JP3676637B2 - Method for producing α-glucosidase inhibitor - Google Patents

Description

【００３０】
【発明の効果】
本発明では、煮沸した大豆を、培養系の塩分が５重量％以下となる条件でアスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属のかびで固形培養して得られた培養物から５０℃以上の水でα−グルコシダーゼ阻害剤を抽出することにより、摂取し易く、臭いもなく、強い阻害活性を示すα−グルコシダーゼ阻害剤が製造できる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing an α-glucosidase inhibitor which has strong inhibitory activity and can be used for pharmaceuticals, foods, health foods and the like, has no odor, and is easy to take.
[0002]
[Prior art]
An α-glucosidase inhibitor inhibits α-glucosidase localized in the microvilli of the small intestine, and suppresses a rapid increase in blood glucose level and subsequent increase in insulin level after Diabate Medicine, 10 , 688 (1993). In order to suppress the metabolism of carbohydrates (especially starch-derived oligosaccharides, sucrose, etc.) in humans and non-human animals, for example, it exhibits an effect of suppressing blood sugar rise and is derived from hyperglycemia and hyperglycemia. It is useful for improving various diseases such as obesity and diabetes. In addition, foods produced by adding an α-glucosidase inhibitor are suitable for dietary abnormal patient foods, and are also suitable for healthy people as metabolic metabolic preventive foods.
[0003]
The present applicant previously reported in Japanese Patent Application No. 10-239326 that touchi (bean drum), which is also used as an ingredient in Chinese cuisine, has a strong α-glucosidase inhibitory activity. Touchi is a solid culture of steamed soybeans in a high salinity of 10% by weight or more, and the touchi contains 10% by weight or more of salt. The thing which suppressed as much as possible is desired.
[0004]
[Problems to be solved by the invention]
However, when the solid culture is performed while suppressing the salt content, there is a problem that a odor like natto is generated from the culture because bacteria such as natto propagate.
[0005]
[Means for Solving the Problems]
As a result of diligent research on the above problems, even a culture obtained by solid-culturing steamed soybeans using molds of the genus Aspergillus under the condition that the salinity of the culture system is 5% by weight or less, It was found that the odor of the inhibitor can be eliminated by extracting it with water at 50 ° C. or higher, and the inhibitory activity can be further improved, and the present invention has been completed.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be specifically described below.
The soybean used in the present invention is a raw material for miso, soy sauce, food oil, tofu, natto, frozen tofu, sprout, etc., and any of raw soybean, dried soybean, soybean flour, etc. can be used. First, soybeans are immersed in water and then steamed or boiled. As conditions for such immersion, soybeans are immersed in water at 10 to 30 ° C. for 1 to 18 hours. Moreover, as conditions for steaming or boiling, the soybeans are put into a steamer, covered and sent with steam from below until the weight becomes 1.02 to 2 times that of soybeans before cooking (after soaking). Steam for about 2 to 2 hours or boil for 30 to 360 minutes in boiling water.
[0007]
Soybeans originally contain about 0.1% by weight of salt in nature, and even steamed or boiled soybeans contain about 0.05% by weight of salt, so they may enter the next molding process as they are, but solid culture In order to improve the stability, the salt content 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 the extraction operation is carried out later, the salt content is mixed in the extract and becomes extremely salty or awkward when ingested.
[0008]
Examples of the salt content include sodium chloride, calcium chloride, potassium chloride, calcium sulfate, etc. The salt content is obtained by diluting the sample with ion-exchanged water and using a digital salinity meter (“SS-31A” manufactured by Sekisui Chemical Co., Ltd.). And measure it.
[0009]
Solid culture is performed after steaming or boiling, but it is first carried out with mold. Such molds are used in the production of soy sauce, moromi, touchi, miso, etc., and molds belonging to the genus Aspergillus are used . Aspergillus nidulans ATCC10074, Aspergillus glaucus (Aspergillus oryzae IFO4135), Aspergillus oryzae IFO4135 Spergillus indicus ATCC 15054), Aspergillus sulphureus ATCC 11904, Aspergillus niger 69 (Aspergillus sogier), Aspergillus sogier
Specifically, “Hi Soya”, “Mild S”, “Three Diamond”, “Diamond C”, “Usumura Saki”, “Treasure Fungus”, “White Soy Sauce Fungus”, “Improved Shochu Fungus” (All of the above are commercially available products such as made by Higuchi Matsunosuke Shoten Co., Ltd.).
The amount of fungi to be added is not particularly limited, but is about 0.0001 to 10% by weight with respect to soybeans. As the method of mold application, methods such as sprinkling mold on steamed or boiled soybeans, or contacting molds remaining in a container or fermentation chamber once contacted with the soybeans.
