KR100477039B1 - Novel microorganism producing 1-deoxynojirimycin and its composition containing the same - Google Patents

Abstract

The present invention relates to a novel microorganism producing 1-deoxynojirimycin having a hypoglycemic and antiviral activity and a composition containing the same, wherein the microorganism is an α-glycosidase. ) Can be significantly inhibited and 1-deoxynojirimycin can be produced to provide pharmaceuticals, feed additives and dietary supplements useful for the prophylactic treatment of diabetes disease.

Description

Novel microorganism producing 1-deoxynojirimycin and its composition containing the same

The present invention relates to a novel microorganism producing 1-deoxynojirimycin and a composition containing the same, more particularly 1-deoxy nojirimycin having antiviral and hypoglycemic activity and a novel production of the same A composition containing the microorganism Bacillus subtilis MORI as an active ingredient and a preparation thereof.

Alkaloids, which are separated from many kinds of plants and microorganisms, mostly contain several hydroxyl groups (OH) in the sugar structure, and exhibit structural features in which the oxygen of sugar molecules is replaced with nitrogen. More than 100 alkaloids are known to date, and they are structurally piperidines, pyrrolidines, indolizidines, pyrrolizidines, nortropanes. It can be divided into five series (Asano, et al ., Tetrahedron, 11 , pp 1645-1680, 2000).

Among the piperidine series, 1-deoxynojirimycin (DNJ) having a structure of hexasaccharide is a representative substance, which is first synthesized from L-sorbofuranose ( Paulsen et al ., Chem Ber. , 100 , p802, 1967) or reduction of nozirimycin (Inouye et al ., Tetrahydron , 24 , pp2125-2144, 1968). (Schmidt et al ., Naturwissenshaften , 66 , pp584-584, 1979), Streptomyces (Inouye et al ., J. Antibiot. Ser.A, 19 , pp288-292, 1966: Ishida et al ., J. Antibiot Ser A , 20 , pp66-71, 1967: Murano and Miyata, Agric Biol. Chem . 44 , pp219-221, 1980), and more recently, Morus alba L. or silkworm (Bombyx mori L. (Asano et al. , J. Agric. Food Chem . 49 , pp 4208 ~ 4213, 2001). 1-deoxynozirimycin has alpha-glycosidase present in the small intestine of mammals and an insulin-independent type that leads to post-prandial hyperglycemia with long-lasting inhibition of glucose influx into the blood in the small intestine. Attention has been drawn to the treatment of diabetes (Yagi et al ., Nippon Nogeikagaku Kaishi , 50 , pp571 to 572, 1976). Piperidine-based alkaloids are also known as 1-deoxymannojirimycin, fagomine, and 3- epi- fagomine.

Pyrrolidine family, which has the structure of pentasaccharide, has lower inhibitory activity against small intestine sucrase or maltase in rabbits, but it is beta-glucosidase (β-glucosidase). ) And glucoamylase have been reported to be excellent (Yosikunin et al ., Agric. Biol. Chem. , 52 , pp 121-128, 1988). The indolizidine family is an alkaloid compound having one pentose structure and one hexasaccharide structure, and swainsonine and castanospermine are representative materials. The pyrrolizidines family is an alkaloid compound having two pentose structures. The representative substance is australine, and the nortropanes family is an alkaloid family newly formed by the discovery of carlystegin. to be.

Alkaloids such as these have an inhibitory function against glucosidase, which plays an important role in the production of glycoproteins and glycoproteins in our body, and thus the industrial application of these substances is expected. There is a therapeutic agent for diabetes that inhibits glucosidase and thus inhibits the intestinal absorption of monosaccharides. Diabetes patients are increasing all over the world, and in Korea, the number of patients with diabetes, obesity, and hyperglycemia has increased steadily since the end of 1980s. Among diabetes, more than 95% of patients with non-insulin dependent diabetes mellitus (NIDDM, Type II diabetes) account for oral diabetes drugs, such as 1-deoxynojirimycin, since the 1970s. (Yagi et al ., Nippon Negeikagaku Kaishi , 50 , pp571-557, 1976: Schmidt et al ., Naturwissenshaften, 66 , pp584-585, 1979). In addition, alkaloids have been found to be effective in antiviral, anticancer and cancer metastasis, and various drugs using the same are sold as drugs through clinical trials.

Currently, as a method of producing 1- deoxynojirimycin , a method of extracting it from plants such as Morus alba L. (Yagi et al ., Nippon Negeikagaku Kaishi , 50 , pp571 ~ 572, 1976: Matsumura et al . , Jpn.Patent kokoku , 59 , p27338, 1984: Evans et al ., Phytochemistry , 24 , pp1953-1955, 1985; Kite et al ., Biochem.Syst.Ecol . , 19 , pp441 ~ 445, 1991: Yamada et al , Shoyakugaku Easshi , 47 , pp47-55, 1993), Bacillus (Frommer et al ., Ger. Patent Often, 265863 [CA, 91, 18359]) or Streptomyces (Murao and Miyata, Agric. Biol. Chem. , 44 , pp291-221, 1980: Ezure et al ., Agric. Biol Chem. , 19 , pp2159 ~ 2165, 1985: Hardick et al ., Tetrahedron , 30 , pp6285 ~ 6296, 1992) Production by fermentation using microorganisms, chemical synthesis using D-glucose as precursor (Paulsen et al ., Chem. Ber., 100, 802, 1967), synthesis and enzymes using glucosylamine Synthesis (Wong et al ., Tetrahydron Lett., 32, 4867-4870, 1991) is known. Extraction from plants has been actively studied to date and most alkaloids known to date have been extracted from plants (Watson et al ., Phytochemistry , 56 , pp265-295, 2001). However, the industrial use of the method of extracting from plants has problems such as low content of 1-deoxynojirimycin, complicated purification process among nojirimycin and deoxymannonojirimycin of similar structure, and chemical 1-deoxy Nozirimycin synthesis is also less economical because of the complex steps and high chemical requirements.

