase inhibitor which has a strong inhibitory activity, has no odor and is easy to take, which can be used for pharmaceuticals, foods, health foods and the like.

[0002]

BACKGROUND OF THE INVENTION α- glucosidase inhibitors, inhibit α- glucosidase localized in microvilli of the small intestine, to suppress spikes and elevated insulin levels subsequent postprandial blood glucose, Diabate Medicine, 10 , 688 (1993). In humans and non-human animals, it shows an inhibitory effect on carbohydrates (especially, starch-derived oligosaccharides, sucrose, etc.). And it is useful for improvement of various diseases such as obesity and diabetes caused by hyperglycemia. Further, the food produced by adding the α-glucosidase inhibitor is suitable for a patient with a metabolic disorder, and is also suitable for a healthy person as a diet for preventing a metabolic disorder.

As an α-glucosidase inhibitor derived from food, for example, Japanese Patent Application Laid-Open No. 9-65836 discloses an enzymatic hydrolyzate of an animal protein or a vegetable protein. Discloses tea polyphenols.

However, Japanese Patent Application Laid-Open No. 9-65836 describes
The α-glucosidase inhibitor described in Japanese Unexamined Patent Application Publication No. H11-146556 must be ingested in large quantities as a food in order to exhibit activity, and in the technology disclosed in Japanese Patent Application Laid-Open No. HEI 5-17364, purification of polyphenol is complicated. In addition, it was necessary to ingest a large amount of tea which is usually consumed.

[0005]

The inventors of the present invention have conducted intensive studies to find such inhibitors from foods, and as a result, have found that a culture obtained by fixedly cultivating cowpea beans has a strong inhibitory activity. Usually, since the fixed culture is cultured under a high salt content, it has been found that the fixed culture is not suitable for daily consumption as a food, especially as a health food. Thus, when solid culture of cowpea beans was carried out while suppressing the salt content, there was a problem that odor was generated from the culture, probably because bacteria and yeast propagated.

[0006]

As a result of diligent research on such problems, steamed or boiled cowpea beans are
Even if the culture is obtained by solid cultivation using mold under the condition that the salt content of the culture system is reduced to 5% by weight or less,
By extracting it with water at 50 ° C. or higher, it was found that the odor of the inhibitor could be eliminated and the inhibitory activity could be further improved, thus completing the present invention.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below. Examples of cowpea beans used in the present invention include adzuki beans (also known as “shoes”), cowpea (also known as “nagasasa” and “hata-sage”), mungbean (also known as “yenari” and “aoazuki”), and take care (also known as “tsuruazuki” and “bakaazuki”).
Powders, grains, and the like obtained by processing such cowpea beans are used. In carrying out the production method of the present invention, first, the above cowpea beans are immersed in water, and then steamed or boiled. Soak in water for 1-18 hours. The conditions of steaming or boiling are as follows: cowpea beans are put in a steamer, covered, and steam is sent from below to weigh 1.02 to 2 times compared to cowpea beans before steaming (after immersion). Cook for about 0.2 to 2 hours or double in boiling water for 3 times.
Boil for 0-360 minutes.

Cowpea beans originally contain 0.1% by weight or less of salt in nature, and steamed or boiled cowpea beans contain 0.01% by weight or less of salt. However, in order to ensure the stability of the solid culture, salt may be added in a range where the salt content of such a system is 5% by weight or less (preferably 1% by weight or less). If the salt content exceeds 5% by weight, even if an extraction operation is performed later, the salt content is mixed into the extract and becomes extremely salty or unsatisfactory at the time of ingestion.

[0009] The salt content of the culture system is measured by adding ion-exchanged water to the culture, stirring the mixture, appropriately diluting the supernatant, and using a digital salt meter (“SS-31A” manufactured by Sekisui Chemical Co., Ltd.). Ask. Sodium chloride,
Examples thereof include calcium chloride and potassium chloride.