[0010]
Start solid culture after mold. The solid culture temperature is about 10 to 50 ° C. (preferably 25 to 45 ° C.) and the humidity is 80 to 100 RH% (preferably 90 to 100 RH%), and is performed for 12 to 120 hours (preferably 48 to 96 hours).
Further, if necessary, the culturing step may be performed by leaving the obtained culture at 5 to 40 ° C. for about 2 to 120 days.
After completion of fermentation, if necessary, wash with water to remove mold attached to the soybean surface.
[0011]
Next, the culture is extracted with water at 50 ° C. or higher, preferably with water at 60 ° C. or higher. If the temperature of the water is less than 50 ° C., the inhibitory activity is not improved, and the odor component generated in the culture cannot be removed, which is inappropriate.
[0012]
In order to extract with the above water at 50 ° C. or higher, 1 to 30 times weight (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, it can be extracted at 100 ° C. or more for 0.2 to 5 hours using an autoclave or the like. The extraction method is not particularly limited, but usually stirring extraction or immersion extraction is used.
The obtained extract is solidified by filtration, centrifugation, membrane separation, etc., then decolorized with diatomaceous earth as necessary, and then pulverized by methods such as concentration, drying, freeze drying, spray drying, etc. It is preferable to do this.
[0013]
Since the α-glucosidase inhibitor thus obtained has a blood glucose elevation-inhibiting action, it is a non-toxic carrier such as a liquid carrier such as water, ethanol, ethylene glycol or polyethylene glycol, or a solid carrier such as starch or cellulose. Diluted and used as ampoules, granules, tablets, pills, capsules, syrups, etc., health foods for patients with metabolic disorders or preventive drugs, diabetes preventive drugs, anti-obesity foods, diet foods be able to. Furthermore, by taking the above-mentioned preparation containing the α-glucosidase inhibitor of the present invention before meals, during meals, after meals, between meals, etc., an increase in blood glucose concentration due to eating can be suppressed.
[0014]
Since the inhibitor of the present invention has an inhibitory activity 2 to 10000 times that obtained when touchi is extracted with water at less than 50 ° C., the intake is preferably 0.001 to 10 g / day as a dry powder. In particular, 0.01-3 g / day is preferable.
[0015]
Since the α-glucosidase inhibitor obtained by the production method of the present invention has a low salt content and has no odor, it can be added to the following foods, for example.
(1) Agricultural and fishery products Harusame, koshian, konjac, bread, noodles (instant noodles, pasta, raw noodles, dried noodles), rice cakes, cereal foods, processed soybean products (tofu, soymilk, natto, frozen tofu), processed fishery products , (Crab flavor) salmon, (fish meat) ham, (fish meat) sausage, (fish meat) winner, sprinkle, tea paste, egg-containing foods (soup, salmon, etc.), canned food (sea chicken, oil sardine, yakitori), Retort food (curry, stew, spaghetti), miso soup, soup (2) dairy milk, processed milk, lactic acid bacteria beverage, butter, cheese, condensed milk, powdered milk (3) seasoning miso, soy sauce, umami (flavor) seasoning, ( Powder) Natural seasoning, sauce, dressing, grilled meat sauce, mirin, curry, stew, spices, spices, yogurt [0016]
(4) Health food (dietary supplement)
(1) Food containing saponin (food containing ginseng root, food containing sorghum) (2) Food containing sugar (oligosaccharide (food containing fructooligosaccharide, food containing isomaltooligosaccharide, food containing galactooligosaccharide), polysaccharide (food containing shiitake) Mucopolysaccharides, protein-containing foods, chondroitin sulfate-containing foods, garlic mushroom (reishi) -containing foods, chitin, chitosan-containing foods)
(3) Mineral-containing foods (calcium-containing foods, alfalfa-containing foods, pruned extract foods, β-carotene-containing foods)
(4) Oils and fats-containing foods Vitamin E-containing oils and fats [wheat (wheat, pigeon) germ oil, soybean germ oil, rice germ oil] Eicosapentaenoic acid-containing foods, soybean lecithin-containing foods, γ-linolenic acid-containing foods (Evening Primrose Oil, Borage Oil), docosahexaenoic acid-containing food (5) protein-containing food, soy protein-containing food, casein, whey protein, koji processed food (6) taurine kaki processed food, shijimi processed food, green mussel processed food [0017]
(5) Other processed food, food for abnormal amino acid metabolism, liquid food (disease food)
In addition, it can be added to foods containing a large amount of sugar, such as the following, but the effects of the present invention may not be clearly expressed. It is preferable that the sugar content be as low as possible or added to a low sugar content using an artificial sweetening amount.