Currently, the most economically attracting method is production using microbial fermentation, and microorganisms producing 1-deoxynojirimycin using industrial fermentation techniques are known as Bacillus subtilis and Bacillus amyl. Bacillus amyloliquifaciens , Bacillus polymyxa (Schmidt et al ., Naturwissenschaften , 66 , pp584-585, 1979), Bacillus subtilis var niger (Hardick et al . , Biochem ., 49 , pp6707-6717, 1993), Streptomyces lavendula e (Inouye et al ., J. Antibiot.Ser.A , 19 , pp288-292, 1966; Tetrahedron , 24 , pp2125 2144, 1968: Ishida et al ., J. Antibiot. Ser A , 20 , pp66-71, 1967: Murao and Miyata, Agric. Biol. Chem. , 44 , pp219-221, 1980: Ezure et al ., Agric Biol.Chem . , 49 , pp1119-1125, 1985), Streptomyces subrutili s ) (Hardick et al ., Terahedron , 48 , pp 6285-6296, 1992). The above method is attracting attention as an efficient method in terms of economics such as a reduction in production cost and a shortening of the purification process, but the researches on alkaloids having high polarity during the search for culture medium are still insufficient compared to other microbial studies on alkaloids. This is not easy (Watson et al ., Phytochemistry , 56 , pp 265-295, 2001).

Therefore, the present invention efficiently produces 1-deoxynojirimycin to isolate and identify a new microorganism Bacillus subtilis MORI that can be applied to industrial medicines and health functional foods, and feeds using the microorganisms. The present invention has been completed by refining the refined drug substance including additives and foods.

It is an object of the present invention to provide a pharmaceutical composition containing a novel microorganism, Bacilus subtilis MORI, and 1- deoxynojirimycin having a hypoglycemic activity and antiviral efficacy produced by the same, To provide a feed additive.

In accordance with the above object, the present invention Bacillus subtilis MORI (depositing institution: Korea Microbiological Conservation Center, Deposit) characterized in that it produces 1-deoxynojirimycin having hypoglycemic and antiviral activity Date: 02/02/2002, Accession No .: KCCM-10450.

Bacillus subtilis MORI according to the present invention is a novel strain of the genus Bacillus isolated and identified from α-glucosidase of swine intestine.

Separation methods, bacteriological properties and uses of Bacillus subtilis MORI are as follows and will be described in detail below.

Bacillus subtilis MORI of the present invention isolates the α-glucosidase from the swine small intestine, and then isolates the microorganisms that inhibit the activity of this enzyme; Final strains showing maltase and α-glucosidase inhibitory activity were selected; As a result of the identification of the selected highly active microorganism strains by 16s rDNA analysis, it showed more than 98% homogeneity with Bacillus subtilis , and the microorganism was named Bacillus subtilis MORI and the Korea Microbiological Center (KCCM) 2002. It was deposited on 02 December.

Strain characteristics are as follows.

A type of microorganism showing activity when cultured at 37 ° C. for 48 hours in YM agar plate medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose, 1.8% agar). The shape is velvety, Gram-positive bacillus, slightly motility, spore-forming ability. The morphology of the colonies, which appeared as the form of bacteria in the medium under the same conditions, constitutes a velvety colony with a diameter of 5.0 to 15.5 mm, and the color tone is white to light brown. Physiological properties are grown at a growth temperature of 20 to 60 ℃, preferably 30 to 55 ℃ or less, a growth pH of pH 3.0 to 11.0, preferably 4.5 to 10, the effect of oxygen is permeable anaerobic, sugar L-arabinose, D-mannose, sorbitol, N-acetylglucosamine, iculin, maltose, saccharose, glycogen, ribose, amigdaline, salicycin, trehalose, D-rapinose, D-fructose, mannitol It is positive for α-methyl-D-glucoside, arbutin, cellobiose, inline, amidone, and β-genthiobiose, and in activity against various enzymes, α-glucosidase, β-glucosidase and maltase include There is activity and no activity on β-galactosidase.

In addition, as a result of analyzing the cellular fatty acid of the microorganism, it was confirmed that the iso (iso-) and anteiso-group (anteiso-) structure is contained a lot, the main structure of the quinone compound has a menaquinone-7.

After the growth of the microorganisms, the culture was analyzed by high performance liquid chromatography (HPLC). As a result, it was confirmed that 1 g / L of 1-deoxynojirimycin was contained in the culture of Bacillus subtilis MORI.

As confirmed above, Bacillus subtilis MORI isolated and identified in the present invention produces high concentrations of 1-deoxynojirimycin having hypoglycemic and antiviral activity, so that it can be used as a feed, nutraceutical and pharmaceutical preparation. have.

The present invention provides a pharmaceutical composition for lowering blood glucose, which contains 1-deoxynojirimycin produced by the novel Bacillus subtilis MORI as an active ingredient and contains a pharmaceutically acceptable carrier or excipient.

The 1-deoxy nozirimycin is incubated with Bacillus subtilis MORI, the cells were removed and repeatedly passed through an ion exchange resin 90%, preferably at least 98% pure purified 1-deoxyno Contains ziamycin.

