KR100734975B1 - 1- 10 1-deoxynojirimycin-producing bacillus subtilis s10 strain and composition comprising same - Google Patents

Abstract

The present invention relates to a novel Bacillus subtilis S10 strain producing 1-deoxynojirimycin (DNJ) and a composition containing the culture medium of the strain as an active ingredient. The Bacillus subtilis S10 strain of the present invention isolated and identified as an α-glucosidase inhibitory strain produces a large amount of 1-deoxynojirimycin that exhibits hypoglycemic, antiviral and anticancer effects. For the purpose of the above effects can be usefully used to prepare feeds, food, cosmetics, pharmaceuticals and the like.

Description

Bacillus subtilis S10 strain producing 1-deoxynojirimycin and a composition containing the same as an active ingredient {1-DEOXYNOJIRIMYCIN-PRODUCING BACILLUS SUBTILIS S10 STRAIN AND COMPOSITION COMPRISING SAME}

1 is a dendogram comparing the 16S rDNA nucleotide sequence of the Bacillus subtilis S10 strain of the present invention with the nucleotide sequences on GenBank,

2 is a result of thin layer chromatography analysis of 1-deoxynojirimycin isolated and purified from Bacillus subtilis S10 strain of the present invention,

3 is a result of HPLC analysis of 1-deoxynojirimycin isolated and purified from Bacillus subtilis S10 strain of the present invention,

Figure 4 is a result of comparing the molecular weight of 1-deoxy nojirimycin and silkworm-derived 1-deoxy nojirimycin isolated and purified from the Bacillus subtilis S10 strain of the present invention,

5 is a result of measuring the activity inhibition of α-glucosidase according to the concentration change of 1-deoxy nozirimycin isolated and purified from Bacillus subtilis S10 strain of the present invention,

6 is a result of investigating the inhibition of α-glucosidase activity of Bacillus subtilis S10 strain of the present invention over time,

Figure 7 is the result of investigating the hypoglycemic effect of the Bacillus subtilis S10 strain of the present invention in streptozotocin-induced diabetic mice.

The present invention relates to a novel Bacillus subtilis S10 strain producing 1-deoxynojirimycin (DNJ) and a composition containing the culture medium of the strain as an active ingredient.

1-deoxynojirimycin (DNJ) is a representative substance of the piperidines family having a hexasaccharide monocycle structure (Asano et al., Tetrahedron 11: 1645-1680, 2000). It is an alkaloid substance that contains several hydroxyl groups (OH) and has a structural characteristic in which oxygen is substituted with nitrogen.

This material is synthesized from L-sorbo furanose (Paulsen et al., Chem Ber. 100: 802, 1967) or reduction of nozirimycin (Inouye et al., Tetrahydron 24: 2125-2144, 1968 By chemical methods such as Bacillus (Schimidt et al., Naturwissenshaften 66: 584, 1979), Streptomyces (Inouye et al., J. Antibiot. Ser.A, 19: 288-292, 1966: Ishida et al., J. Antibiot. Ser.A, 20: 66-71, 1967: Muano and Miyata, Agric Biol. Chem. 44: 219-221, 1980) and the like ( Morus alba L. ) Or silkworm ( Bombyx mori L.) (Asano et al., J. Agric. Food Chem . 49: 4208-4213, 2001).

1-Deoxynozirimycin rapidly increases blood sugar levels after meals by inhibiting the activity of α-glucosidase, which breaks down starch, oligosaccharides and maltose into the blood and digests them in the digestive tract of mammals. Prevent it from happening. In particular, 1-deoxy nozirimycin has a high inhibitory activity against α-glucosidase in the small intestine of the mammal and inhibits glucose inflow from the small intestine to the blood, thus causing insulin-independent diabetes mellitus for a long time. Insulin Independent Diabetes Mellitus) has been attracting attention (Yagi et al., Nippon Nogeikagaku Kaishi , 50: 571-572, 1976). In addition, as the anti-viral, anti-cancer and cancer metastasis inhibitory effect of 1-deoxy nojirimycin has been found, the development of various medicines using the same has been actively made.