[0010] Next, solid culture is performed after steaming or boiling. Such molds are used for the production of soy sauce, moromi, torch, miso and the like, and include Aspergillus (Aspergillus).
s) Fungi of the genus genus are preferred, for example Aspergillus albus IFO
4039), Aspergillus candidas (Asp
ergillus candidus IFO438
9), Aspergillus needslance (Aspergi)
llus nidulans ATCC 10074),
Aspergillus glaucus
glaucus ATCC10059), Aspergillus oryzae
IFO4135), Aspergillus flavus (As
pergillus flavus IFO583
9), Aspergillus indica
lrus indicus ATCC 15054), Aspergillus sulphleus (Aspergillus).
sulphurus ATCC 11904), Aspergillus niger (Aspergillus nig).
er IFO4343), Aspergillus sojae IFO420
0), Aspergillus tamari (Aspergillus
stamariATCC12669) and the like. Specifically, "High Sawyer", "Mild
S ”,“ Three Diamonds ”,“ Diamond C ”,“ Usumurasaki ”,“ Treasure Bacteria ”,“ Bacteria for White Soy Sauce ”,“ Bacteria for Improved Shochu ”(all of which are manufactured by Higuchi Matsunosuke Shoten Co., Ltd.) Goods. The amount of mold added when molding is not particularly limited.
About 10% by weight. Examples of the method of mold application include sprinkling mold on steamed or boiled cowpea beans, and bringing the cowpea beans into contact with molds remaining in a container or fermentation chamber once cultured.

After molding, solid culture is started. Solid culture temperature is about 10 to 50 ° C (preferably 25 to 45 ° C),
Humidity 80-100% RH (preferably 90-100% RH
H) for 12 to 120 hours (preferably 48 to 96 hours). Further, the obtained culture is optionally
The aging step may be performed by leaving the mixture at ４０40 ° C. for about 2 to 120 days. After the fermentation, if necessary, washing is performed with water to remove mold attached to the cowpea.

Next, the culture is extracted with water at 50 ° C. or more, preferably with water at 60 ° C. or more. If the temperature of the water is lower than 50 ° C., no improvement in the inhibitory activity is observed, and furthermore, the odor components generated in the culture cannot be removed, which is inappropriate.

In order to extract with water at 50 ° C. or higher, 1 to 30 times (preferably 2 to 15 times) weight of water is added to the culture, and the temperature is raised. The extraction time may be about 5 to 20 hours at an extraction temperature up to 90 ° C, and about 1 to 10 hours when it exceeds 90 ° C. If necessary, extraction can be performed at 100 ° C. or higher for 0.2 to 5 hours using an autoclave or the like. Although there is no particular limitation on the extraction method, usually stirring extraction or immersion extraction is used. The obtained extract is subjected to clarification filtration, centrifugation, membrane separation, and the like to remove solids, and then, if necessary, decolorized with activated carbon or terra alba, and then concentrated to dryness, freeze-dried, spray-dried, or the like. Pulverization is preferred.

Since the thus obtained α-glucosidase inhibitor has an action of suppressing a rise in blood glucose, it is nontoxic to liquid carriers such as water, ethanol, ethylene glycol and polyethylene glycol and solid carriers such as starch and cellulose. Diluted with a pharmaceutically acceptable carrier, and used as pharmaceuticals such as ampoules, granules, tablets, pills, capsules and syrups, as a health food, as a diet or prophylactic for metabolic disorders, as a preventive for diabetes, or as an anti-obesity diet, diet Can be used as food. Furthermore, by taking the above-mentioned preparation containing the α-glucosidase inhibitor of the present invention before, during, after or between meals, an increase in blood glucose concentration due to eating can be suppressed.

[0015] The intake amount is 0.0
It is preferably from 01 to 10 g / day, particularly preferably from 0.01 to 3 g / day.

The α-glucosidase inhibitor obtained by the production method of the present invention has a low salt content and no odor, and thus can be added to, for example, the following foods. (1) Agriculture and fishery products Harusame, Koshian, konjac, bread, noodles (instant noodles, pasta, raw noodles, dried noodles), mochi, cereal foods, soybean products (tofu, soymilk, natto, frozen tofu), fishery products [kneaded products , (Crab flavor) kamaboko, (fish meat) ham, (fish meat) sausage, (fish meat) wiener, sprinkle, tea sauce), egg-containing food (soup, bowl, etc.), canned (sea chicken, oil sardine, yakitori), Retort food (curry, stew, spaghetti), miso soup, soup (2) Dairy products Milk, processed milk, lactic acid beverage, butter, cheese, condensed milk, powdered milk (3) Seasoning miso, soy sauce, umami (flavor) seasoning, ( Powder) Natural seasonings, sauces, dressings, grilled meat sauce, mirin, curry, stew, spices, spices, yogurt

