A novel Bacillus coagulans strain CC producing α-glucosidase inhibitor

Abstract

The novel Bacillus coagulans CC strain (KCTC14267BP) of the present invention is a novel strain that can survive under anaerobic conditions and produces and supplies an α-glucosidase inhibitor in the intestine. This allows the effective concentration of α-glucosidase and its main component, 1-deoxynojirimycin, to be maintained at a constant level in the small intestine, where the enzyme acts. This maximizes the inhibition of monosaccharide absorption, thereby contributing to weight loss and blood glucose reduction. Furthermore, because the α-glucosidase inhibitor and its main component, 1-deoxynojirimycin, are produced and supplied in the intestine, no additional fermentation or processing costs are required beyond the cost of spore production, thereby reducing production costs. Furthermore, since the α-glucosidase inhibitor does not suffer from fermentation odor or other problems, it can be used in various forms of food, feed, and pharmaceutical compositions.

Description

[Technical field]

[0001] The present invention relates to a new strain, Bacillus coagulans CC (KCTC14267BP), which produces a large amount of an α-glucosidase inhibitor in the intestine, and uses thereof. [Background technology]

[0002] When carbohydrates are digested in the body, they are broken down into disaccharides such as maltose, then broken down into monosaccharides such as glucose by an enzyme called α-glucosidase, and absorbed into the bloodstream. If the action of α-glucosidase is inhibited during this process, the breakdown of disaccharides slows down, making it possible to suppress the absorption of sugars. Therefore, α-glucosidase inhibitors significantly suppress postprandial hyperglycemia, making them effective in preventing or improving diabetes, obesity, etc.

[0003] α-glucosidase inhibitors are distributed in some plants and microorganisms. Known examples of plant-derived α-glucosidase inhibitors include aza sugars contained in mulberry leaves, isoflavones and flavone glycosides contained in soybeans, while known examples of microbial-derived α-glucosidase inhibitors include pseudooligosaccharides derived from Actinoplanes strains, 1-deoxynojirimycin produced by microorganisms such as Streptomyces or Bacillus, and tris bases. As pharmaceuticals, several types of α-glucosidase inhibitors are already on the market as treatments for diabetes.

[0004] In recent years, numerous studies have been conducted to verify the effectiveness of α-glucosidase inhibitors derived from natural products such as mulberry leaf extracts and natto, as typified by 1-deoxynojirimycin (Non-Patent Document 005). Although these primary processed products or extracts are mass-produced, they have in common factors that reduce palatability, such as odor and color, limiting the diversification of the products. In addition, these products use secondary metabolites produced by plants or microorganisms, so the concentration of the active ingredient is low, and they have the disadvantage that the desired purpose cannot be achieved unless a certain amount is taken several times a day.

[0005] On the other hand, facultative anaerobic bacteria can grow under anaerobic conditions such as in the intestines of animals. Therefore, it is expected that facultative anaerobic bacteria capable of producing α-glucosidase inhibitors will grow in the intestines, continuously produce α-glucosidase inhibitors, and supply them to the intestinal environment. The Bacillus subtilis strain used to produce α-glucosidase inhibitors using microorganisms is an aerobic bacterium, and is difficult to grow in the intestines, so it is difficult to supply α-glucosidase inhibitors to the intestines. As a result of closely watching the technologies related to α-glucosidase inhibitors so far and conducting repeated research with the aim of mass-producing α-glucosidase inhibitors in the intestines that are highly safe and have a high ability to reach the intestines by spore formation, the present invention has been completed by successfully isolating a strain that mass-produces α-glucosidase inhibitors in the intestines. Summary of the Invention [Problem to be solved by the invention]

[0006] The inventors of the present invention carefully searched a wide range of natural samples to select microorganisms that have strong α-glucosidase inhibitory activity and can grow in the intestine. As a result, they isolated and identified a novel Bacillus coagulans CC (KCTC14267BP) strain, which is facultatively anaerobic, forms spores, and is safe from microorganisms that exhibit α-glucosidase inhibitory activity, and mass-produces α-glucosidase inhibitors and their main component, 1-deoxynojirimycin, in the intestines of animals. They confirmed that the fermented product or vegetative cells containing spores of the microorganism can be usefully used in foods, medicines, feed, etc. for the purposes of weight loss, blood sugar reduction, and relief of diabetes and constipation. Therefore, an object of the present invention is to provide technical details regarding Bacillus coagulans CC (KCTC14267BP), a novel strain that mass-produces an α-glucosidase inhibitor. [Means for solving the problem]

[0007] In order to achieve the above-mentioned technical objectives, the present invention provides a novel Bacillus coagulans CC strain (Bacillus coagulans CC, Accession No. KCTC14267BP) having an improved ability to produce an α-glucosidase inhibitor.

