KR20070028997A - A strain having high productivity for alpha glucosidase inhibitor and a method for preparing the strain - Google Patents

Abstract

A microorganism capable of producing large quantity of alpha glucosidase inhibitor and a method for preparing the same microorganism are provided to mass-produce alpha glucosidase inhibitor which inhibits degradation of oligosaccharide by alpha glucosidase, thereby reducing blood sugar level. The microorganism capable of producing large quantity of alpha glucosidase inhibitor, Bacillus subtilis DC-15(KCTC 10763BP) is provided, wherein Bacillus subtilis DC-15(KCTC 10763BP) is isolated from fermented soybean(Cheonggukjang). The method for preparing the Bacillus subtilis DC-15(KCTC 10763BP) comprises the steps of: suspending the fermented soybean(Cheonggukjang) sample in distilled water and heating the suspension; culturing the suspension on LB agar medium and isolating colonies grown; inoculating the colonies to LB broth medium and culturing them; and centrifuging the cultured medium at 5000 to 7000g for 5-15 minutes, measuring the alpha glucosidase-inhibiting activity of the supernatant, and selecting the microorganism showing the highest alpha glucosidase-inhibiting activity.

Description

A strain having high productivity for alpha glucosidase inhibitor and a method for preparing the strain}

1 is an electron micrograph of a strain according to the present invention.

The present invention relates to a new strain for producing a large amount of alpha glucosidase inhibitor and its use, and more particularly, to Bacillus subtilis di-15 strain which is isolated and cultured from Cheonggukjang and produces a large amount of alpha glucosidase inhibitor. The present invention relates to a method for producing alpha glucosidase and functional food using the strain, and a pharmaceutical composition, functional food and cosmetic composition comprising the alpha glucosidase.

Alphaglucosidase inhibitors are substances that inhibit the degradation of oligosaccharides by alphaglucosidase and are distributed in some plants and microorganisms. With alpha-glucosidase inhibitors of microbial origin, such as the capital city of Actinoplanes strains origin oligosaccharide (pseudooligosaccharide), 1- deoxy Nojiri azithromycin (1-deoxynojirimycin) which is produced in the bacterium Streptomyces sheath (Streptomyces) and Bacillus (Bacillus) Known and derivatives thereof have been developed and marketed as a drug for lowering blood sugar.

A large number of alphaglucosidase inhibitors are currently in clinical trials or already available as pharmaceuticals (Kmuse et al., 1982; Rhinehalt et al., 1987; Yoshikuni, l988), and the typical glucose lowering alphaglucose already on the market. The inhibitors of acidase include acarbose, a substance found in 1972 of the capital metabolites, a secondary metabolite produced by fermentation of Bayer's Actinoplanes strain. Glucobay, the main ingredient of Acarbose (Rury et al., 1999), Migitol, a derivative of 1-deoxynojirimycin (Asano, l994) isolated from a strain of Streptomysis in 1978. miglitol and emiglitate are miglitol (DeBuono et al, l993), and also vogliose (Matsuo et al., l992; Saito et al., derivative of valiolanin from Takeda) al, l998). In addition, substances that have reached the stage of clinical trials include the polypeptide tendamistat and the monosaccharide emiglitate. The alpha-glucosidase inhibitors have been much isolated from the culture medium of a microorganism, in particular liquid Martino Plastic Ness (Actinoplanes), ampoules La Ella (Ampullariella) and streptomycin Incubation Clear (Streptoporangium) in the bacteria and Aspergillus across switch (Aspergillus) and Cloud also Production of alpha glucosidase inhibitors of fungi in the cladosorium has been reported.

Most of these alphaglucosidase inhibitors are carbohydrate derivatives, which have a cyclohexene ring substituted on the active moiety and a 4,6-di-deoxy-4-amino-D-glucose structure, the amino group of which is alpha glucose. The hydroxyl group of an aze inhibits its hydrolytic activity on the oxygen binding of the substrate (Heiker, 1982).

In addition, polyphenols of various origins are not specific to alpha glucosidase, but have been reported to effectively inhibit the activity of alpha glucosidase and thus have an effect on suppressing blood glucose elevation (Hara and Honda, l990; Honda and Hara, 1993; Matsumoto, 1993).

Accordingly, by inhibiting the activity of alpha glucosidase, various inhibitors have been developed to obtain effects such as hypoglycemia and obesity treatment, and the need for economic mass production of such inhibitors is gradually increasing.