The hypoglycemic composition of the present invention comprises 0.5 to 50% by weight of the compound relative to the total weight of the composition.

Compositions comprising a compound of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The compositions comprising the compounds according to the invention are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions according to conventional methods. The carriers, excipients and diluents that may be used in the composition comprising the extract may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, 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. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, 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, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of the compound of the present invention may vary depending on the age, sex, and weight of the patient, but generally, an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, may be administered once or several times a day. Can be. In addition, the dosage of the extract can be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

Since the compound of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for a long time for the purpose of prevention.

The present invention provides a feed additive containing the novel Bacillus subtilis MORI or 1-deoxynojirimycin produced thereby as an active ingredient.

The additive of the present invention may be prepared as a feed additive and food by mixing the cells and the culture medium obtained by culturing the Bacillus subtilis MORI with an excipient, or by spray drying or freeze drying.

The additive or food prepared as such contains 0.5 to 5 g / l, preferably 2 to 4 g / l of 1-deoxynojirimycin, and the additive may be used in an amount of 0.1 to 2% by weight in the feed. . Regardless of the type or type of feed, it can be applied to all fermented feeds, silage feeds, powdered feeds and pellet feeds.

The present invention provides a health functional food comprising the novel Bacillus subtilis MORI or 1-deoxynojirimycin produced thereby as an active ingredient and a food acceptable additive.

Similarly to the above feed additives, 1-deoxy nozirimycin contains 0.5 to 5 g / l, preferably 2 to 4 g / l, and the Bacillus subtilis MORI is fermented in soybean to itself or dried product. Can be used as food.

Compositions comprising the strains and compounds of the present invention can be used in a variety of drugs, food and beverages for the prevention of diabetes diseases. Examples of the food to which the present 1-deoxynojirimycin may be added include various foods, beverages, gums, teas, vitamin complexes, dietary supplements, and the like, which are powders, granules, tablets, capsules, or beverages. Available in form.

The strain or compound of the present invention may be added to food or beverage for the purpose of preventing diabetes disease. At this time, the amount of the extract in the food or beverage is generally added to the health food composition of the present invention to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 10g based on 100ml, preferably It can be added at a ratio of 0.3 to 1 g.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates are conventional monosaccharides such as disaccharides such as glucose, fructose and the like, such as maltose and sucrose, and polysaccharides such as dextrin, cyclodextrin and the like. Sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, 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. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical 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.

The present invention is described in more detail based on the following examples and experimental examples, but the present invention is not limited thereto.

Example 1 Isolation of Enzymes from Porcine Small Intestine

Immediately after the slaughter, the foreign body in the small intestine was washed with tap water using a detergent, then carefully scraped off the mica and suspended in 0.1M phosphate buffer solution (pH 6.8), centrifuged (10,000 rpm × 20 minutes), and then the supernatant. Was taken. Precipitate the protein by adding 80% ammonium sulfate to the supernatant, and precipitate the precipitate obtained by centrifugation at 10,000 rpm for 20 minutes at 4 ° C using dialysis membrane (MW 12,000), and then lyophilize to use as an enzyme source. It was.

Experimental Example 1. Protein analysis of enzyme source

In order to determine the protein content of the enzyme source obtained in Example 1, the following assay was performed.

9 ml of Bradford (Bradford, Sigma-Aldrich, USA) reagent was added to 1 ml of the diluted sample and reacted at room temperature for 15 minutes, and then absorbance was measured at 595 nm using an absorbance spectrometer (UV-1202, Shimadzu, Japan). The protein content of the sample was measured, and calf serum albumin (100 µg / ml) was used as a standard sample.

As a result, the protein content of the coenzyme sample was found to contain 30% protein at 30 µg / mg.

Experimental Example 2. α-glucosidase confirmation experiment

In order to detect α-glucosidase from the enzyme source obtained in Example 1, the following experiment by electrophoresis was performed.

Electrophoresis was performed using the Native-PAGE method (Davis B., Ann. New York Acad. Sci . Pp 121-404, 1964) and 7.5% polyacrylamide gel. The sample was adjusted with a phosphate buffer solution (pH 6.8) so as to have a concentration of 10 mg / ml, and 20 µl of the prepared sample was sufficiently mixed with 40% sugar and 0.001% bromophenol blue, and then dripped. Electrophoresis was energized with Tris-glycine buffer (pH 8.3) at a constant current of 30 mA at 4 ° C. for 90 minutes. After electrophoresis, the gel was divided into two portions, Coomassie Brilliant Blue, Bio-Rad, US) R-250 staining detects protein and the other is dissolved in 0.1M phosphate buffer (pH 6.8) 2.3 mM 4-methylumbelliprylyl-alpha-D-glucose (4-Methylumbelliferyl-α-D-glucose , Sigma-Aldrich, USA) was subjected to enzymatic reaction at 37 ° C. for 1 hour, and then α-glucosidase was identified using an ultraviolet analyzer (Ultraviolet transilluminator, TL-33E, UVP Inc., USA). .

After electrophoresis, Coomassie Brilliant Blue R-250 staining and enzyme staining were performed in parallel to confirm that the same protein was found on the same plate (see FIG. 1).

Experimental Example 3. Digestase Confirmation Experiment

In order to detect the disaccharide enzyme from the enzyme source obtained in Example 1, the following experiment was performed.