However, since 1-deoxynojirimycin is an expensive natural raw drug, it is not easily used in daily life, and although it is expected to lower blood sugar by ingesting it through some mulberry leaf tea or silkworm powder, Due to its rigid structure, 1-deoxynojirimycin is not easily eluted, and silkworm powder has a problem in that it is difficult to ingest due to aversion or rejection and must be consumed separately from the daily diet. On the contrary, microorganisms such as the genus Bacillus have the advantage of being able to produce large amounts of 1-deoxy nozirimycin due to its excellent growth rate, easy strain improvement, and convenient fermentation and production management.

Accordingly, the present inventors have sought to select such microorganisms, and as a result, we have isolated a novel Bacillus subtilis S10 strain that produces a large amount of 1-deoxynozirimycin among microorganisms showing α-glucosidase inhibitory activity. The 1-deoxy nozirimycin identified and cultured from or isolated from the microorganism strain can be usefully used as a microbial additive for feed, food, cosmetics, medicines, etc. for the purpose of lowering blood sugar, antiviral and anticancer effects. By confirming that the present invention was completed.

Accordingly, it is an object of the present invention to provide a Bacillus subtilis strain that produces 1-deoxynojirimycin.

Another object of the present invention is to provide a composition containing a culture solution of Bacillus subtilis strains as an active ingredient.

In order to achieve the above object, the present invention provides a novel Bacillus subtilis S10 strain (Accession No .: KCCM 10650P), which produces 1-deoxynojirimycin, which exhibits hypoglycemic, antiviral and anticancer effects.

Bacillus subtilis S10 strain of the present invention can be obtained from the soil, specifically isolates the microorganisms that inhibit the α-glucosidase activity from the soil, among them a potent and α-glucosidase inhibitory ability at the same time After selection of strains with good production of oxynojirimycin and safe as foods, the final selection is performed by confirming that they have at least 99% homology with Bacillus subtilis by 16S rDNA analysis. The present inventors named the microorganism Bacillus subtilis S10 ( Bacillus subtilis S10) and deposited it on April 1, 2005 at Korea Microorganism Conservation Center (KCCM) (Accession No .: KCCM 10650P).

The Bacillus subtilis S10 strain is a Gram-positive bacillus that forms a velvety colony of 0.4 to 16 mm in diameter, white to brownish hue, slightly motile, and spore-forming.

Physiologically, the Bacillus subtilis S10 strain grows under conditions of 20 to 60 ° C., preferably 30 to 55 ° C., pH 3.0 to 11.0, preferably pH 4.5 to 10, and is anaerobic, L-arabi North, D-mannose, sorbitol, iakuline, maltose, saccharose, glycogen, ribose, D-glucose, salicycin, trehalose, D-rapinose, D-fructose, mannitol, arbutin, cellobiose, inulin, Sugars such as amidone and β-gentiobioses can be used.

In addition, the microorganisms mass produce 1-deoxynojirimycin, as confirmed by analyzing its culture by high performance liquid chromatography (HPLC). As described above, Bacillus subtilis S10 isolated and identified as an α-glucosidase inhibitor strain according to the present invention produces high concentrations of 1-deoxynojirimycin having hypoglycemic activity, antiviral and anticancer activity. It is expected that it may be usefully used as a microbial agent for cosmetics, pharmaceuticals and the like.

Accordingly, the present invention provides a pharmaceutical composition for lowering blood sugar, antiviral or anticancer, containing the culture medium of the Bacillus subtilis S10 strain as an active ingredient and including a pharmaceutically acceptable carrier or excipient.

1-deoxynojirimycin produced by the strain of the present invention is a compound known to have little toxicity and side effects, inhibits the activity of α-glucosidase and inhibits glycemic elevation or inhibits glycoprotein synthesis necessary for virus growth. By blocking the infection of animal viruses, or inhibiting cancer and cancer metastasis, the composition containing the culture medium of the strain according to the present invention as an active ingredient can be used with confidence even in long-term use.

After culturing Bacillus subtilis S10, the 1-deoxynojirimycin can be purified by 90% or more, preferably 98% or more purely by repeatedly passing the culture medium from which the cells are removed through an ion exchange resin.

Culture of the Bacillus subtilis S10 strain of the present invention or 1-deoxynojirimycin produced therefrom is included in an amount of 0.1 to 50% by weight relative to the total weight of the pharmaceutical composition.

Compositions of the present invention may include carriers, excipients and / or diluents commonly used in the manufacture of pharmaceutical compositions, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, Starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and Mineral oil.

The compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions according to conventional methods. In this case, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used are prepared. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as calcium carbonate, starch, sucrose, lactose, gelatin in the active ingredient. Etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups.In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, microgol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention may generally be administered in an amount of 0.01 to 500 mg / kg body weight, preferably 0.1 to 100 mg / kg body weight, once or several times a day, but this is the route of administration, the severity of the disease, and the patient. The dose may be increased or decreased depending on the sex, weight, age, etc. of the present invention, but the above dosage does not limit the scope of the present invention in any aspect.

The present invention also provides a feed additive containing the culture of Bacillus subtilis S10 as an active ingredient.

The feed additive according to the present invention may be prepared by mixing the probiotic prepared by spray drying or freeze drying the culture solution of Bacillus subtilis S10 or 1-deoxynojirimycin isolated and purified from the culture solution with an excipient. The feed additive according to the present invention is useful for blocking pathogenic viral infections in livestock since it inhibits glycoprotein synthesis necessary for virus propagation.

The feed additive thus prepared contains 1-deoxynojirimycin at 0.5 to 5 g / l, preferably 2 to 4 g / l, which can be added to the feed in an amount of 0.1 to 2% by weight It can be applied to all fermented feeds, silage feeds, powdered feeds and velvet feeds, regardless of the type or type of feed.

The present invention also provides a food additive containing a culture of Bacillus subtilis S10 as an active ingredient.

The food additive according to the present invention may be added to foods or beverages for the purpose of preventing diabetes diseases through lowering blood sugar, inhibiting infection of pathogenic viruses, or inhibiting cancer or cancer metastasis, and may be provided as functional foods or health supplements. The food additives may be added as they are or may be appropriately mixed with other foods or food ingredients according to conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, that is, the prevention, health promotion or therapeutic treatment, and the same as the above-mentioned feed additive, 0.1 to 5 g / L of 1-deoxynojirimycin, preferably Preferably 2 to 4 g / l. In addition, the Bacillus subtilis S10 may be fermented into soybeans, grains, or the like or may be used as a dry matter.

There is no particular limitation on the type of food to which the food additive of the present invention can be applied, and dairy products, various soups, including meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, ice cream, Drinks, teas, drinks, alcoholic beverages and vitamin complexes, and the like, and includes the functional foods and health supplements in the conventional sense. The amount of the food additive according to the present invention may generally be added at 0.01 to 15% by weight of the total food weight in the case of food, and at a ratio of 0.02 to 1 g based on 100 ml in the case of a beverage.

The food additive of the present invention contains various nutrients, carbohydrates, vitamins, minerals (electrolytes), flavoring agents, coloring agents and neutralizing agents, pectic acid and its components, except for containing the culture solution of Bacillus subtilis S10 as an active ingredient in the indicated ratio. Salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like, may optionally be contained. These components may be used independently or in combination of two or more components, and the ratio of these components is not so important but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation and Identification of α-glucosidase Inhibiting Strains

<1-1> Isolation of α-glucosidase inhibitor

In order to select the α-glucosidase inhibitor strains, 4,000 strains were isolated from 1,000 soil samples collected from Gyeonggi-do, Chungcheong-do and Jeolla-do, and among them, spores were formed, which is likely to be a bacterium of Bacillus. Only high strains were selected and then the α-glucosidase inhibitory activity was examined as follows.

First, sterilized corn by adding 10 g of corn starch, 5 g of soybean meal, 5 g of yeast extract, 0.5 g of diphosphoric acid and 0.5 g of ammonium sulfate to 1 L of distilled water and adjusting the pH to 7.5 Starch broth (corn starch broth, CSB) was prepared. Inoculate 1 loop each of the strains into a 250 ml Erlenmyer flask containing 100 ml CSB medium, shake culture at 180 rpm in a 37 ° C. incubator for 7 days, and then use the culture solution obtained therefrom. Α-glucosidase inhibitory activity was measured as follows.