[0008] In one embodiment of the present invention, the strain may comprise the 16s rRNA base sequence of SEQ ID NO:1, but is not limited thereto.

[0009] In one embodiment of the invention, the strain may be, but is not limited to, anaerobic.

[0010] In one embodiment of the present invention, the strain may be, but is not limited to, one that is excreted from the organism within 2 to 4 weeks after administration of the strain.

[0011] In one embodiment of the present invention, the strain may be, but is not limited to, one that produces an α-glucosidase inhibitor in the intestine.

[0012] In one embodiment of the present invention, the α-glucosidase inhibitor produced in the intestine may be, but is not limited to, one that is excreted from the organism within 2 to 4 weeks after administration of the strain.

[0013] In one embodiment of the present invention, the α-glucosidase inhibitor may be, but is not limited to, 1-deoxynojirimycin.

[0014] The present invention also provides a food composition for preventing or improving obesity, which contains a novel Bacillus coagulans CC strain (Bacillus coagulans CC, accession number KCTC14267BP), its culture medium, culture filtrate or fermentation product as an active ingredient.

[0015] The present invention also provides a feed composition for preventing or improving obesity, which contains a novel Bacillus coagulans CC strain (Bacillus coagulans CC, accession number KCTC14267BP), its culture medium, culture filtrate or fermentation product as an active ingredient.

[0016] The present invention also provides a pharmaceutical composition for preventing or treating obesity, comprising a novel Bacillus coagulans CC strain (Bacillus coagulans CC, Accession No. KCTC14267BP), its culture medium, culture filtrate or fermentation product as an active ingredient.

[0017] The present invention also provides a food composition for preventing and improving hyperglycemia or diabetes, which contains a novel Bacillus coagulans CC strain (Bacillus coagulans CC, accession number KCTC14267BP), its culture medium, culture filtrate or fermentation product as an active ingredient.

[0018] The present invention also provides a feed composition for preventing and improving hyperglycemia or diabetes, which contains a novel Bacillus coagulans CC strain (Bacillus coagulans CC, accession number KCTC14267BP), its culture medium, culture filtrate or fermentation product as an active ingredient.

[0019] The present invention also provides a pharmaceutical composition for preventing and treating hyperglycemia or diabetes, comprising a novel Bacillus coagulans CC strain (Bacillus coagulans CC, accession number KCTC14267BP), its culture medium, culture filtrate or fermentation product as an active ingredient.

[0020] Finally, the present invention provides a method for preventing or treating obesity, comprising the step of administering the pharmaceutical composition for preventing or treating obesity. Effect of the Invention

[0021] The novel Bacillus coagulans CC (KCTC14267BP) strain of the present invention is a new strain that can live under anaerobic conditions, and by producing and supplying an α-glucosidase inhibitor in the intestine, the effective concentration of α-glucosidase and its active ingredient, 1-deoxynojirimycin, can be kept constant in the small intestine where they act, and the absorption of monosaccharides can be inhibited to the maximum extent, resulting in weight loss and blood sugar reduction. The Bacillus coagulans CC strain (KCTC14267BP) of the present invention does not adhere to the intestine and exhibits the characteristic of living temporarily, and the strain or the α-glucosidase inhibitor produced by the strain, which is administered as needed, and the active ingredient, 1-deoxynojirimycin, can be excreted after a certain period of time, making it safe to use.

