JP4738571B2 - Method for producing α-glucosidase inhibitor - Google Patents

Description

【００３３】
【発明の効果】
本発明では、蒸煮あるいは煮沸した穀類を、培養系の塩分が５重量％以下となる条件で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]
As an α-glucosidase inhibitor derived from food, for example, JP-A-9-65836 discloses an enzyme hydrolyzate of animal protein or plant protein, and JP-A-5-17364 discloses tea polyphenol. Is disclosed.
[0004]
However, the α-glucosidase inhibitor described in JP-A-9-65836 must be ingested in large quantities as food in order to exhibit activity, and in the technique disclosed in JP-A-5-17364, However, the purification of polyphenols is complicated, and it is necessary to consume a large amount of tea that is usually consumed.
[0005]
[Problems to be solved by the invention]
Therefore, as a result of intensive studies to find such an inhibitor from food, the present inventor has found that the culture in which cereals are fixedly cultured has strong inhibitory activity, but the fixed culture is usually cultured under high salinity. Therefore, it was proved unsuitable for daily consumption as a food, particularly as a health food. Therefore, when solid culture of cereals was performed while suppressing the salt content, there was a problem that odors were generated from the culture because of the propagation of bacteria and yeast.
[0006]
[Means for Solving the Problems]
As a result of earnest research on such problems, even if it is a culture obtained by solid-culturing steamed or boiled cereals using mold under conditions of 5% by weight or less with a small amount of salt in the culture system, It was found that the odor of the inhibitor can be eliminated by extracting the product with water at 50 ° C. or higher, and the inhibitory activity can be further improved, thereby completing the present invention.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be specifically described below.
Examples of cereals used in the present invention include awa, enba, barley, acne, wheat, rice, glutinous rice, buckwheat, corn, mushrooms, corn, rye, and pigeons. Oatmeal, wheat, wheat flour, soft flour, medium flour, strong flour, brown rice, half-puffed rice, rice with seven parts, milled rice, germ-milled rice, fortified rice, alpha rice, upper fresh flour, white flour, full layer flour, corn mill, Corn grips, corn flour, rye flour, etc. can be used.
In carrying out the production method of the present invention, the above cereal is first immersed in water and then boiled or boiled. The conditions for such immersion are as follows: Soak for 18 hours. Moreover, as conditions for cooking or boiling, put the cereal in a steamer, cover it and send water vapor from the bottom until the weight becomes 1.02 to 2 times that of the cereal before cooking (after soaking) Cook for about 0.2 to 2 hours or boil in boiling water for 30 to 360 minutes.
[0008]
Cereals originally contain 0.1% by weight or less of salt in nature, and steamed or boiled cereals contain 0.01% by weight or less of salt, so you may enter the next molding process as is, 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 wt% or less (preferably 1 wt% 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.
[0009]
The salinity of the culture system is obtained by adding ion-exchanged water to the culture and stirring, diluting the supernatant as appropriate, and measuring using a digital salinometer (“SS-31A” manufactured by Sekisui Chemical Co., Ltd.). Examples of the salt to be added include sodium chloride, calcium chloride, potassium chloride and the like.
[0010]
Next, solid culture is performed after steaming or boiling, but first the mold is applied. Such molds are used in the production of soy sauce, moromi, touchi, miso, etc., and preferably belong to the genus Aspergillus, for example, Aspergillus albus IFO4039, Aspergillus candidus (Aspergillus Oculus), Neissance (Aspergillus nidulans ATCC10074), Aspergillus glaucus (Aspergillus glaucus ATCC10059), Aspergillus oryzae IFO4135, Aspergillus flavus (Ospergalus Insulins) Spergillus indicus ATCC 15054), Aspergillus sulphureus ATCC 11904, Aspergillus niger (Aspergillus IFI 4343), Aspergillus sogier (Aspergillus sojue)
Specifically, “Hi Soya”, “Mild S”, “Three Diamond”, “Diamond C”, “Usumura Saki”, “Treasure Fungus”, “White Soy Sauce Fungus”, “Improved Shochu Fungus” Commercial products such as those manufactured by Matsunosuke Shojiguchi Co., Ltd. are mentioned.
The amount of fungi to be added is not particularly limited, but is about 0.0001 to 10% by weight with respect to cereals. Examples of the method of mold application include methods of sprinkling mold on steamed or boiled cereals, or contacting the cereal with molds remaining in a container or fermentation chamber once cultured.
[0011]
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 grain surface.
[0012]
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.
[0013]
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.
After removing the solid content of the resulting extract by clarification filtration, centrifugation, membrane separation, etc., decolorize with activated carbon or clay as necessary, and then concentrate, dry, freeze dry, spray dry, etc. Preferably it is powdered.
[0014]
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.
[0015]
As an intake, as dry powder, 0.001-10 g / day is preferable, and 0.01-3 g / day is especially preferable.
[0016]
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, Konnyaku, Bread, Noodles (immediate 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 [0017]
(4) Health food (dietary supplement)
(1) Saponin-containing foods (Ginseng root-containing foods, Ezokogi-containing foods)
(2) Sugar-containing foods [oligosaccharides (fructo-oligosaccharide-containing foods, isomalt-oligosaccharide-containing foods, galactooligosaccharide-containing foods), polysaccharides (shiitake-containing foods, mucopolysaccharides, protein-containing foods, chondroitin sulfate-containing foods, mannentake (reishi) ) Contained 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 [0018]