100 μl of the sample (1 mg / ml) dissolved in 0.1 M phosphate buffer (pH 6.8) and 400 μl of maltose, sugar, cellobiose, and lactose of 10 mM each were enzymatically reacted at 37α for 45 minutes and then at 90 ° C. The reaction was stopped by heat treatment for 5 minutes. 10 μl of each reaction solution was added dropwise to a TLC plate (Merck, Silica gel F254) and developed in a mixed solvent (butanol: propanol: acetic acid: water = 5: 3: 3: 1.5). Developed together. The developed TLC plate was dried in an oven at 60 ° C., followed by spraying with an arylene-diphenylamine reagent (2 ml arylene, 100 ml methanol, 2 g diphenylamine, 10 ml 85% phosphoric acid), and at 104 ° C. for 30 minutes. Color development was performed to investigate the production of glucose.

As a result, glucose was thickened in the sample reacted with maltose and coenzyme solution, but weakly observed in the sample reacted with sugar, and glucose was not found in the sample reacted with cellobiose and lactose. Accordingly, it was confirmed that α-glucosidase was contained in the protein in the coenzyme sample, and the titer of the maltase was excellent (see FIG. 2).

Experimental Example 4. Measurement of α-glucosidase titer of enzyme source

In order for the enzyme source to measure the titer of α-glucosidase, the following experiment was performed.

It was dissolved the enzyme source sample in a 0.1M phosphate buffer solution (pH 6.8) 12mM p- substrate by the previously prepared serial dilutions nitrophenyl -α-D- glucopyranoside (p -nitrophenyl-α-D- glucopyronoside, Sigma- Aldrich, USA) was added and reacted at 37 DEG C for 45 minutes, and then 200mM sodium carbonate was added to stop the reaction. The amount of p -nitrophenol produced by measuring the enzyme reaction solution at 405 nm was measured, and the amount of p -nitrophenol produced at a concentration of 1 μM for 1 minute was calculated as the enzyme titer of 1 unit.

As a result of the experiment, it was confirmed that it has 7.7 units of α-glucosidase activity per mg of protein.

Experimental Example 5. Selection of α-glucosidase inhibitor producing strain

In order to select strains that inhibit α-glucosidase, the following experiment was performed.

It strains genus Streptomyces (Streptomyces sp.) 28 and a strain Bacillus sp isolated from cereals (Bacillus sp.) Was used as the 200 strain, the standard α- glucosidase as production strain Bacillus subtilis (Bacillus to the comparative experiment subtilis KCCM 11314), Bacillus amyl as liqueur Pacific Enschede (Bacillus amyloliquefaciens KCCM 40764), Streptomyces sub rutile-less (Streptomyces subrutilis KCCM 40337), Streptomyces Raven circumference (Streptomyces lavendulae KCCM 32799, KCCM 12294 , KCCM 12293), Streptomyces Myces Nojiriensis ( Streptomyces nojiriensis KCCM 12307) was used from Korea Microbial Conservation Center (KCCM).

The strains were shaken for 5 days at 37 ° C. and 150 rpm in a liquid medium containing 1% glucose, 0.5% soybean meal and 0.3% yeast extract, and the culture medium was heat-treated at 100 ° C. for 5 minutes and then centrifuged. (7,000 rpm x 20 minutes) and the obtained supernatant was used as a sample for the search for α-glucosidase.

After dissolving the enzyme extract isolated from pig intestine at a concentration of 1 mg / ml in buffer, 100 μl of this solution, 400 μl of 10 mM maltose and 200 μl of the culture medium were added thereto, reacted for 45 minutes at 37 ° C., and heat-treated at 90 ° C. for 5 minutes. The reaction was stopped. The reaction solution was added dropwise to the TLC plate by 10 µl and developed through a mixed solvent (butanol: propanol: acetic acid: water = 5: 3: 3: 1.5), followed by the development of glucose developed by an arylene-diphenylamine reagent. Glucose-producing strains were selected based on their production.

As a result, 14 strains which inhibit glucose production in maltose were selected.

Experimental Example 6. Selection of α-glucosidase inhibitor production strain by HPLC

In order to confirm the production of 1-deoxy nojirimycin of the strain culture liquid having excellent maltose degradation inhibition through HPLC analysis, the following experiment was performed.

For HPLC analysis, 10 µl of 9-flurenylmethyl chloroformate (Fluka, Switzerland) was added to 10 µl of the culture and reacted at 20 ° C. for 20 minutes, and 0.1M glycine was added to stop the reaction. 950 μl of 0.1% acetic acid was added to the reaction solution, the mixture was adjusted to 1 ml, filtered by 0.2 μm injection filtration, and then injected into the column at 10 μl. The column was Phenomenex Luna C18 (4.60 × 250 mm). ID, 5 μm), the solvent was eluted with acetonitrile-0.1% acetic acid (1: 1, v / v), the dissolution rate was 1 ml / min, the detection was FL3000 fluorescence detector (excitation 254 nm, emission 322 nm, FL3000, Hewlett) -Packard, USA), the analysis program was used for ChromeQuestTM (ChromQuestTM Version 2.51, ThermoQuest, USA) respectively. Also, as the standard sample polyhydroxylated alkaloids, 2-0, which was isolated from Muberry alba by Asano laboratory at Hokuriku University, completed ¹ H and ¹³ C-NMR analysis. -α-D-galactopyranosyl-1-deoxynojiri7mycin (2-O-α-D-galactopyranosyl-1-deoxynojirimycin, 2α-Gal-DNJ), 1,4-dideoxy-1,4 -Imino-D-arabinitol (1,4-dideoxy-1,4-imino-D-arabinitol, DAB), 1,4-dideoxy-1,4-imino- (2-0-β- D-glucopyranosyl) -D-arabinitol (1,4-dideoxy-1,4-imino- (2-O-β-D-glucopyranosyl) -D-arabinitol, 2β-Glc-DAB), pagomin (fagomine), 3- epi- fagomine, calystegin B2 and N-methyl-1-deoxynojirimycin, N-Me -DNJ) was used.