100 μl of culture supernatant and 900 μl of distilled water were added to a 15 ml test tube, followed by 900 μl of 0.1 M phosphate buffer and 12 mM p-nitrophenyl-alpha-glucopyranoside (substrate). 1 ml of p-nitrophenyl-α-glucopyranoside (Sigma) was added. 100 µl of α-glucosidase (100 unit / ml, Sigma) was added thereto, followed by enzymatic reaction for 45 minutes in a 37 ° C. constant temperature water bath. At this time, 100 μl of 0.1 M phosphate buffer solution was added instead of α-glucosidase, and 100 μl of distilled water was used instead of the culture supernatant. After 45 minutes, 1 ml of 200 mM sodium carbonate was added to stop the reaction. Then, the enzyme reaction solution was prepared by using a spectrophotometer (UV / vis spectrophotometer, Shimadzu Co., Ltd.) at 405 nm. The absorbance value (S) of the experimental group reacted by addition, the absorbance value (B) of the test zone reacted by adding a buffer solution instead of the enzyme, and the absorbance value (C) of the control group reacted by adding distilled water instead of the sample solution were measured. Α-glucosidase inhibitory ability of each strain was calculated according to Equation 1 below.

From this, as shown in Table 1 below, strains showing 85% or more α-glucosidase inhibitory ability were selected.

In the present invention, among the strains showing α-glucosidase inhibitory ability of 85% or more in Table 1 , S10 strains which were found to be excellent in food safety belong to GRAS (Generally Recognizable As Safe), which is an edibility criterion, was finally selected.

<1-2> Identification of Selected α-Glucosidase Inhibiting Strains

In Example <1-1>, 16S rDNA sequencing was performed to identify S10 strains selected as α-glucosidase inhibitor strains.

First, single colonies generated by culturing the selected strains in YM plate medium were taken, and genomic DNA was isolated using AccuPrep Genomic DNA Extraction kit (BIONEER). After confirming the separated genomic DNA by electrophoresis, it was used as a template and PCR (GeneAmp PCR System 2700) was performed using 27F of SEQ ID NO : 1 and 1492R of SEQ ID NO: 2 as a primer pair. At this time, PCR was performed by repeating the reaction of 95 seconds for 30 seconds (modification), 55 ° C for 1 minute (annealing), and 72 ° C for 1 minute (extension). PCR products obtained therefrom were subjected to electrophoresis to confirm that 16S rDNA of about 1,500 bp size was amplified. The amplified 16S rDNA was purified using an AccuPrep PCR Purification kit (BIONEER), and then analyzed for 16S rDNA sequences using an automated DNA sequencer (ABI 3100, Applied Biosystem). It was. At this time, four types of primers were used as primers for sequencing , as well as 27F and 1492R of SEQ ID NOs: 1 and 2 , 530F of SEQ ID NO : 3 , and 1100R of SEQ ID NO: 4 . The sequences analyzed therefrom were combined using Cluster X software and then advanced blast search (NCBI) provided by The National Center for Biotechnology Information (http: //www.ncbi.nlm.nih). Advanced Blast search, Altschul et al., Nucleic Acids Research 25: 3389-3402, 1997) were used to compare sequences on Genbank, and finally dendrograms were determined by neighbor-joining analysis. Created.

As a result, the S10 strain having excellent α-glucosidase inhibitory activity has a 16S rDNA nucleotide sequence represented by SEQ ID NO: 5 , and this sequence was found to show 99% homology with Bacillus subtilis ( 1 ). Thus, the strain was named Bacillus subtilis S10 and was deposited in Korea Microorganism Conservation Center (KCCM) as of April 1, 2005 (Accession Number: KCCM 10650P).

Example 2 Biological Properties of α-Glucosidase Inhibiting Strains

The biological properties of Bacillus subtilis S10 identified as α-glucosidase inhibitors in the above were investigated as follows.

First, the Bacillus subtilis S10 strain was inoculated into YM (YM) plate medium containing 0.3% enzyme extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose and 1.8% agar and for 48 hours at 37 ° C. Colonies formed by culturing were observed. As a result, the strain was a Gram-positive bacillus that forms a velvety colony with a size of 0.4 to 16 mm in diameter, and has a white to brown tint and some mobility and sporulation ability. In addition, the optimum culture conditions of the Bacillus subtilis S10 strain exhibited anaerobic anaerobicity in the range of 20 to 60 ℃, pH 3.0 to 11.0.