[0022] When producing α-glucosidase inhibitors using microorganisms, the production costs, such as fermentation costs and purification costs, are high and a lot of labor is required. However, when Bacillus coagulans CC strain (KCTC14267BP) is used, the α-glucosidase inhibitor and its active ingredient, 1-deoxynojirimycin, are produced and supplied in the intestine, so there is no need for additional fermentation or processing costs other than the cost of spore production, which reduces production costs. In addition, since there are no problems such as fermentation odor, the product can be applied to various types of food, feed, and pharmaceutical compositions. [Brief description of the drawings]

[0023] [Figure 1] This is the result of HPLC analysis of 1-deoxynojirimycin, the main component of the α-glucosidase inhibitor produced by Bacillus coagulans CC strain. [Diagram 2] This is a graph showing the effect of administration of Bacillus coagulans CC strain spores on mouse body weight when fed a high-carbohydrate diet, showing a standard diet group (○), a high-carbohydrate diet group (□), and a high-carbohydrate diet + CC spore administration group (■). [Diagram 3] Figure 2 shows the results of observing fat distribution in the chest and abdomen of mice in the normal diet group (Normal-Diet), high-carbohydrate diet group (HC-Diet), and high-carbohydrate diet + CC spore administration group (HC-Diet + CC Spore) at the 11th week through CT scans. [Figure 4] This is a graph showing the effect of administration of Bacillus coagulans CC strain spores on mouse body weight when fed a high-fat diet, and shows a standard diet group (○), a high-fat diet group (□), and a high-fat diet + CC spore administration group (■). [Diagram 5] Figure 4 shows the results of CT scans taken at the 11th week to examine fat distribution in the chest and abdomen of mice in the normal diet group (Normal-Diet), high-fat diet group (HF-Diet), and high-fat diet + CC spore administration group (HF-Diet + CC Spore). [Figure 6]Graph showing the inhibitory activity of α-glucosidase in 1 g of mouse feces, showing the high-carbohydrate diet + CC spores administration group (gray) and the high-fat diet + CC spores administration group (black). CC spores were administered for 7 weeks, and then the mice were fed a high-carbohydrate diet without spores or a high-fat diet. The arrow indicates the time when CC spore administration was stopped. [Figure 7] Graph showing distribution of Bacillus coagulans CC strains excreted in feces of mice fed a high-carbohydrate diet with CC spores (gray) and a high-fat diet with CC spores (black). CC spores were administered for 7 weeks, after which mice were fed a high-carbohydrate diet without spores or a high-fat diet. The arrow indicates the time when CC spore administration was stopped. [Figure 8] FIG. 1 is a schematic diagram showing the taxonomic position of Bacillus coagulans CC strains. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention provides a novel Bacillus coagulans CC strain (Bacillus coagulans CC, accession number KCTC14267BP) having an improved ability to produce an α-glucosidase inhibitor.

[0025] In one embodiment of the present invention, the strain is obtained by isolating bacteria that form heat-resistant spores from hay samples and selecting a strain that can grow facultatively anaerobically and produce a high amount of an α-glucosidase inhibitor.

[0026] In one embodiment of the present invention, the strain may contain 16s rRNA represented by the base sequence of SEQ ID NO:1.

[0027] In one embodiment of the present invention, the strain can simultaneously have anaerobic properties for producing an α-glucosidase inhibitor in high yield in the intestine and aerobic properties for producing an α-glucosidase inhibitor in vitro.

[0028] In one embodiment of the present invention, the strain can form spores, exhibits the property of living temporarily in the intestine without adhering thereto, and can be excreted from the intestine after administration as necessary, so that it can be used safely. Preferably, the strain can be excreted from the body of the organism 2 to 4 weeks after administration to the organism, and more preferably, can be excreted after 1 week.

[0029] In one embodiment of the present invention, the strain can produce the α-glucosidase inhibitor in the intestine, so that the effective concentration of the α-glucosidase inhibitor can be maintained constant in the intestine, and the α-glucosidase inhibitor produced in the intestine can be excreted, so that it can be used safely. Preferably, the α-glucosidase inhibitor can be excreted from the organism 2 to 4 weeks after the strain is administered to the organism.

[0030] For this reason, the composition containing the strain can be administered by ingestion, and the α-glucosidase inhibitor can be produced in high yield in the intestine, and can be excreted from the body after a certain period of time, so that it can be effectively used for the purpose of weight loss and blood sugar reduction. The oligosaccharides that are not broken down by the action of the α-glucosidase inhibitor produced in large amounts in the intestine reach the large intestine and function as prebiotics as food for microorganisms in the intestine, which can relieve constipation, so that it can be used for the purpose of alleviating constipation.