The present invention provides a new strain that produces a large amount of alpha glucosidase inhibitors in order to meet such demands, and to provide a variety of drugs, food and cosmetic compositions using such strains have.

The present invention in one embodiment for achieving the above technical problem,

Provided are Bacillus subtilis di-15 strains that are isolated from Chonggukjang and produce large amounts of alpha glucosidase inhibitors.

In another embodiment,

Suspension of the Cheonggukjang samples in distilled water, and heat-treated in a constant temperature water bath;

Plating the heat-treated suspension on an agar-containing LB plate medium and culturing in an incubator to separate the cells that form a colony;

Inoculating and culturing the isolated strain in LB liquid medium; And

After centrifuging the culture, and measuring the alpha glucosidase inhibitory activity from the supernatant provides a method for producing a Bacillus subtilis Dici-15 (KCTC 10763BP) strain comprising the step of selecting the strain with the highest inhibitory activity. .

In another embodiment,

Provided is a method for preparing an alpha glucosidase inhibitor using a Bacillus subtilis dici-15 (KCTC 10763BP) strain according to the present invention.

In another embodiment,

Provided is a method for producing a functional food using Bacillus subtilis Dish-15 (KCTC 10763BP) strain according to the present invention.

In another embodiment,

It provides a pharmaceutical composition comprising alpha glucosidase prepared by the method according to the invention.

In another embodiment,

Provided is a functional food comprising alpha glucosidase produced by the method according to the invention.

In addition, the present invention in another embodiment,

Provided is a cosmetic composition comprising an alpha glucosidase inhibitor prepared by the method according to the invention.

Hereinafter, the present invention will be described in more detail.

The present inventors have confirmed that the new strain Bacillus subtilis di-15, isolated from Korean traditional soybean fermented food, Cheonggukjang, has the ability to produce a large amount of an alpha glucosidase inhibitor, and thus, the present invention has been completed.

Specifically, the present invention Bacillus subtilis var. DC-15, KCTC 10763BP isolated from the strain of Cheonggukjang, a traditional Korean fermented soybean meal using rice straw, method for producing the strain, from the strain An alphaglucosidase inhibitor produced, and a pharmaceutical composition, functional food and cosmetic composition comprising the alphaglucosidase inhibitor.

According to the present invention, since it is possible to produce a large amount of alpha glucosidase inhibitor by a simple and economical method, using the produced alpha glucosidase, high value-added blood sugar lowering, development of products such as anti-obesity functional foods, etc. This becomes possible.

The present inventors collect a variety of Cheonggukjang produced and marketed in various parts of the Republic of Korea in order to obtain a microorganism producing an alpha glucosidase inhibitor having a variety of functions and applications as described above, excellent productivity of the alpha glucosidase inhibitor Was searched and separated.

As a result of the search, it was isolated from Bacillus isolated from 12 types of natto, a soybean fermented food marketed in Japan, to isolate an excellent Bacillus strain that produces a large amount of alpha glucosidase inhibitor that is not produced, which was Bacillus subtilis Di-15. var. DC-15), and for convenience, the strain was named DC-15 ( Bacillus sp. DC-15), and as of January 18, 2005, the Korea Biotechnology Research Institute Gene Bank (KCTC, Yuseong-gu, Daejeon Metropolitan City). Eoeun-dong 52) was deposited with an accession number of KCTC 10763BP. The isolated strain was obtained from Cheonggukjang, which has been used for a long time and proved to be safe. Therefore, the alphaglucosidase inhibitor produced by the strain is added to pharmaceutical compositions, functional foods, and cosmetic compositions in an appropriate and proper manner. Can be used.

In another embodiment, the present invention provides a method for preparing the strain, which comprises suspending the Cheonggukjang sample in distilled water and heat-treating in a constant temperature bath; Plating the heat-treated suspension on an agar-containing LB plate medium and culturing in an incubator to separate the cells that form a colony; Inoculating and culturing the isolated strain in LB liquid medium; And centrifuging the culture, and then selecting the highest inhibitory activity strain by measuring alpha glucosidase inhibitory activity from the supernatant.

Preferably, the heat treatment is carried out at 60 ℃ to 70 ℃ for 20 to 30 minutes, the incubator temperature in the culture of the heat-treated suspension is 20 ℃ to 40 ℃, the culture time is 1 day to 3 days.