The biochemical properties of the strain were investigated using an API 50 CH kit (BioMerieux) for identifying Bacillus bacteria, inoculated with the strain on the same YM agar medium and incubated at 37 ° C. for 2 days, 5 ml of colonies formed. Was suspended in 0.85% NaCl saline at high concentration. This was again suspended in 5 ml dilution to McFarland standard (BioMerieux) turbidity 2 and then 10 ml of CHB medium (0.2% ammonium sulfate, 0.05% yeast extract, 0.1% tryptone) contained in the API 50 CH kit. , 0.018% phenol red, 1% (v / v) mineral base in phosphate buffer, pH 7.8) and finally the McF turbidity 2 was adjusted. CHB medium suspension was dispensed into each well of CH50 strips (20 strips) by 140 μl, and then mineral oil was dispensed until the scale of each well was convex and incubated at 37 ° C. The sugar availability of the strain was examined by observing the color change of the phenolphthalein indicator at 12 hour intervals.

As a result, the Bacillus subtilis S10 strain of the present invention is L-arabinose, D-mannose, sorbitol, iaculin, maltose, saccharose, glycogen, ribose, D-glucose, salicycin, trehalose, D- Raffinose, D-fructose, mannitol, arbutin, cellobiose, inulin, amidone and β-gentiobio were positive.

Example 3 Isolation and Purification of 1-deoxynojirimycin

Ion-exchange resin, thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and liquid from the culture of Bacillus subtilis S10 isolated and identified as α-glucosidase inhibitor 1-deoxynojirimycin (DNJ) was isolated and purified through liquid chromatography-mass spectrometer (LC-MS) analysis as follows.

First, Bacillus subtilis S10 strains were cultured in CSB medium for 1 day to make seed for inoculation. 3 L of CSB medium was added to 5 L fermenter (Jar fermenter, KFC), sterilized at 121 ° C. for 20 minutes, inoculated with the seed prepared as described above, and incubated under conditions of 37 ° C., 300 rpm, and 0.5 vvm for 5 days. .

After adsorbing 10 ml of the culture solution on a column (20 × 400 mm, H ⁺ form) filled with Amberlyst 15 (Sigma, A-5647), an ion-exchange resin, secondary Washing with distilled water to remove the pigment. The eluate obtained by eluting this column with 0.5 N ammonia water was concentrated with a vacuum concentrator (Buchi). The resulting concentrate was added to a column (20 × 400 mm, OH ⁻ form) filled with Doex 1 × 2-100 (Sigma-aldrich, 21,739-7), adsorbed, and eluted with secondary distilled water. A fraction was obtained. Each fraction obtained in the separation process was added dropwise to TLC plate (Silica gel 60 F254, Merck) and then developed under solvent conditions of propanol-acetic acid-water (4: 1: 1). It was. The plates were developed using a chlorine-o-tolidine reagent to compare the Rf values of each fraction. The fractions having the same Rf value as the 1-deoxynojirimycin standard sample were collected and concentrated under reduced pressure, and then again packed with Amberlite (Amberlite CG-50) (Sigma-aldrich, 21,635-6) (20 × 400 mm). , NH ₃ ⁺ form) and eluted with secondary distilled water to obtain a fraction. Each fraction was analyzed again using the TLC method as described above, and only the fractions containing 1-deoxynojirimycin were collected and concentrated under reduced pressure.

Figure 2 shows the results of the TLC analysis, lane 1 is a 1-deoxy nojirimycin standard sample, lanes 2 to 18 in the even unit received fractions received in 10 ml in a column filled with amber 3 μl in TLC It was dripped down. As a result, it can be seen that 1-deoxynojirimycin isolated and purified from the Bacillus subtilis S10 strain of the present invention is detected at the same Rf value 0.46 as the standard sample.

10 μl of the concentrate prepared above for HPLC analysis, 10 μl of 0.4 M potassium borate buffer (pH 8.5) and 20 μl of 10 mM 9-fullenylmethyl-chloroformate, FMOC-Cl) was added to an eppendorf tube and reacted at 20 ° C. for 20 minutes to stop the reaction by adding 10 μl of 0.1 M glycine (glycine). 950 μl of 0.1% acetic acid was added to the reaction solution to adjust the volume to 1 ml, and then filtered through a 0.2 μm syringe filter (nylon filter, Nalgene). Μl was used for HPLC analysis. The column was a C ₁₈ capsule pack (C ₁₈ Capcell Pak MG, 4.60 × 250 mm, inner diameter 5 μm), and the solvent was acetonitrile-0.1% acetic acid (1: 1, v / v). Eluted at an elution rate of 1 mL / min. The luminescence of the eluate was detected using a FL3000 fluorescence detector (excitation: 254 nm, emission: 322 nm), which was analyzed using the SMC21 Quick analysis program.

