JP2002027978A - Method for producing α-glucosidase inhibitor - Google Patents

Abstract

(57) [Problem] To provide a method for producing an α-glucosidase inhibitor which is easy to ingest, has no odor and exhibits strong inhibitory activity. A target substance is extracted with water at 50 ° C. or higher from a culture obtained by solid-culturing steamed or boiled kidney beans using a mold with a salt content of a culture system of 5% by weight or less. .

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 an inhibitor from foods, and as a result, have found that a culture obtained by fixedly cultivating a kidney bean 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. So when I tried solid culture of kidney beans with reduced salt content,
There is a problem that odor is generated from the culture due to propagation of bacteria and yeast.

[0006]

As a result of diligent studies on such problems, steamed or boiled kidney beans are solid-cultured using mold under the condition that the salt content of the culture system is reduced to a small amount and 5% by weight or less. It has been found that by extracting the resulting culture with water at 50 ° C. or higher, the odor of the inhibitor can be eliminated and the inhibitory activity can be further improved, and the present invention has been completed.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below. Examples of the kidney beans used in the present invention include kidney beans (also known as May cowpea, tertiary beans, and vegetable beans), safflower beans (also known as Hanamame and Hanasasage), and bean beans (also known as Rama bean, Aoi bean and Tsuki bean). Any of powders, grains and the like obtained by processing kidney beans can be used. In carrying out the production method of the present invention, the beans are first immersed in water, and then steamed or boiled. The conditions for such immersion are as follows: Soak in water for 1-18 hours. The conditions for steaming or boiling were as follows: put kidney beans in a steamer, cover the vessel, and send steam from below to weigh 1% less than beans before steaming (after immersion).
Steam for about 0.2 to 2 hours until it becomes 02 to 2 times,
Boil for 30-360 minutes in boiling water.

Since beans of the genus Bean originally contain 0.1% by weight or less of salt in nature, and steamed or boiled beans of the genus Bean contain 0.01% by weight or less of salt,
Although the process may proceed to the next molding step as it is, in order to ensure the stability of the solid culture, a salt may be added in such a range that the salt content of the 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] Then, after steaming or boiling, solid culture is performed. Such molds are used for the production of soy sauce, moromi, torch, miso and the like, and include Aspergillus (Aspergill).
us) are preferred, for example Aspergillus
Albus (Aspergillus albus IF
O4039), Aspergillus candidas (As
pergillus 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
stamari ATCC 12669) 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 to be added is not particularly limited.
It is about 1 to 10% by weight. Examples of the mold-making method include a method in which mold is sprinkled on steamed or boiled kidney beans, and a method in which mold beans remaining in a vessel or a fermentation chamber once cultured are brought into contact with the beans.

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 adhered to the beans.

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 kidney beans was added to 10 liters of water at 25 ° C.
After soaking for 1100 g in weight, put in a steamer and steamed at 100 ° C for 1 hour, and cooked 1900 g of kidney beans [salt content is below the detection limit (0.01%)]
I got The steamed kidney 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 uniformly sprinkled and added at 35 ° C. and 100% humidity. The cells were solid-cultured at RH for 96 hours. After completion of the culture, the mold on the surface of the culture is washed with water, and
Air-dried at 24 ° C. for 24 hours to obtain 760 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 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 the mixture was centrifuged (1000 × g). Activated carbon 10 was added to the obtained supernatant.
After adding 0 g, stirring and decoloring, filtration was performed to obtain 3 liters of extract. Thereafter, the mixture was concentrated to dryness using a rotary evaporator to obtain 215 g of an inhibitor. 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, easy to ingest with the flavor of kidney beans. X: Salty or haricot beans peculiar smell 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 mold ("Mild S" manufactured by Higuchi Matsunosuke Shoten Co., Ltd.). The same steamed kidney bean as in the same example was grown at 35 ° C.
The mold was solid-cultured at 100% 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 800 g of the culture. After immersing the culture in 3 liters of water for 1 hour, the immersion liquid was placed in an autoclave,
Extracted at 125 ° C. for 1 hour. Thereafter, the same treatment as in Example 1 was carried out to obtain 3 liters of the extract, which was concentrated to dryness with a rotary evaporator to obtain 232 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 The same culture as in Example 1 was carried out except that the temperature of the solid culture was 37 ° C., to obtain 760 g of a culture. The culture was placed in a pottery vessel, covered, and post-fermented at 35 ° C. for 40 days to obtain 750 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 217 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 extract was treated in the same manner as in Example 1 to obtain 3 liters of the extract, and concentrated to dryness with a rotary evaporator to obtain 109 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 safflower beans were used instead of kidney beans to obtain 690 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 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 200 g of an inhibitor. Inhibitory activity of such inhibitors, odor,
The ease of ingestion was evaluated as in Example 1.

Example 6 Solid culture was performed in the same manner as in Example 1 except that bean bean was used instead of kidney bean to obtain 710 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 extract was treated in the same manner as in Example 1 to obtain 3.8 liters of the extract, and concentrated to dryness with a rotary evaporator to obtain inhibitor 20.
6 g were obtained. 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 kidney beans were immersed in 10 liters of water for 3 hours, and then put in a steamer and steamed at 100 ° C. for 1 hour.
00g of steamed kidney beans (salt content 0.01% or less)
Was added, and 200 g of salt was further added (salt content: 10%), followed by fermentation in the same manner as in Example 1 to obtain 910 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 liters of the extract, which was concentrated to dryness with a rotary evaporator to obtain 360 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 2 750 g of the culture obtained in Example 1 was immersed in 3 liters of water for 1 hour and extracted at 40 ° C. for 1 hour. Thereafter, the extract was treated in the same manner as in Example 1 to obtain 2.8 liters of the extract, and concentrated to dryness using a rotary evaporator to obtain 87 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 92 ○ ○ Example 2 93 ○ ○ Example 3 91 ○ ○ Example 4 91 ○ ○ Example 5 90 ○ ○ Example 6 92 ○ ○ Comparative Example 1 91 × × Comparative Example 2 13 △ × * Concentration of the test substance in the reaction system 10 mg / ml

[0033]

According to the present invention, a steamed or boiled kidney 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.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) A61K 35/78 A61K 35/78 Y A61P 3/04 A61P 3/04 3/10 3/10 // (C12N 9/99 (C12N 9/99 C12R 1:66) C12R 1:66) F term (reference) 4B018 LB01 LB02 LB05 LB06 LB07 LB08 LB09 MD57 ME03 MF01 MF13 4C087 AA02 AA04 BC06 CA10 CA11 CA47 NA14 ZC35 4C088 AB59 AC04 AD18 BA09 NA37 CA37 ZC35

Claims (2)

[Claims] 1. A steamed or boiled kidney bean,
A method for producing an α-glucosidase inhibitor, comprising solid-culturing the salt content of a culture system under 5% by weight or less using a fungus, and extracting the resulting culture with water at 50 ° C. or higher. 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).