In addition, the volume of the LB liquid medium in which the isolated strain is inoculated is preferably 1 ml to 10 ml, and the incubation temperature and time after inoculation are preferably 20 ° C. to 40 ° C. and 24 to 72 hours, respectively.

Morphological and physiological characteristics of the strains produced by the above method are as follows.

That is, the strain according to the present invention forms a colony having a milky white rough surface when cultured in LB agar plate medium, and is a Gram-positive bacterium with active growth of cells under aerobic conditions of 25 ° C to 30 ° C. There is a characteristic that the cell growth does not occur at the culture temperature.

In another embodiment, the present invention provides a method for preparing an alpha glucosidase inhibitor using the strain prepared as described above.

Method for producing an alpha glucosidase inhibitor according to the invention, the step of centrifuging the culture medium of the strain; And obtaining an alpha glucosidase inhibitor from the supernatant obtained from the centrifugation.

Preferably, the centrifugation is performed at 5000g to 7000g for 5 minutes to 15 minutes.

In another embodiment, the present invention provides a method for producing a functional food using the strain prepared as described above, wherein the functional food is preferably Cheonggukjang.

In the case of preparing the cheonggukjang using the strain according to the present invention, after inoculating and culturing the strain according to the present invention in boiled soybeans, it can be prepared according to the conventional cheonggukjang manufacturing method.

Cheonggukjang prepared according to the present invention was prepared by selecting strains with very high activity of alpha glucosidase inhibitors, and thus the activity is excellently higher than that of conventional Cheonggukjang. That is, no activity of alpha glucosidase inhibitor was found in 12 types of natto, a soybean fermented food marketed in Japan, and the conventional Cheonggukjangs sold in Korea also showed low and uneven alpha glucosidase inhibitor activity. It shows a markedly different alpha glucosidase activity from Chungkookjang according to the invention. This is because conventional conventional Cheonggukjang is prepared by mixed culture of strains or pure culture using alpha glucosidase inhibitor inactive strains, whereas Chunggukjang according to the present invention selects only strains with particularly high alpha glucosidase activity and pure cultures. This is because the production of Cheonggukjang.

In addition, the alpha glucosidase inhibitor prepared as described above may be added to various medicines, food and cosmetics, etc. Therefore, in another embodiment of the present invention, the pharmaceutical composition, functional food and cosmetics comprising the alpha glucosidase inhibitor To provide a composition.

For example, when preparing a pharmaceutical composition comprising an alpha glucosidase inhibitor according to the present invention, various pharmaceutically acceptable additives may be included, and such additives include conventional excipients, disintegrants, binders, One kind or two or more kinds of lubricants, suspending agents and the like may be selectively used. For example, when the composition of the present invention is prepared in a solid form such as tablets or hard capsules, microcrystalline cellulose, lactose, low-substituted hydroxycellulose and the like can be used as excipients, sodium starch glycolate, anhydrous as a disintegrant. Calcium monohydrogen phosphate may be used. As the binder, polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose and the like can be used, and the lubricant can be selected from magnesium stearate, silicon dioxide, talc and the like. In addition, as the suspending agent, surfactants (eg, sorbitan esters, polysorbates, etc.) commonly used in the pharmaceutical field may be used.

The pharmaceutical compositions can be formulated in a variety of forms, including solid and liquid forms, which include tablets, capsules (hard or soft), solutions, suspensions, emulsions, syrups and the like.

The pharmaceutical composition may be administered by various administration methods including oral or parenteral, and the dosage may be adjusted to an appropriate amount depending on the condition such as age, age and sex and the severity of the disease, such dosage Can be easily adjusted by those skilled in the art who are clinically engaged in the treatment or prevention of patients.

Alpha glucosidase inhibitors according to the present invention may be added to various types of functional foods. Non-limiting examples of such functional foods include confectionery, gums, beverages, and the like. In addition, alpha glucosidase inhibitors according to the invention can also be used in the preparation of cosmetic compositions. In this case, it is possible for a person skilled in the art to add various kinds of additives within an appropriate content range.

When preparing a functional beverage or cosmetic composition using the alpha glucosidase inhibitor according to the present invention, the culture of Cheonggukjang according to the present invention is extracted with water, centrifuged, and concentrated to add the supernatant. Can be. In this case, in addition to the alpha glucosidase inhibitor, the extract of the Chungkookjang culture may contain additives such as water-soluble peptides and polygamma glutamic acid to impart functionality other than the alpha glucosidase inhibitor.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the present invention by way of example, the scope of the present invention is not limited only to the following examples.