1-deoxy Nojiri azithromycin (1-deoxynojirimycin) of the Bacillus amyl Quebec Pacific Enschede (Bacillus amyloliquefaciens, KCCM 40764) strain as Streptomyces spp Raven dulrae Raven dulrae (Streptomyces lavendulae subsp. Lavendulae, KCCM 12293) to produce a strain Standard strain, among the isolates Four strains of Streptomyces sp. Analysis of 10 strains of Bacillus sp. ( Bacillus sp. ) By HPLC showed that finally MORI strains with excellent inhibition and producing 1-deoxynojirimycin were selected (see FIGS. 3, 4 and Table 1).

 Maltase Inhibitory Activity and 1-Deoxynojirimycin Production Strain Maltase Inhibition 1-deoxynojirimycin production Standard strain KCCM 40764 ++++ ○ KCCM 12293 +++ ○ Bacteria in Streptomyces SS-0247 +++ × SS-2922 +++ × SS-3825 +++ × SS-4299 +++ × Bacteria of the genus Bacillus HG-17 ++ × HG-19 +++ × HG-24 +++ × HG-28 ++ × HG-42 + × HG-54 + × HG-58 ++ × HG-64 ++ × HG-91 ++++ ○ HG-131 ++ × +: Weak inhibition, ++: moderate inhibition, +++: strong inhibition, ++++: very strong inhibition ○: 1-deoxynojirimycin production, ×: 1-deoxynojirimycin production

Experimental Example 7. Identification of strains through sugar analysis

In order to identify the selected strain through the sugar analysis, the following experiment was performed.

To sympathize, 50 CHB ^?? Kit (BioMeriux Inc.) was used, and the strains were incubated for 2 days at 37 ° C. in YM agar medium (Difco Inc., USA), and the colonies were suspended in a high concentration in 5 ml of dilution and then diluted in McParland Turbidity 2 in 5 ml of dilution. In suspension, 10 ml CHL medium (BioMerieux, France) was inoculated and finally adjusted to McF 2 turbidity. Pour 5 ml of sterile water into each section of the CH50 container for moisture control, insert the inoculated medium to the bottom scale of each hole of CH50, and dispense mineral oil until the scale of each hole becomes convex. The reaction was recorded as +,-while incubating at 37 ° C and identified using the API (Biomeriux) analysis program (see Table 2).

As a result of sugar availability analysis of MORI bacteria, Bacillus amyloliquefaciens And Bacillus subtilis ( Bacillus subtilis ) showed a similarity of 99% and 95%, respectively, thereby confirming that the MORI bacteria Bacillus sp.

temperament Usability temperament Usability temperament Usability Glycerol - Erythritol - D-Arabinose - L-Arabinose + Ribose + D-Xylose - L-Xylose - Aconite - β-methyl-xyloxide - Galactose - D-glucose + D-fructose + D-Mannose + L-Solbos - Rhamnos - Dulcitol - Inositol - Mannitol + Sorbitol + α-methyl-D-mannoside - α-methyl-D-glucoside + N-acetylglucosamine + Amigdalin + Arbutin + Eculine + Salinity + Cellobiose + Maltose + Lactose - Melibiose - Saccharose + Trehalose + Inline + Melezitos - D-Raffinose + Amidodon + Glycogen + Xylitol - β-gentiobiose + D-Turanos - D-Rixos - D-tagatose - D-fucose - L-fucose - D-Arabitol - L-Arabitol - Gluconate - 2-cetogluconate - 5-cetogluconate - Control -  +: Production,-: non-production The above data shows more than 99% identity with the genus Bacillus.

Experimental Example 8. Analysis of Total Fatty Acid Composition

In order to analyze the total fatty acid composition of the strain, the following experiment was performed using gas chromatography (GC, HP 6890).

As a standard fatty acid, standard fatty acid provided by Hewlett-Packard (HP) was used, and the strain was cultured at 150 rpm for 28 hours at 28 ° C. using 50 ml of savoraoud (DIFCO, USA) medium. The cultured cells were obtained through filtration. The obtained cells were collected in a test tube, and then 1 ml of Reagent 1 (Table 3) was added for saponification. The cells were bathed at 100 ° C. for 30 minutes, cooled in running water, and reacted with Reagent 2 (Table 3) for methylation. ) Was added 2 ml, and the mixture was reacted at 80 ° C. for 10 minutes and cooled in running water. After adding 1.25 ml of Reagent 3 (Table 3) for extraction, the mixture was shaken slowly at room temperature for 10 minutes, and then the supernatant was transferred to a new test tube. The washing was shaken slowly for 5 minutes by adding 3 ml of Reagent 4 (Table 3). 500 μl of saturated sodium chloride solution was added for separation. The separated supernatant was taken and used as a sample for analysis. The sample was analyzed using GC and analyzed through Sherlock (Sherlock, MIDI).

The total fatty acid of the cell is an essential component of the cell lipid and consists of about 10 to 24 hydrocarbons, and it is an important criterion for classifying microorganisms because they exist in various forms depending on their length, the position of the double bond, and the substituent. As a result of confirming the intracellular fatty acid structure of MORI bacteria, it was found that the main fatty acid contained many iso- and anteiso-branched structures (see FIGS. 5, 6 and Table 3). ).