(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)
[0019]
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, other known α-glucosidase inhibitors such as valienamine and aminocyclitol may be used in combination.
[0020]
【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 1000 g of rice was immersed 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 cook 1800 g of cooked rice [salt content is the detection limit (0. 01%) or less]. Such cooked rice is placed on a bamboo basket in a fermentation room, and 10 g of commercially available mold for soy sauce (“Mild S” manufactured by Matsunosuke Hamaguchi Co., Ltd.) is added to the mixture evenly. Solid culture was carried out at RH for 96 hours. After completion of the cultivation, mold on the surface of the culture was washed with water and air-dried at 80 ° C. for 24 hours to obtain 750 g of the culture.
The culture was immersed in 3 liters of water for 1 hour, and then the immersion liquid was placed in an autoclave and extracted at an internal temperature of 125 ° C. for 1 hour. Large residues were removed using a sieve from the resulting extract. The obtained filtrate and the filtrate obtained by adding 3 liters of water again to the residue and filtering the mixture were combined and centrifuged (condition 1000 × g). 100 g of activated carbon was added to the resulting supernatant and stirred to remove the color, followed by filtration to obtain 3 liters of extract. Thereafter, the mixture was concentrated and dried with a rotary evaporator to obtain 225 g of an inhibitor. The inhibitory activity, odor, and ease of intake of such inhibitors were evaluated as follows.
[0021]
(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. .
[0022]
-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
[0023]
(smell)
The odor of the obtained inhibitor was evaluated as follows.
○ ... It is completely odorless.
Δ: Smell slightly.
X ... Strong smell.
[0024]
(Ease of consumption)
○ ・ ・ ・ It is not salty, and the umami taste is easy to take out.
×: Salty or cereal-specific odor is strong, and taking 1g or more is painful.
[0025]
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 steamed rice as in the same example was solid-cultured at 35 ° C and humidity 100% RH for 72 hours. 790 g was obtained.
The culture was immersed in 3 liters of water for 1 hour, and then 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 carried out to obtain 3 liters of an extract, which was concentrated and dried by a rotary evaporator to obtain 245 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 3
Except that the temperature of solid culture was changed to 37 ° C. in Example 1, the same culture was performed to obtain 745 g of culture. The culture was placed in a pottery vessel, capped and post-fermented at 35 ° C. for 40 days to obtain 740 g of the 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 125 ° C. for 1 hour. Thereafter, the same treatment as in Example 1 was carried out to obtain 3.8 liters of an extract, which was concentrated and dried by a rotary evaporator to obtain 237 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]
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 3 liters of an extract, which was concentrated and dried by a rotary evaporator to obtain 142 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.
[0028]
Example 5
A solid culture was carried out in the same manner as in Example 1 except that wheat was used instead of rice to obtain 700 g of the culture. The culture was immersed in 3 liters of water for 1 hour, and then 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 carried out to obtain 3.8 liters of an extract, which was concentrated and dried with 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.
[0029]
Example 6
In Example 1, 730 g of the culture was obtained by solid culture in the same manner except that wheat was used instead of rice. The culture was immersed in 3 liters of water for 1 hour, and then 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 carried out to obtain 3.8 liters of an extract, which was concentrated and dried with a rotary evaporator to obtain 220 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.
[0030]
Comparative Example 1
After 1000 g of rice was immersed in 10 liters of water for 3 hours, it was placed in a steamer and steamed at 100 ° C. for 1 hour to obtain 1800 g of steamed rice (salt content of 0.01% or less). 200 g of salt was added to the cooked rice (10% salinity) and then fermented in the same manner as in Example 1 to obtain 860 g of culture.
The culture was extracted in the same manner as in Example 1, treated in the same manner to obtain 3.3 liters of extract, and concentrated to dryness on a rotary evaporator to obtain 335 g of a solid. The inhibitory activity, odor, and ease of ingestion of the solid were evaluated in the same manner as in Example 1.
[0031]
Comparative Example 2
750 g of the culture obtained in Example 1 was immersed in 3 liters 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 2.8 liters of an extract, which was concentrated and dried by a rotary evaporator to obtain 98 g of a solid. The inhibitory activity, odor, and ease of ingestion of such solids were also evaluated.
[0032]
[Table 1]

[0033]
【The invention's effect】
In the present invention, an α-glucosidase inhibitor is extracted with water at 50 ° C. or higher from a culture obtained by solid-culturing steamed or boiled cereals with mold under a condition that the salinity of the culture system is 5% by weight or less. Thus, 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 rice or wheat using a Aspergillus fungus under a condition where the salinity of the culture system is 5% by weight or less, at a culture temperature of 10-50 ° C. and a humidity of 80-100% RH, 12-120 A method for producing an α-glucosidase inhibitor, characterized by solid culture for a period of time and extraction from the obtained culture with water at 50 ° C. or higher.