Example 1. Isolation and Identification of Strains According to the Invention

1.1 Isolation of Strains

About 200 kinds of Cheonggukjang, a traditional soybean fermented food produced by traditional soybean fermentation method using rice straw, were obtained from all over the country and used as samples. Each sample was added to a small amount of sterile distilled water and suspended, followed by heat treatment for 20 minutes in a 60 ° C. constant temperature water bath to form endogenous spores of Bacillus bacteria, and then the LB plate medium containing 2% agar (1% tryptone). , 0.2% sugar, 0.5% yeast extract and 0.5% NaCl, pH7.0) and incubated for 2 days in a 30 ℃ incubator. After incubation, the cells forming the colonies were isolated. In order to investigate the production amount of the alpha glucosidase inhibitor obtained by the present invention, the isolated strain was inoculated in 5 ml LB liquid medium and incubated at 30 ° C. for 48 hours. The culture medium was centrifuged to measure alpha glucosidase inhibitory activity from the supernatant and the strain having the highest inhibitory activity was selected.

1.2 Morphological and Biochemical Properties of Isolated Strains

(1) Morphological Characteristics of Isolated Strains

The isolated high activity of the alpha glucosidase inhibitor strain formed a milky white colony in LB agar plate medium, the edges of the cells had a sawtooth shape and the surface of the colony showed a wrinkled appearance. In the temperature effect on the cell growth, the growth was good in the range of 25 ℃ to 40 ℃, cell growth was not confirmed at 50 ℃ or more. As a result of microscopic observation in the logarithmic phase of cell growth using LB liquid medium, it was a gram-positive short rod-like appearance similar to that of the comparative strain Bacillus subtilis, and an electron micrograph of the strain is shown in FIG. 1. The strain was Gram positive and the cell size was approximately 0.6-0.7 × 1.5-1.7 μm.

(2) Biochemical Properties of Isolated Strains

The biochemical properties of strains according to the present invention were investigated using a method for analyzing the taxonomic characteristics of the strains of genus Bacillus (Bergey's Manual of Systematic Bacteriology, 2002) and the API50CHB kit (bioMerieux, France). The strain according to the present invention is a gram-positive cylinder, formed in the center of spore cells, has a reducing power of nitrate, and does not produce indole. It can also degrade casein and starch, produce catalase, and grow only under aerobic conditions. On the other hand, glycerol, L-arabinose, D-xylose, D-galactose, D-glucose, D-mannitol, sucrose, maltose and the like can be used.

Detailed morphological and biochemical properties of the strains according to the invention are shown in Table 1 below.

 characteristic  Inventive strain  Gram dyeing  positivity  Shape and size  Rod shape 0.6 ~ 0.7 × 1.6 ~ 1.9 ㎛  Spores  Forming  Spore's Location  Cylindrically located at the center of the cell  Motility (electron micrograph)  positivity  Growth temperature  25 to 40 ℃  Growth in NaCl 7%  positivity  Growth in aerobic conditions  positivity  Growth in anaerobic conditions  voice  Boze-Pro Scowzer Exam  positivity

 Nitrate reduction  positivity  Indole formation  voice  Catalase  positivity  Starch decomposition  positivity  Casein decomposition  positivity  Propionic Acid Availability  voice  Acid production from glycerol  positivity  Acid production from L-arabinose  positivity  Acid production from D-xylose  positivity  Acid production from D-galactose  positivity  Acid production from D-glucose  positivity  Acid production from D-mannitol  positivity  Acid production from D-maltose  positivity  Acid production from D-sucrose  positivity  Acid production from D-lactose  positivity

(3) Identification of Isolated Strains

The strain of the present invention exhibits a property of producing a large amount of an inhibitor of alpha glucosidase, unlike a strain of the genus Bacillus. Therefore, the strain of the present invention is classified as a new strain belonging to Bacillus subtilis and named as' Bacillus subtilis var. DC-15 ', and for convenience, the name of the strain is' B. 15 '( Bacillus sp. DC-15) was deposited on January 18, 2005 with the Korea Biotechnology Research Institute Gene Bank (KCTC, 52 Ueun-dong, Yuseong-gu, Daejeon) and received the accession number KCTC 10763BP.