  Reagent Composition of Total Fatty Acid Composition Analysis Reagent Furtherance Reagent 1 (Saponification) 45 g sodium hydroxide 150 ml methanol 150 ml deionized distilled water Reagent 2 (methylated) 325 ml 6N hydrochloric acid 275 ml methanol Reagent 3 (extraction) 200 ml hexane 200 ml methyl- tert butyl ether Reagent 4 (base wash) 10.8 g sodium hydroxide 150 ml deionized distilled water

Experimental Example 9. Quinone Analysis

In order to perform quinone analysis of the strain, the following experiment was performed.

The cells were incubated and recovered by centrifugation (5,000 rpm x 20 minutes), washed twice with 50 mM phosphate buffer (pH 7.0), and the collected cells were washed with 20 ml of extractant (chloroform: methanol = 2: 1). After stirring for 5 minutes, the concentration was repeated three times. The concentrated sample was dissolved in 2 ml of nucleic acid and 2 ml of water, the nucleic acid layer was separated and concentrated three times, and the final concentrated sample was dissolved in a small amount of acetone. The dissolved sample was developed using TLC (Merck, Kiesel-gel 60 F254) with a developing solvent (hexane: diethyl ether = 85:15) to confirm the developing part with an ultraviolet analyzer (Ultraviolet transilluminator). Dissolved in a small amount of acetone and centrifuged (5,000rpm × 10 minutes) was repeated three times to collect the supernatant. The collected supernatant was concentrated with nitrogen gas and used as an HPLC analysis sample. According to HPLC analysis, the column was spheroid (Spherisorb 5 μm ODS2, Waters, 4.6 × 250 mm), the solvent composition was methanol: isopropyl ether (3: 1) for ubiquinone analysis, and methanol: isopropyl ether (for menaquinone analysis). 4: 1) was used, the elution rate was 1 ml / min, detection was performed using an ultraviolet detector, ubiquinone was detected at 275 nm, and menaquinone at 270 nm. As a standard sample, the ubiquinone series was purchased from Sigma, and the menaquinone series was used by culturing the standard strain and extracting the same method as the sample.

As a result, the bacteria of the genus Bacillus contained menaquinone, which is also present in other Gram-positive bacteria, and the main structure of the quinone compound of the MORI bacterium was found to have a menaquinone-7 structure in 18 components (see FIG. 7). ).

Experimental Example 10 16s rDNA sequencing

For sequencing of the strains, the following experiments were performed.

After preparing genome DNA using Wizard Genomic DNA Prep Kit (Promega, USA), multi-chain reactors (PCR, Minicycler, MJ Research) were denatured at 94 ° C / 1 min, cooled to 60 ° C. 1 min 30 sec, increasing 72 ° C./1 min, primers were subjected to a total of 30 times using 27F (5 ′ AGAGTTTGATCMTGGCTCAG) and 1492r (5 ′ TACGGYTACCTTGTTACGACTT), followed by pGEM T Easy Vector (pGEM T Easy Vector, Promega Inc.) was transformed. White colonies were isolated by smearing on a medium containing X-gal (X-gal), plasmids were isolated, and the sequences were analyzed using an automated DNA analyzer (ABI3700, Applied Biosystems). The sequences were then compared to Genebank's sequences using Advanced Blast search using Cluster X software (NCBI, USA), and the dendrogram It was created by neighboring-joining analysis.

As a result of the experiment, the MORI bacterium was identified as a new microorganism having a homogeneity of 98% or more with the Bacillus subtilis bacteria (see SEQ ID NO: 1 and FIG. 8), and was named Bacillus subtilis MORI.

Experimental Example 11. Electron Microscopy

The shape of the bacteria was observed through a scanning electron microscope as follows.

Pretreatment of the sample for electron microscopic observation was carried out in four stages: washing, fixing, drying and ion coating. First, colonies incubated for 2 days in YM (Difco Co., Ltd.) agar medium were washed three times using platinum sterilized with sterile water, and 5 ml of 25% glutaaldehyde (glutaraldehyde) was added and fixed at 4 ° C. for 12 hours. After completion of the fixation, the cells were washed three times with 0.1 M phosphate buffer (pH 6.0), and only the cells were recovered, sliced into cover glass, and dried for 3 hours using a freeze dryer (Tokyo Kikakikai, Japan). Gold coating was observed with an electron microscope (see FIG. 9).

Experimental Example 12 Purification of 1-deoxynojirimycin and Polyhydroxylated alkaloids

In order to purify 1-deoxynojirimycin and alkaloids produced by Bacillus subtilis MORI, the following experiments were performed.

A 50-L fermenter was prepared with 30-L culture medium consisting of 2.5% starch, 1% corn immersion, 0.5% yeast extract, 0.5% soy flour, 0.5% potassium phosphate, 0.1% iron sulfate (pH 7.5), and then sterilized. 1 L was inoculated and incubated for 5 days while maintaining the agitation rate 230rpm, aeration 0.3vvm at 37 ℃. After completion of the culture, the recovered culture solution was concentrated under reduced pressure and centrifuged (10,000 rpm x 20 minutes) to remove the cells and protein components, and then purified using an ion exchange resin. Using four kinds of ion exchange resin method 1-deoxynojirimycin and alkaloids in Bacillus subtilis MORI cultures were isolated. 20 L of the culture solution was adsorbed to Amberlyst (Amberlyst 15, 10 × 90 cm, H ⁺ type), eluted with 0.5 N ammonia water, and concentrated under reduced pressure to obtain 50 g of fraction (1). After absorption of this fraction (1) 30g on Amberlite (Amberlite CG-50, 4 × 70㎝, NH 3 + type), and eluted with distilled water. As a result of the separation of the new fraction, fraction (2) was obtained, and eluted with 0.5N ammonia water to separate 1-deoxynojirimycin with 89.6% purity.