(6) Confectionary cake, mousse, (powder) dessert, ice cream, candy, chocolate, gummi, candy, cookies, wafers, jelly (7) Beverage soft drink (carbonated drink, fruit drink, sports drink, nutrition drink), taste Beverages (coffee, cocoa, wort)
[0018]
As addition amount in said (1)-(7), 0.01-80 weight% is preferable as a dry powder with respect to the said foodstuff, and 1-70 weight% is especially preferable.
Furthermore, sweeteners, preservatives, dispersants, colorants, antioxidants and the like can be used in combination as long as the effects of the present invention are not impaired. Furthermore, you may use together alpha-glucosidase inhibitors, such as another known alpha-glucosidase inhibitor, such as valienamine and aminocyclitol.
[0019]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples. In the following description, “%” means “% by weight” unless otherwise specified.
Example 1
After soaking 1000 g of soybeans in 10 liters of water at 25 ° C. for 3 hours to a weight of 1100 g, it was placed in a steamer and steamed at 100 ° C. for 1 hour to obtain 1300 g of boiled beans (with a salt content of 0.05%). . 10 g of commercially available soy sauce mold (“Mild S” manufactured by Higuchi Matsunosuke Shoten Co., Ltd.) is sprinkled on top of the bamboo basket in the fermentation room and added evenly over the boiled beans at 35 ° C. and humidity of 100 RH%. Solid culture was performed for 72 hours. After completion of the cultivation, the soybean was washed with water to wash the mold on the soybean surface, and air-dried at room temperature for 24 hours to obtain 820 g of a culture.
The culture was immersed in 4 liters of water for 1 hour, and then the immersion liquid was placed in an autoclave and extracted at 1.4 kg / cm ² (gauge pressure) and an internal temperature of 135 ° C. for 1 hour. Large residues were removed using a sieve from the resulting extract. The obtained filtrate was combined with the filtrate obtained by adding 4 liters of water again to the residue and filtered, and centrifuged (condition 1000 × g). 100 g of diatomaceous earth was added to the resulting supernatant and stirred to remove the color, followed by filtration to obtain 7.8 liters of extract. Thereafter, the mixture was concentrated and dried with a rotary evaporator to obtain 320 g of an inhibitor. The inhibitory activity, odor, and ease of intake of such inhibitors were evaluated as follows.
[0020]
(Inhibitory activity)
Preparation of di (three) sugar hydrolase (α-glucosidase) from rat small intestine The rat small intestine (jejunum), which had been stored frozen, was thawed, and the mucous membrane was collected by extrusion with forceps. A 0.1 M potassium phosphate buffer solution (pH 7.0) containing 5 times the weight of 5 mM ethylenediaminetetraacetic acid was added to the mucous membrane and homogenized while cooling. Thereafter, the mixture was centrifuged (4 ° C., 21000 × g, 60 minutes), and 0.1 M potassium phosphate buffer (pH 7.0) containing 1% Triton X-100 so that the obtained precipitate had a weight of 5 times. And solubilization treatment (4 ° C., 60 minutes) was performed. This was subjected to ultracentrifugation (4 ° C., 110000 × g, 90 minutes), and the supernatant was dialyzed against 0.01 M potassium phosphate buffer (pH 7.0) (4 ° C., 24 hours) to obtain an enzyme solution. .
[0021]
-Measurement of enzyme (α-glucosidase) activity A commercially available kit was used for enzyme activity, and sucrose was used as a substrate.
The standard reaction solution composition is 60 mM substrate solution (sucrose dissolved in 0.1 M potassium phosphate buffer pH 6.3) 0.7 ml, test substance solution (each fractional component water, organic solvent completely After removal, it was dissolved in 50% dimethyl sulfoxide aqueous solution (0.2 ml) and the enzyme solution was 0.1 ml (1.0 ml in total). This was reacted at 37 ° C. for 15 minutes, and the reaction was stopped using 1.5 ml of 2M Tris-HCl buffer (pH 7.0) to prepare a test solution.
Next, after adding 50 μl of a test solution (ethyl acetate etc. distilled off) to 200 μl of a color developing reagent [glucose B test Wako (manufactured by Wako Pure Chemical Industries)] per 96-well microplate and incubating at 37 ° C. for 30 minutes The absorbance at 490 nm was measured with a microplate reader (BIO RAD, MODEL550). The absorbance when 0.1 M potassium phosphate buffer (pH 6.3) was added instead of the substrate solution was defined as a blank value, and the value obtained by subtracting this value was defined as A _490s . The absorbance when 50% by weight dimethyl sulfoxide aqueous solution was added instead of the test solution was taken as a control value (A _490c ), and α-glucosidase inhibitory activity was determined by the following equation. The measurement was performed twice, and the average value was taken as the measured value.