(4) Healthy foods (dietary supplements) Saponin-containing foods (pancreatic ginseng root-containing foods, eleuthero-containing foods) Sugar-containing foods [oligosaccharides (fructooligosaccharide-containing foods,
Isomaltooligosaccharide-containing foods, galactooligosaccharide-containing foods), polysaccharides (shiitake-containing foods, mucopolysaccharides, protein-containing foods, chondroitin sulfate-containing foods, Mannentake (reishi) -containing foods, chitin, chitosan-containing foods)] Mineral-containing foods ( Foods containing calcium, foods containing alfalfa, foods containing prune extract, foods containing β-carotene) Foods containing fats and oils Foods containing fats and oils containing vitamin E [wheat (wheat, barley) germ oil, soybean germ oil, rice germ oil], foods containing eicosapentaenoic acid, Soy lecithin-containing food, γ-linolenic acid-containing food (evening primrose oil, borage oil), docosahexaenoic acid-containing food Protein-containing food Soy protein-containing food, casein, whey protein, carp processed food Taurine, persimmon processed food, processed seafood, green ai Shellfish processed food

(5) Others Processed foods for turkey, foods for abnormal amino acid metabolism, liquid foods (disease foods) In addition, they can be added to foods containing a large amount of sugar as described below, but the effects of the present invention are not In some cases, it is not clearly expressed, and when it is added to the following food products, it is preferable to add the sugar content as low as possible during the production of the food product or to reduce the sugar content using artificial sweetness. (6) Confectionery cake, mousse, (powder) dessert, ice cream,
Candy, chocolate, gummy, candy, cookies, wafers, jelly (7) Beverages Soft drinks (carbonated drinks, fruit drinks, sports drinks, nutritional drinks), favorite drinks (coffee, cocoa, wort)

The amount of addition in the above (1) to (7) is 0.01 to 8 as a dry powder with respect to the food.
0% by weight is preferable, and 1 to 70% by weight is particularly preferable.
Further, a sweetener, a preservative, a dispersant, a coloring agent, an antioxidant and the like can be used in combination as long as the effects of the present invention are not impaired. Further, other known α-glucosidase inhibitors such as varienamine and aminocyclitol may be used in combination.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be specifically described below with reference to examples. In the following description, “%” means “% by weight” unless otherwise specified. Example 1 1000 g of adzuki bean was immersed in 10 liters of water at 25 ° C. for 3 hours to make the weight 1100 g, and then put in a steamer for 10 hours.
Steamed at 0 ° C. for 1 hour to obtain 2,000 g of steamed adzuki beans (salt content is below the detection limit (0.01%)). The steamed red beans are arranged on a bamboo basket in a fermentation room, and 10 g of a commercially available mold for soy sauce production (“Mild S” manufactured by Higuchi Matsunosuke Shoten Co., Ltd.) is evenly sprinkled and added.
Solid cultivation was carried out for 96 hours at 100 ° C. and 100% humidity. After completion of the culture, the mold on the surface of the culture was washed with water and air-dried at 80 ° C. for 24 hours to obtain 800 g of the culture. After immersing the culture in 4 liters of water for 1 hour, the immersion liquid was put into an autoclave,
Extraction was performed at an internal temperature of 125 ° C. for 1 hour. A large residue was removed from the resulting extract using a sieve. The obtained filtrate was combined with the filtrate obtained by adding 4 liters of water to the residue again, and then centrifuged (condition: 1000 × g).
did. 100 g of activated carbon was added to the obtained supernatant, stirred, decolorized, and filtered to obtain 4 liter of extract. After that, it was concentrated to dryness with a rotary evaporator and the inhibitor was 240 g.
I got The inhibitory activity, odor, and ease of ingestion of the inhibitor were evaluated as follows.