As a result, as shown in FIG . 3 , it was confirmed that 1-deoxynojirimycin was purified and purified from the Bacillus subtilis S10 strain according to the method of the present invention.

In addition, 1-deoxynojirimycin isolated and purified from the culture medium of the Bacillus subtilis S10 strain as described above was diluted 100-fold with distilled water, adjusted to 100 mM concentration, and then liquid chromatography-mass spectrometer (400 μl / min). Molecular weight was measured by direct injection into a liquid chromatography-mass spectrometer, LC-MS, agilent 1100 LC / MSD trap classic Agilent). At this time, 1-deoxy nojirimycin (provided by the Rural Development Administration, Department of Agriculture and Biological Activities) isolated and purified from silkworms was used as a comparison group.

As a result, it can be seen that the 1-deoxynojirimycin isolated and purified from the Bacillus subtilis S10 strain of the present invention has the same molecular weight as that of the silkworm-derived 1-deoxynojirimycin previously reported at 164. ( FIG. 4 ).

<Example 4> α-glucosidase inhibitory ability according to the concentration of 1-deoxy nojirimycin

In Example 3, 1-deoxynozirimycin isolated and purified from the Bacillus subtilis S10 strain of the present invention was diluted to an appropriate concentration, followed by 1-deoxyno according to the method described in Example <1-1>. The inhibitory effect of α-glucosidase according to the concentration of girimycin was investigated.

As a result, as shown in FIG . 5 , α-glucosidase activity was inhibited in a concentration-dependent manner, and a hemihydrate enzyme inhibition concentration (EC ₅₀ ) was confirmed at about 0.7 mM.

<Example 5> α-glucosidase inhibitory ability according to the change of incubation time

As in Example <1->, 1.5 L of CSB medium was prepared, inoculated with Bacillus subtilis S10 strain, and incubated in a 5 L fermenter (Jar fermentor) for 2 days at 37 ° C. and 280 rmp. Culture medium samples were taken to measure changes in carbon source, nitrogen source, and personnel. In addition, cell growth was measured by calculating the dry weight of the collected culture sample, α-glucosidase inhibitory ability was measured by performing the collected culture sample according to the method described in Example <1-1>.

As a result, as shown in Figure 6 , as the culture proceeds, the carbon source, nitrogen source, personnel, etc. were reduced, the cell growth reached a logarithmic growth in about 20 hours. Also, when cell growth reached log phase, α-glucosidase inhibitory activity showed the highest value.

Example 6 In Vivo in vivo Hypoglycemic effect

In vivo of 1-deoxynojirimycin isolated and purified from culture medium of Bacillus subtilis S10 strain in Example 3 The hypoglycemic effect was tested in hyperglycemic mice.

First, three-week-old male and female ICR mice were acclimated for one week with sufficient feed and water in an animal feeding room maintained at a temperature of 23 ± 1 ° C. and a humidity of 55 ± 5%, and then crossed. 60 mg / kg streptozotocin (streptozotocin, STZ, Sigma, S-0130) was added 40 μl of 0.1 M citric acid buffer (pH 4.5) to induce 3 days old mice into persistent hyperglycemia. Was injected once into the abdominal cavity and at 3 weeks of age, only mice with a blood glucose level of 200 mg / dL or more were selected and used for the experiment.