α-glucosidase inhibitory activity (%) = [(A _490c −A _490s ) / A _490c ] × 100
[0022]
(smell)
The odor of the obtained inhibitor was evaluated as follows.
○ ... It is completely odorless.
Δ: Slightly natto smell.
× ... Strong natto smell.
[0023]
(Ease of consumption)
○ ... It is not salty and it is easy to take in soy flavor.
X: Salty or soy-specific odor is strong, and taking 1 g or more is painful.
[0024]
Example 2
After completion of Example 1, in the same fermentation chamber as Example 1, instead of mold (“Mild S” manufactured by Matsunosuke Higuchi Co., Ltd.), the culture adhered to the bamboo basket used in Example 1 In the mold, the same boiled bean as in the same example was solid-cultured for 72 hours at 35 ° C. and humidity of 100 RH. After the cultivation, the soybean was washed with water, the mold on the soybean surface was washed with water, and air-dried at room temperature for 12 hours 820 g of culture was obtained.
The culture was immersed in 4 liters of water for 1 hour, and then the immersion liquid was placed in an autoclave and extracted at 1.4 kg / cm ² (gauge pressure) at 135 ° C. for 1 hour. Thereafter, the same treatment as in Example 1 was performed to obtain 7.8 liters of an extract, which was concentrated and dried by a rotary evaporator to obtain 350 g of an inhibitor. The inhibitory activity, odor, and ease of ingestion of such an inhibitor were evaluated in the same manner as in Example 1.
[0025]
Example 3
The culture was conducted in the same manner as in Example 1 except that the solid culture temperature was 37 ° C., to obtain 820 g of a culture. The culture was placed in a pottery vessel, capped and post-fermented at 35 ° C. for 40 days to obtain 780 g of culture.
The culture was immersed in 4 l of water for 1 hour, and then the immersion liquid was placed in an autoclave and extracted at 1.4 kg / cm ² (gauge pressure) at 135 ° C. for 1 hour. Thereafter, the same treatment as in Example 1 was carried out to obtain 7.8 liters of an extract, which was concentrated and dried by a rotary evaporator to obtain 290 g of an inhibitor. The inhibitory activity, odor, and ease of ingestion of such an inhibitor were evaluated in the same manner as in Example 1.
[0026]
Example 4
Instead of the autoclave in Example 1, extraction was performed using a pan at an extraction temperature of 60 ° C. Thereafter, the same treatment as in Example 1 was performed to obtain 7.8 liters of an extract, which was concentrated and dried by a rotary evaporator to obtain 200 g of an inhibitor. The inhibitory activity, odor, and ease of ingestion of such an inhibitor were evaluated in the same manner as in Example 1.
[0027]
Comparative Example 1
After 1000 g of soybeans were immersed in 10 liters of water for 3 hours, they were placed in a steamer and steamed at 100 ° C. for 1 hour to obtain 1800 g of boiled beans (salt content of 0.1%). After adding 200 g of salt to the boiled beans (10% salt content), fermentation was carried out in the same manner as in Example 1 to obtain 960 g of culture.
The culture was extracted in the same manner as in Example 1, treated in the same manner to obtain 8 liters of extract, and concentrated to dryness with a rotary evaporator to obtain 400 g of inhibitor. The inhibitory activity, odor, and ease of ingestion of such an inhibitor were evaluated in the same manner as in Example 1.
[0028]
Comparative Example 2
820 g of the culture obtained in Example 1 was immersed in 4 l of water for 1 hour and then extracted at 40 ° C. for 1 hour. Thereafter, the same treatment as in Example 1 was performed to obtain 7.8 liters of an extract, which was concentrated and dried by a rotary evaporator to obtain 60 g of an inhibitor. The inhibitory activity, odor, and ease of ingestion of such inhibitors were also evaluated.
[0029]

[0030]
【The invention's effect】
In the present invention, boiled soybeans are inhibited by α-glucosidase inhibition with water at 50 ° C. or higher from a culture obtained by solid culture of Aspergillus fungi under conditions where the salinity of the culture system is 5% by weight or less. By extracting the agent, an α-glucosidase inhibitor that is easy to ingest, has no odor, and exhibits strong inhibitory activity can be produced.

Claims (1)

Steamed or boiled soybeans are solid-cultured with Aspergillus fungi under a condition where the salinity of the culture system is 5% by weight or less, and extracted from the resulting culture with water of 50 ° C or higher. A process for producing an α-glucosidase inhibitor characterized by