[0031] In one embodiment of the invention, the strain may be present in the form of spores, and may be present at 10 6 ~10 14 The microorganism may include, but is not limited to, individual spores.

[0032] In one embodiment of the present invention, the α-glucosidase inhibitor produced by the strain may be, but is not limited to, 1-deoxynojirimycin.

[0033] The term "fermented product" as used herein can include processed products derived from the fermented product itself, such as the fermented product cultured in a liquid / solid medium, a filtrate (centrifuged supernatant) obtained by filtering or centrifuging the fermented product to remove the bacterial strain, or a precipitate containing the bacteria.

[0034] The term "cultivation" as used herein means growing microorganisms under moderately artificially controlled environmental conditions.

[0035] The Bacillus coagulans CC strain can grow in a conventional medium, for example, nutrient broth medium. The medium contains nutrients required by the microorganism to be cultured, i.e., the microorganism to be cultured, and may further contain a specific target substance. The medium is also called a culture vessel or culture solution, and is a concept that includes all of natural media, synthetic media, and selective media. The strain can be cultured according to a conventional culture method.

[0036] The medium used for the culture must be a normal medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc., and must be adjusted in an appropriate manner to meet the requirements of a specific strain, while adjusting temperature, pH, etc. In addition to using glucose as the main carbon source, usable carbon sources include sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials can be used individually or as a mixture. Usable nitrogen sources include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; amino acids such as glutamic acid, methionine, and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition products, and defatted soybean foil (meal) or its decomposition products. These nitrogen sources can be used alone or in combination. The medium can contain potassium monophosphate, potassium diphosphate, and the corresponding sodium-containing salts as phosphate sources. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as phosphate sources. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the above substances.

[0037] Also, a suitable precursor for the culture medium can be used. The raw materials can be added to the culture in a suitable manner in the culture process in a batch manner, a fed-batch culture manner or a continuous manner, but are not limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture.

[0038] The present invention also provides a food composition, feed composition and pharmaceutical composition for preventing or improving obesity, which contain the novel Bacillus coagulans CC strain (KCTC14267BP), its culture medium, culture filtrate or fermentation product as an active ingredient.

[0039] The food composition, feed composition and pharmaceutical composition for preventing or improving obesity contain the above-mentioned novel Bacillus coagulans CC strain (KCTC14267BP), and therefore, the description of the contents overlapping with the above-mentioned strain of the present invention will be omitted in order to avoid excessive complexity of this specification due to the description of the overlapping contents.

[0040] In addition to containing the active ingredient, the food composition of the present invention can contain various flavoring agents or natural carbohydrates as additional ingredients, like a conventional food composition.

[0041] Examples of the natural carbohydrates are monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as normal sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. The flavoring agent may suitably be natural flavoring agents (thaumatin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition, and used as a functional food or added to various foods. Examples of foods to which the composition of the present invention may be added include beverages, meat, chocolate, food, confectionery, pizza, ramen, other noodles, gums, candies, ice cream, alcoholic beverages, vitamin complexes, and dietary supplements.

[0042] In addition to the extract as an active ingredient, the food composition may contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, coloring agents and enhancers (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. Furthermore, the food composition of the present invention may contain fruit pulp for preparing natural fruit juice, fruit juice drinks, and vegetable drinks.