Example 2 Preparation of Cheonggukjang Using Strains According to the Present Invention and Determination of Alpha Glucosidase Inhibitor Activity of Prepared Cheonggukjang

After inoculating the boiled soybean strains of the present invention, it was incubated for 72 hours at 30 ℃. Stirring was performed twice a day for smooth supply of oxygen during the culture. After the incubation, it was autoclaved to deactivate the alpha glucosidase and protease produced by the strain of the present invention, and then, 10 g of Cheonggukjang was added 10 times of 50 mM phosphate buffer solution (pH 7.0) and homogenizer (homogenizer). ) Was finely crushed and then extracted by shaking at 4 ° C. for 10 hours or more. Cheonggukjang suspension was centrifuged to determine the alphaglucosidase inhibitory activity of the supernatant. Alpha glucosidase inhibitory activity was measured as follows. That is, 50 μl of 1M phosphate buffer solution (pH 7.0), 50 μl of pig small intestine source alphaglucosidase coenzyme diluted to a predetermined concentration, and 50 μl of alpha glucosidase inhibitor were mixed and pre-warmed at 37 ° C. for 3 minutes. The reaction was performed by adding 500 μl of substrate, 0.075% maltose solution. Subsequently, after the reaction for 30 minutes, the reaction was stopped by heating in boiling water for 5 minutes, and after cooling sufficiently, 100 μl of this was added to 1 ml of the kit for measuring the amount of glucose and left at 37 ° C. for 5 minutes, followed by a spectrophotometer. Alpha glucosidase inhibitory activity was measured by measuring the absorbance at 500 nm. In this case, the inhibition rate was calculated according to the following formula using water as a control instead of the inhibitor.

% Inhibition = (A-B) / A × 100

Where A is the absorbance of the control and B is the absorbance of the sample containing the inhibitor.

Cheonggukjang according to the present invention and 16 conventional conventional Cheonggukjang commercially available in Korea and Japan were extracted by the above method, and diluted 3200-fold to determine the alphaglucosidase inhibition rate by the above method is shown in Table 2 below.

 sample  Alphaglucosidase inhibition rate (%)  Bacillus subtilis dish-15 Cheonggukjang  32  Korea Commercial Cheonggukjang A  0  Korea Commercial Cheonggukjang B  0  Korea Commercial Cheonggukjang C  0  Korea Commercial Cheonggukjang D  0  Japan market natto E  0  Japan's market natto F  0  Japan's market natto G  0  Japan market natto H  0  Japan's market natto I  0  Japan market natto J  0  Japan market natto K  0  Japan market natto L  0  Japan market natto M  0  Japan's market natto O  0  Japan market natto P  0  Natto Q in Japan  0

According to the present invention, it is possible to obtain a strain producing a large amount of alpha glucosidase inhibitor, the strain according to the present invention can be usefully used for the production of various functional foods, such as Cheonggukjang, etc., from the strain according to the invention The prepared alpha glucosidase may be usefully used for the preparation of high value-added blood sugar lowering agents, anti-obesity functional foods, and the like.

Claims (10)

Bacillus subtilis di-15 strain isolated from Chonggukjang and producing a large amount of alpha glucosidase inhibitor. The strain of claim 1, deposited with accession number KCTC 10763BP. Suspension of the Cheonggukjang samples in distilled water, and heat-treated in a constant temperature water bath; Plating the heat-treated suspension on an agar-containing LB plate medium and culturing in an incubator to separate the cells that form a colony; Inoculating and culturing the isolated strain in LB liquid medium; And After centrifuging the culture, the method for producing Bacillus subtilis di-15 (KCTC 10763BP) strain comprising the step of selecting the highest inhibitory activity by measuring the alpha glucosidase inhibitory activity from the supernatant. Centrifuging the culture medium of the strain according to claim 1; And obtaining an alpha glucosidase inhibitor from the supernatant obtained from the centrifugation. The method of claim 4, wherein the centrifugation is performed at 5000g to 7000g for 5 to 15 minutes. Method for producing a functional food using the strain according to claim 1 or 2. 7. The method of claim 6, wherein the functional food is Cheonggukjang. A pharmaceutical composition comprising an alpha glucosidase inhibitor prepared by the method according to claim 4. Functional food comprising an alpha glucosidase inhibitor prepared by the method according to claim 4. Cosmetic composition comprising an alpha glucosidase inhibitor prepared by the method according to claim 4.

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