(Inhibitory activity) Preparation of di- (tri) sugar hydrolase (α-glucosidase) from rat small intestine The frozen rat small intestine (jejunum) is thawed, and the mucous membrane is extruded with forceps. Collected. A 0.1 M potassium phosphate buffer (pH 7.0) containing 5 times the weight of 5 mM ethylenediaminetetraacetic acid was added to the mucous membrane, and homogenized while cooling. Thereafter, centrifugation (4 ° C., 21000
× g, 60 minutes), and a 0.1 M potassium phosphate buffer (pH 7.0) containing 1% Triton X-100 was added to the obtained precipitate so as to have a 5-fold weight, and solubilization treatment (4 ℃,
60 minutes). This is ultracentrifuged (4 ° C, 1100
00 × g, 90 minutes), and the supernatant is dialyzed against 0.01 M potassium phosphate buffer (pH 7.0) (4 ° C., 24 hours)
And used as an enzyme solution.

Measurement of enzyme (α-glucosidase) activity The enzyme activity was measured using a commercially available kit.
Source was used. Standard reaction solution composition is 60 mM substrate solution
(Sucrose in 0.1 M potassium phosphate buffer pH
0.7 ml dissolved in 6.3) and a test substance solution
After completely removing the water and organic solvent of each fraction component,
Dissolved in 50% dimethyl sulfoxide aqueous solution) 0.2m
1, 0.1 ml of the above enzyme solution (1.0 ml in total). This
This was allowed to react at 37 ° C. for 15 minutes, followed by 2M Tris-HCl buffer.
(PH 7.0) Stop the reaction with 1.5 ml and test
Liquid. Next, one hole is generated in a 96-well microplate.
Color reagent [Glucose B Test Wako (Wako Pure Chemical Industries)] 2
Add 50 μl of test solution to 00 μl (ethyl acetate and the like were distilled off)
) And incubated at 37 ° C. for 30 minutes,
Micro plate reader (BIO RAD, MOD
The absorbance at 490 nm was measured using EL550). Substrate solution
0.1 M potassium phosphate buffer (pH 6.
The absorbance at the time of adding 3) is defined as a blank value.
A minus the value_490sAnd 50% by weight instead of test liquid
The absorbance when adding dimethyl sulfoxide aqueous solution
Troll value (A _490c) And α-glucosida by the following formula
The inhibitory activity was determined. Measure twice and measure the average
Value. α-glucosidase inhibitory activity (%) = [(A_490c−
A_490s) / A_490c] × 100

(Odor) The odor of the obtained inhibitor was evaluated as follows.・ ・ ・: It is completely odorless. Δ: Smell slightly. ×: Smell strong.

(Ease of ingestion) ・ ・ ・: Not salty, and the taste of cowpea beans is easy to ingest. ×: Salty or cowpea peculiar odor is strong and it is painful to take in 1 g or more.

Example 2 After the completion of Example 1, in the same fermentation chamber as in Example 1, the bamboo basket used in Example 1 was attached instead of the mold ("Mild S" manufactured by Higuchi Matsunosuke Shoten Co., Ltd.). The same steamed red bean as in the same example was grown at 35 ° C and a humidity of 1
The mold was solid-cultured at 00% RH for 72 hours. After completion of the culture, the mold on the surface of the culture was washed with water and air-dried at 80 ° C. for 24 hours to obtain 780 g of the culture. After the culture was immersed in 4 liters of water for 1 hour, the immersion liquid was placed in an autoclave,
Extracted at C for 1 hour. Thereafter, the extract was treated in the same manner as in Example 1 to obtain 4 liters of the extract, and concentrated to dryness with a rotary evaporator to obtain 234 g of an inhibitor. The inhibitory activity, odor, and ease of ingestion of the inhibitor were evaluated in the same manner as in Example 1.

Example 3 Culture was carried out in the same manner as in Example 1 except that the temperature of the solid culture was changed to 37 ° C., to obtain 745 g of a culture. The culture was placed in a pottery vessel, covered, and post-fermented at 35 ° C. for 40 days to obtain 740 g of the culture. After immersing the culture in 4 liters of water for 1 hour, the immersion liquid was placed in an autoclave and
Extracted at 25 ° C. for 1 hour. Thereafter, the extract was treated in the same manner as in Example 1 to obtain 3.8 liters of the extract, and concentrated to dryness using a rotary evaporator to obtain 220 g of an inhibitor. The inhibitory activity, odor, and ease of ingestion of the inhibitor were evaluated in the same manner as in Example 1.