[0043] The functional food composition of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. In the present invention, the term "health functional food composition" refers to a food prepared and processed using raw materials and ingredients having useful functions for the human body as specified in the Health Functional Food Law No. 6727, and is intended to be ingested for the purpose of obtaining useful effects for health applications such as regulating nutrients for the structure and function of the human body and physiological effects. The health functional food of the present invention can contain ordinary food additives, and the suitability of the food additive is determined according to the specifications and standards for the item in accordance with the general provisions and general test methods of the Food Additives Official Standards approved by the Food and Drug Administration, unless otherwise specified. Examples of the items listed in the "Food Additives Standards" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon color, licorice extract, crystalline cellulose, sorghum color, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations. For example, a tablet-type health functional food can be prepared by granulating a mixture of the active ingredient of the present invention mixed with an excipient, a binder, a disintegrant, and other additives by a conventional method, and then compressing the mixture by adding a lubricant or the like, or by directly compressing the mixture. In addition, a tablet-type health functional food may contain an umami seasoning or the like as necessary. Among capsule-type health functional foods, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention mixed with an excipient or other additive into a conventional hard capsule, and soft capsules can be prepared by filling a mixture of the active ingredient of the present invention mixed with an excipient or other additive into a capsule base such as gelatin. The soft capsule may contain, if necessary, a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc. The health functional food in pill form may be prepared by molding a mixture of the active ingredient of the present invention and an excipient, a binder, a disintegrant, etc., by a conventionally known method, and may be coated with white sugar or other coating agents, or may be surface-coated with materials such as starch and talc, if necessary.The health functional food in granular form can be prepared by mixing the active ingredient of the present invention with excipients, binders, disintegrants, etc., and preparing the mixture into granules by conventional known methods, and can contain flavorings, umami seasonings, etc. as necessary.

[0044] The feed composition of the present invention shows effects such as suppressing obesity and lowering blood sugar, and is expected to have an effect of improving the health condition of animals. The feed composition of the present invention can be prepared in the form of fermented feed, mixed feed, pelleted form, silage, etc. The fermented feed can be prepared by fermenting organic matter by adding the Bacillus coagulans CC of the present invention, and the mixed feed can be prepared by mixing various types of general feed with the spores and fermented products of the Bacillus coagulans CC strain of the present invention. The pelleted feed can be prepared by applying heat and pressure to the mixed feed in a pellet manufacturing machine, and the silage can be prepared by fermenting green feed with the microorganism of the present invention. The wet fermented feed can be prepared by collecting and transporting organic matter such as food waste, mixing it with an excipient for sterilization and moisture control in a certain ratio, fermenting it at a temperature suitable for fermentation for 24 hours or more, and adjusting the moisture content to about 70%. The fermented and dried feed can be prepared by further drying the wet fermented feed and adjusting the moisture content to about 30% to 40%.

[0045] The feed composition of the present invention may further contain ingredients added to conventional feed. Examples of ingredients added to such feed may include grain flour, meat flour, legumes, etc. The grain flour may be one or more selected from rice flour, wheat flour, barley flour, and corn flour. The meat flour may be one or more selected from chicken, beef, pork, and ostrich meat that have been powdered. The legumes may be one or more selected from soybeans, kidney beans, peas, and black beans.

[0046] The feed composition of the present invention may contain at least one selected from the group consisting of nutrients and inorganic substances to increase the nutritional value of the feed, in addition to the grain flour, meat powder, and beans that are added to conventional feed as described above, and may contain at least one selected from the group consisting of antifungal agents, antioxidants, anticoagulants, emulsifiers, and binders to prevent deterioration of the feed quality.

[0047] The pharmaceutical composition according to the present invention can be prepared in a form in which the active ingredient is mixed with a pharma- ceutically acceptable carrier. Here, the pharma-ceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. The pharma-ceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0048] The pharmaceutical composition of the present invention can be formulated into powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., by conventional methods and used.

[0049] When preparing a formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, or surfactant that is commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing the active ingredient with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, liquids for internal use, emulsions, syrups, etc., and can contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin.

[0050] The pharmaceutical compositions may be formulated in a variety of oral dosage forms.

[0051] Dosage forms for oral administration include, for example, tablets, pills, hard and soft capsules, liquids, suspensions, emulsions, syrups, granules, etc., and these dosage forms may further contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salt and / or polyethylene glycol) in addition to the active ingredient. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and may optionally contain disintegrants or effervescent mixtures such as starch, agar, alginic acid or its sodium salt and / or absorbents, colorants, flavorings and sweeteners. The dosage forms may be prepared by a general mixing, granulating or coating method.

[0052] The present invention also provides a food composition, feed composition, and pharmaceutical composition for preventing or improving hyperglycemia and diabetes, which contain the novel Bacillus coagulans CC strain (KCTC14267BP) or its culture medium, culture filtrate, or fermentation product as an active ingredient.