In addition, 20 g of fraction (1) was adsorbed onto DOEX (Dowex 1 × 2-100, 4 × 70 cm, OH ⁻ type), eluted with distilled water, and then Amberlite (Amberlite CG-50, 2 × 70 cm, NH). After adsorption to the ₃ ⁺ form) and eluted with distilled water, the 1-deoxy Nojiri rapamycin was separated by a 94% purity.

2.04 g of the sample containing 1-deoxynojirimycin and concentrated under reduced pressure was finally purified using a doex resin, and as a result, 1.8% or more of 1-deoxynojirimycin was obtained 1.8g (Fig. 10). Reference).

Experimental Example 13. Enzyme Activity Test

In order to analyze the glucosidase inhibitory activity of 1-deoxynojirimycin isolated and purified in the culture medium of Bacillus subtilis MORI bacteria, the following experiment was performed.

Enzymes and substrates used in the experiment were purchased from Sigma, and pig small intestine maltase was used. α-glucosidase and swine small intestine maltase were 12 mM p -nitrophenyl-α-D-glucopyranoside, and β-glucosidase was 12 mM p -nitrophenyl-β-D-glucopyranoside, β-gal The lactosidase was reacted for 45 minutes by adding the inhibitor concentration at the optimum pH and temperature of each enzyme using 18 mM p -nitrophenyl-β-D-galactopyranoside, respectively, and then reacting with 200 mM sodium carbonate. Stopped. The resulting p-nitrophenol was determined from the amount of p 405㎚ 1μM for 1 minute was defined titers generating nitrophenol 1 enzyme unit, define the activity of the enzyme concentration of the inhibitor to inhibit 50% EC ₅₀ It was.

Inhibitory concentrations of α-glucosidase, β-glucosidase, β-galactosidase, and swine intestinal maltase against 1-deoxynojirimycin isolated and purified in the culture medium of the Bacillus subtilis MORI strain were examined. As a result, it was confirmed that strong inhibitory activity against α-glucosidase and small intestine maltase (see Table 4).

α-glucosidase (EC ₅₀ ) β-glucosidase (EC ₅₀ ) β-galactosidase (EC ₅₀ ) Maltase (EC ₅₀ ) Amount of deoxynojirimycin 2 x 10 ^-5 M (3.26 ppm) 2 × 10 ^-3 M (326ppm) NI 2 × 10 ^-6 M (0.326 ppm)

Experimental Example 14. Toxicity Test

1. Oral administration

ICR mice and Sprague Dawley were divided into four groups of 10 mice each, and orally administered 1-deoxynojirimycin of the present invention at doses of 500, 725, 1000 and 5000 mg / kg, respectively. After two weeks of toxicity, none of the four groups died, and no apparent symptoms were found.

2. Intraperitoneal administration

ICR-based mice (weight 25 ± 5 g) and Sprague dooli were divided into four groups of 10 mice each to intraperitoneally administer 1-deoxynojirimycin of the present invention at doses of 25, 250, 500 and 725 mg / kg, respectively. After 24 hours, no one died in all 4 groups and no symptoms were observed.

As a result of the experiment, Bacillus subtilis MORI of the present invention and 1-deoxy nojirimycin produced by it was confirmed that there is little acute toxicity and is safe.

Bacillus subtilis MORI of the present invention and 1-deoxy nojirimycin produced therein can be administered in the following formulation, the formulation examples below are merely illustrative of the present invention, whereby the contents of the present invention It is not limited.

1. Preparation of powder

1-deoxynojirimycin 100mg

Corn Starch 100mg

Lactose 100mg

Talc 10mg

The above ingredients are mixed and filled in airtight cloth to prepare a powder.

2. Preparation of Tablets

1-deoxynojirimycin 100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components and tableting according to the manufacturing method of the conventional tablet to prepare a tablet.

3. Preparation of Capsule

1-deoxynojirimycin 100mg

Lactose 50mg

1 mg magnesium stearate

The above ingredients are mixed and compressed into tablets according to a conventional method for preparing capsules to fill gelatin capsules.

4. Manufacture of liquid

1-deoxynojirimycin 100mg

10 g of isomerized sugar

10g per book

Lemon flavor

Purified water

According to the conventional method for preparing a liquid solution, each component is added to the purified water, dissolved, and lemon flavor is added, and then purified water is added to adjust the total amount to 100 ml, and then filled into a brown bottle to prepare a liquid solution.

Formulation Example 5 Preparation of Healthy Food

1-deoxynojirimycin 1000 mg

Vitamin Mixture

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 6 Preparation of Healthy Drink

1-deoxynojirimycin 1000 mg

Revitalization extract 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to a conventional healthy beverage manufacturing method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Formulation Example 7 Preparation of Feed Additives (1)

Bacillus subtilis MORI bacteria were cultured in a liquid medium containing 1.5% starch, 1.0% glucose, 0.5% soy flour and 0.3% yeast extract at 40 ° C. and 150 rpm for 3 to 7 days, preferably 5 days. After completion of the culture, the culture medium was again mixed with sterile soybean meal and solid cultured at 40 ° C. for 2 to 5 days, preferably 3 days, followed by hot air drying at 45 ° C.