Example 4 Extraction was carried out at 60 ° C. using a pot instead of the autoclave in Example 1. Thereafter, the same treatment as in Example 1 was carried out to obtain 3 liters of the extract, which was then concentrated to dryness with a rotary evaporator to obtain 153 g of an inhibitor. The inhibitory activity, odor, and ease of ingestion of the inhibitor were evaluated in the same manner as in Example 1.

Example 5 Solid culture was carried out in the same manner as in Example 1 except that Takeazuki was used instead of adzuki to obtain 730 g of a culture. After the culture was immersed in 3 liters of water for 1 hour, the immersion liquid was placed in an autoclave and extracted at 125 ° C. for 1 hour. Thereafter, the mixture was treated in the same manner as in Example 1 to obtain 3.5 liters of the extract.
Concentrate to dryness with a rotary evaporator and make inhibitor 220g
I got The inhibitory activity, odor, and ease of ingestion of the inhibitor were evaluated in the same manner as in Example 1.

Example 6 Solid culture was carried out in the same manner as in Example 1 except that mung bean was used instead of adzuki to obtain 715 g of a culture. After the culture was immersed in 3 liters of water for 1 hour, the immersion liquid was placed in an autoclave and extracted at 125 ° C. for 1 hour. Thereafter, the same treatment as in Example 1 was performed to obtain 3.2 liters of the extract.
Concentrate to dryness with a rotary evaporator and make 193 g of inhibitor
I got The inhibitory activity, odor, and ease of ingestion of the inhibitor were evaluated in the same manner as in Example 1.

Comparative Example 1 1000 g of adzuki bean was immersed in 10 liters of water for 3 hours, then put in a steamer and steamed at 100 ° C. for 1 hour to obtain 2,000
g of steamed red bean (0.01% or less in salt content) was obtained. 200 g of salt was added to the steamed rice (salt content: 10%) and fermented in the same manner as in Example 1 to obtain 900 g of a culture. The culture was extracted in the same manner as in Example 1 and treated in the same manner to obtain 4.1 L of an extract, which was then concentrated to dryness with a rotary evaporator to obtain 320 g of a solid. The inhibitory activity, odor, and ease of ingestion of the solids were evaluated in the same manner as in Example 1.

Comparative Example 2 800 g of the culture obtained in Example 1 was immersed in 4 liters of water for 1 hour and then extracted at 40 ° C. for 1 hour. Thereafter, the extract was treated in the same manner as in Example 1 to obtain 3.7 liters of the extract, and concentrated to dryness with a rotary evaporator to obtain 80 g of a solid.
The inhibitory activity, odor and ease of ingestion of such solids were also evaluated.

[Table 1] α-Glucosidase Odor Ease of Ingestion Inhibition Activity (%) * Example 1 90 ○ ○ Example 2 91 ○ ○ Example 3 91 ○ ○ Example 4 90 ○ ○ Example 5 92 ○ ○ Example 6 92 ○ ○ Comparative Example 1 88 × × Comparative Example 2 10 △ × * Concentration of the test substance in the reaction system 10 mg / ml

[0033]

According to the present invention, a steamed or boiled cowpea bean is solid-cultured in a mold under the condition that the salt content of the culture system is 5% by weight or less. -By extracting the glucosidase inhibitor, an α-glucosidase inhibitor which is easy to ingest, has no odor, and exhibits strong inhibitory activity can be produced.

Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A61P 3/04 A61P 3/04 3/10 3/10 43/00 111 43/00 111 // (C12N 9/99 ( C12N 9/99 C12R 1:66) C12R 1:66) F term (reference) 4B018 LB01 LB02 LB04 LB05 LB06 LB07 LB08 LB09 MD57 ME03 MF13 4C087 AA01 AA02 BC06 CA10 CA11 MA01 NA14 ZA70 ZC20 ZC35 4C088 AB59 AC04 ZA70 ZC20 ZC35

Claims (2)

[Claims] 1. A steamed or boiled cowpea bean is solid-cultured using a mold under conditions in which the salt content of the culture system is 5% by weight or less, and the obtained culture is extracted with water at 50 ° C. or higher. A method for producing an α-glucosidase inhibitor, characterized in that: 2. The method of claim 2, wherein the mold is Aspergillus.
2. The method for producing an α-glucosidase inhibitor according to claim 1, wherein the agent belongs to the genus (illus).