Changes in blood glucose levels of the culture concentrate samples of Bacillus subtilis S10 strain containing 1-deoxynojirimycin and silkworm powder and acarbose were investigated as follows. 40 g of lyophilized silkworm powder (provided by the Gyeongbuk Yecheon Yangjam Agricultural Cooperative) was added to 800 ml of 80% methanol, ground in an ultrasonic grinding machine for 1 hour, and then bathed in a 60 ° C constant temperature water bath for 3 hours, followed by centrifugation (10,000 rpm × The supernatant obtained by 20 minutes) was concentrated under reduced pressure to 40 mL to prepare a comparative sample. The experimental sample was cultured by shaking the Bacillus subtilis S10 strain at 37 ° C. in 1.5 L CSB medium for 5 days at 37 ° C. for 10 minutes at 100 ° C., centrifuged at 4,000 rpm for 20 minutes, and then separating the supernatant 1 L from this. Was prepared by concentrating to 40 mL using a vacuum concentrator (Buchi). Acarbose (50 mg of glucobai tablets, Bayer Korea Co., Ltd.) contains 50 mg of acarbose per tablet, and each party weighs 0.135 g. Thus, about 6.7 g obtained by grinding 50 acarbose tablets with a mortar and pestle is used as an acarbose sample. Used.

10 mice having a blood glucose level of 200 mg / dL or more were used as a group, and the silkworm powder extract or Bacillus subtilis S10 strain culture concentrate containing 1-deoxynojirimycin in each experimental group was in an amount of 2 ml / kg. Acarbose was orally administered once a day for 7 weeks in an amount of 300 mg / kg. In addition, 2 ml / kg of distilled water was orally administered to the control group. After oral administration, feed was fed for 1 hour, fasted for 2 hours, blood was collected from the tail vein, and the change in blood glucose was observed using a blood glucose meter (Roche).

As a result, the control group to which only distilled water was orally administered showed a rapid increase in blood glucose and high blood sugar, but in the experimental group to which acarbose, silkworm powder extract and Bacillus subtilis S10 strain culture concentrated sample were administered, significantly lower than the control group. Of blood sugar was being maintained. From this, it was confirmed that the Bacillus subtilis S10 strain exhibited a hypoglycemic effect at the same level as the acarbose or silkworm powder known as an α-glucosidase inhibitor ( FIG. 7 ).

Preparation Example 1 Preparation of Microbial Additives 1

Bacillus subtilis S10 producing 1-deoxynojirimycin was inoculated in a medium containing 0.5% corn starch, 0.5% soybean meal, 0.5% yeast extract, 0.1% diphosphate and 0.05% ammonium sulfate And incubated at 37 ° C. for 3 days. The culture solution was simply mixed with steel bark such as skim steel, soybean meal, jade powder in a weight ratio of 1: 1 to 1:10, dried at 40 ° C. or lower, and ground to prepare a probiotic for feed addition. The probiotic thus prepared contained Bacillus subtilis S10 at least 1 × 10 ⁸ cfu / g.

Preparation Example 2 Preparation of Microbial Additives 2

Bacillus subtilis S10 producing 1-deoxynojirimycin was inoculated in a medium containing 0.5% corn starch, 0.5% soy flour, 0.5% yeast extract, 0.1% diphosphate and 0.05% ammonium sulfate at 37 ° C. Incubated for 3 days. The content of the culture was inoculated at a concentration of 10% in a solid incubator in which the skimmed steel, soybean meal, jade powder and molasses were mixed at a weight ratio of 3: 1: 1: 1, respectively, and water was added to adjust the water content to 45 to 60%. This was carried out for 3 days while stirring at 40 ℃ solid fermentation to prepare a feed for probiotic. The probiotic thus prepared contained more than 1 × 10 ⁷ cfu / g Bacillus subtilis S10.

Preparation Example 3 Preparation of Microbial Additives 3

Bacillus subtilis S10 producing 1-deoxynojirimycin was inoculated in a medium containing 0.5% corn starch, 0.5% soy flour, 0.5% yeast extract, 0.1% diphosphate and 0.05% ammonium sulfate at 37 ° C. Incubated for 3 days. To the culture solution was added starch or lactose in the same amount as powdered milk or skim milk (skim milk) and frozen by fast freezing at -70 ℃ or less, and freeze-dried for 36 hours to prepare a probiotic for feed addition. At this time, the temperature of the hot plate to increase the water evaporation efficiency during freeze-drying was 30 ℃, freeze-drying temperature was -60 ℃. The probiotic prepared as described above contained Bacillus subtilis S10 of 1 × 10 ¹⁰ cfu / g or more.