Formulation Example 8 Preparation of Feed Additives (2)

Bacillus subtilis MORI bacteria were cultured in a liquid medium containing 1.5% starch, 1.0% glucose, 0.5% soy flour and 0.3% yeast extract at 40 ° C. and 150 rpm for 3 to 7 days, preferably 5 days. The finished culture was spray-dried under conditions of an input temperature of 150 ° C. and a discharge temperature of 120 ° C. to complete a feed additive containing 1-deoxynojirimycin as an active ingredient, and the content of the active ingredient was 0.5 to 7 g / kg. .

Formulation Example 9. Preparation of 1-deoxynojirimycin-containing food (1)

Bacillus subtilis MORI bacteria were cultured in a liquid medium containing 1.0% glucose, 0.5% soy flour, and 0.3% yeast extract at 40 ° C. and 150 rpm for 2 to 4 days, preferably 3 days, and then water 50 to Inoculated at 60% sterilized soybean at 5% concentration was incubated solid at 40 ℃ for 5 days. The solid fermented culture was pulverized as is to complete a food or food additive, and the content of 1-deoxynojirimycin was 0.3 to 4 g / kg.

Formulation Example 10 Preparation of 1-deoxynojirimycin-containing food (2)

Bacillus subtilis MORI bacteria were cultured in a liquid medium containing 1.0% glucose, 0.5% soy flour, and 0.3% yeast extract at 40 ° C. and 150 rpm for 2 to 4 days, preferably 3 days, and then water 50 to Inoculated at 60% sterilized soybean at 5% concentration was incubated solid at 40 ℃ for 5 days. The solid fermented cultures were hot air dried at 45 to 50 ° C. and then ground to complete a food or food additive, and the content of 1-deoxynojirimycin was 0.3 to 4 g / kg.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, use purpose.

A composition containing the Bacillus subtilis MORI of the present invention and 1-deoxynojirimycin produced thereby, a useful new strain Bacillus subtilis MORI significantly inhibits α-glucosidase and maltase and In addition, 1-deoxynojirimycin produced by it has excellent hypoglycemic activity and antiviral activity, so that it can be effectively used for the prevention and treatment of blood glucose-related diseases, and is useful as a health food and feed composition.

1 is an α-glucosidase (diagram) of an enzyme source sample isolated from the small intestine of pigs,

2 is a reaction diagram of maltose, sugar, cellobiose and lactose of an enzyme source sample,

3 is a maltase inhibition test of the selected strains,

4 is a diagram showing the production of 1-deoxynojirimycin in Bacillus subtilis MORI strain culture,

5 is a Gas Chromatography diagram of the Bacillus subtilis MORI strain,

6 is a total fatty acid composition analysis of Bacillus subtilis MORI strain,

7 is a quinone analysis of the Bacillus subtilis MORI strain,

8 is a dendrogram of the Bacillus subtilis MORI strain,

9 is an electron microscope view of the Bacillus subtilis MORI strain,

10 is an ultraviolet analysis diagram using ion exchange resins of 1-deoxynojirimycin and other alkaloids produced by the Bacillus subtilis MORI strain.

<110> BIOTOPIA.CO., LTD. <120> Novel microorganism producing 1-deoxynojirimycin and its composition containing the same <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1493 <212> DNA <213> Bacillus subtilis MORI <400> 1 agaggtttga tcatggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgag 60 cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa cacgtgggta 120 acctgcctgt aagactggga taactccggg aaaccggggc taataccgga tgcttgtttg 180 aaccgcatgg ttcaaacata aaaggtggct tcggctacca cttacagatg gacccgcggc 240 gcattagcta gttggtgagg taacggctca ccaaggcaac gatgcgtagc cgacctgaga 300 gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag gcagcagtag 360 ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg atgaaggttt 420 tcggatcgta aagctctgtt gttagggaag aacaagtacc gttcgaatag ggcggtacct 480 tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta 540 ggtggcaagc gttgtccgga attattgggc gtaaagggct cgcaggcggt ttcttaagtc 600 tgatgtgaaa gcccccggct caaccgggga gggtcattgg aaactgggga acttgagtgc 660 agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt ggaggaacac 720 cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggagcgaaag cgtggggagc 780 gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa gtgttagggg 840 gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg agtacggtcg 900 caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta 960 attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaat cctagagata 1020 ggacgtcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt 1080 gagatgttgg gttaagtccc gcaacgagcg caacccttga tcttagttgc cagcattcag 1140 ttgggcactc taaggtgact gccggtgaca aaccggagga aggtggggat gacgtcaaat 1200 catcatgccc cttatgacct gggctacaca cgtgctacaa tggacagaac aaagggcagc 1260 gaaaccgcga ggttaagcca atcccacaaa tctgttctca gttcggatcg cagtctgcaa 1320 ctcgactgcg tgaagctgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg 1380 ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac ccgaagtcgg 1440 tgaggtaacc ttttaggagc cagccgccga aggtgggaca gatgattggg gtg 1493

Claims (8)

Novel Bacillus subtilis MORI having the following characteristics, characterized by producing 1-deoxynojirimycin, which exhibits hypoglycemic and antiviral activity (deposit institution: Korea Microbiological Conservation Center, Deposit Sun: 02/02/2002, Accession No .: KCCM-10450): 1) L-arabinose, D-mannose, sorbitol, N-acetylglucosamine, iculin, maltose, saccharose, glycogen, ribose, amigdalin, salicylic, trehalose, D-rapinose, D-fructose in sugar availability Positive for oss, mannitol, α-methyl-D-glucoside, arbutin, cellobiose, inline, amidone, β-gentiobis; 2) It has activity on α-glucosidase, β-glucosidase and maltase, and no activity on β-galactosidase. delete delete delete delete delete delete delete