Preparation Example 4 Preparation of Microbial Additives 4

Bacillus subtilis S10 producing 1-deoxynojirimycin was inoculated in a medium containing 0.5% corn starch, 0.5% soy flour, 0.5% yeast extract, 0.1% diphosphate and 0.05% ammonium sulfate at 37 ° C. Incubated for 3 days. Milk powder, lactose, calcium carbonate and the like were mixed in the culture solution (2: 1: 0.5), and then spray-dried under conditions of an inlet temperature of 150 ° C. and an outlet temperature of 110 ° C. to prepare a probiotic for feed addition. Probiotics prepared as described above contained Bacillus subtilis S10 1 × 10 ⁹ cfu / g or more, and 1-deoxy nozirimycin was also concentrated more than five times confirmed that the α-glucosidase inhibitory ability is enhanced.

Preparation Example 5 Preparation of 1-deoxynojirimycin Pure Crystal

Bacillus subtilis S10 producing 1-deoxynojirimycin was inoculated in a medium containing 0.5% corn starch, 0.5% soy flour, 0.5% yeast extract, 0.1% diphosphate and 0.05% ammonium sulfate at 37 ° C. Incubated for 3 days. The culture solution was mixed with calcium carbonate (culture medium: calcium carbonate = 15: 1) to obtain an ethanol extract fraction of twice or more ethanol in the dried product obtained by spray-drying and concentrating. The ethanol extract fraction was centrifuged to recover the supernatant, adsorbed onto a column filled with Amberlyst 15, washed with secondary distilled water to remove the pigment, and eluted with 0.5 N ammonia water. The eluted solution was concentrated under reduced pressure at 60 ° C., and the concentrated solution was adsorbed onto a column filled with Dow's 1 × 2-100 and eluted with secondary distilled water. The eluate was concentrated under reduced pressure, frozen at −70 ° C. and dried with a freeze dryer to crystallize. As a result of measuring the purity of the crystal by HPLC, it was confirmed that 1-deoxy nojirimycin was purified at least 99%.

Preparation Example 6 Preparation of Health Supplements

Bacillus subtilis S10 producing 1-deoxynojirimycin was inoculated in a medium containing 0.5% corn starch, 0.5% soy flour, 0.5% yeast extract, 0.1% diphosphate and 0.05% ammonium sulfate at 37 ° C. Incubated for 3 days. 60% or more grains such as rice, barley, corn, soybeans and wheat, 40% or more embryos derived from rice, barley, corn or wheat, 60% or more fruit or vegetables such as apples, tangerines or tomatoes, or ginseng It contained 60% or more of plant raw materials such as Angelica, Angelica, pomegranate, and sterilized by adding water containing 0.5% yeast extract and 0.2% diphosphoric acid. The culture solution was inoculated 5% or more and then incubated at 37 ° C. for 3 days, and the culture obtained therefrom was dried and ground at 40 ° C. to obtain a powder. The powder was molded into tablets, pills, capsules, and the like to prepare a dietary supplement. In these products, Bacillus subtilis S10 contained 1 × 10 ⁷ cfu / g or more, in addition to enzymes such as amylase and protease. It was confirmed that it contains.

As described above, Bacillus subtilis S10 of the present invention inhibits the α-glucosidase activity to prevent rapid blood glucose rise, block the infection of animal viruses through the inhibition of glycoprotein synthesis necessary for virus growth and anti-cancer It has excellent ability to produce 1-deoxynojirimycin, which has a cancer metastasis inhibitory effect, and can be useful for preparing feed, food, cosmetics, medicines, etc. for the purpose of α-glucosidase inhibitor, antiviral agent, and anticancer agent. Can be.

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Claims (8)

Bacillus subtilis S10 (Accession No .: KCCM 10650P) which inhibits α-glucosidase activity and produces 1-deoxynojirimycin. A pharmaceutical composition for inhibiting blood sugar elevation, comprising the culture solution of Bacillus subtilis S10 of claim 1 as an active ingredient. The pharmaceutical composition for antiviral containing the culture solution of Bacillus subtilis S10 of claim 1 as an active ingredient. delete Feed or food additive containing a culture of Bacillus subtilis S10 of claim 1 as an active ingredient. The method of claim 5, An additive characterized in that the culture solution of Bacillus subtilis S10 is mixed with a carrier and then contained in the form of a probiotic prepared by spray-drying or freeze-drying. The method of claim 5, An additive, which is used to prevent pathogenic viral infections in livestock. The method of claim 5, An additive, which is used to suppress blood sugar rise.

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