[0053] The food composition, feed composition and pharmaceutical composition for preventing or improving hyperglycemia and diabetes contain the above-mentioned novel Bacillus coagulans CC strain (KCTC14267BP), and therefore, the description of the contents overlapping with the above-mentioned strain of the present invention will be omitted in order to avoid excessive complexity of this specification due to the description of the overlapping contents.

[0054] The present invention provides a method for preventing or treating obesity, comprising the step of administering the pharmaceutical composition for preventing or treating obesity.

[0055] The pharmaceutical compositions of the invention may be administered in a therapeutically effective amount or a pharma- ceutically effective amount.

[0056] The term "pharmacologically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level can be determined according to factors including the type and severity of the individual, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical art.

[0057] The method of administering the pharmaceutical composition is not particularly limited, and examples thereof include application to the skin, intradermal injection, and subcutaneous injection.

[0058] Furthermore, the term "individual" as used herein means a subject in need of a method for preventing, controlling or treating a disease, and more specifically means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc. In some cases, humans may be excluded.

[0059] The present invention will be described in more detail with reference to the following examples. These examples are provided to more specifically explain the present invention, and are not intended to limit the scope of the present invention.

[0060] Example 1: Identification of bacterial strains Microorganisms were isolated from approximately 800 samples of hay collected from various locations in Korea. Each sample was suspended in a small amount of sterile saline, then heat-treated in a thermostatic water bath at 80°C for 20 minutes. The resulting spore liquid was smeared on a BCP Plate Count Agar plate containing 1.5% agar, and cultured anaerobically in an incubator at 55°C for 2 days to isolate bacterial colonies. To examine the amount of α-glucosidase inhibitor produced, the isolated strain was inoculated into 5 ml of medium containing 5% soybean flour and cultured at 37°C for 24 hours with shaking. The culture was centrifuged, and the α-glucosidase inhibitory activity was measured from the supernatant.

[0061] <Example 2> Measurement of α-glucosidase inhibitory activity α-glucosidase was extracted from pig small intestine and partially purified and used as the enzyme source. The α-glucosidase enzyme solution was appropriately diluted with 0.5M phosphate buffer solution (pH 7.0) to 24 units / ml, and p-nitrophenyl α-D-glucopyranoside (pNPG) was used as the substrate. 30 μl of culture supernatant and 50 μl of enzyme were mixed in a test tube and pre-incubated at 37°C for 10 minutes, after which 50 μl of 3 mM pNPG was added and reacted at 37°C for 20 minutes. The reaction was stopped by adding 0.1M sodium carbonate to the reaction solution, and the absorbance at 405 nm was measured with a spectrophotometer to measure the α-glucosidase inhibitory activity. At this time, the control group was one in which water was added instead of the inhibitor, and the inhibition rate was calculated based on the following formula 1.

[0062]

number

[0063] In the above formula, A is the absorbance of the control group and B is the absorbance of the sample containing the inhibitor.

[0064] In this case, the α-glucosidase inhibition unit (AGI unit) was defined as the amount of inhibitor required to inhibit 1% of the α-glucosidase used in the activity measurement.

[0065] Example 3: Selection of strains After primary selection of about 50 kinds of bacteria as α-glucosidase inhibitor producers according to the process of Example 2, the culture solution of the strains that can grow anaerobically at 55°C and do not grow in a medium containing propionic acid was centrifuged, and the α-glucosidase inhibitory activity of the supernatant was measured, and three strains with high α-glucosidase inhibitory activity were secondary selected. Among the three strains, DH-128, which is classified as Bacillus coagulans according to the taxonomic characteristics analysis method according to Bergey's Manual of Systematic Bacteriology (2002), has high α-glucosidase inhibitor productivity, and is easy to form spores, was finally selected (see Table 1).

[0066] [Table 1]

[0067] Example 4: Mutagenesis of DH-128 strain <4.1> Selection and characterization of novel Bacillus coagulans CC DH-128 strain was treated with nitrosoguanidine (100μg / ml) to induce mutations at a mortality rate of 99.9%, and then 200-300 colonies were smeared on BCP Plate Count Agar plates and cultured at 40℃ for 2 days. The colonies produced were randomly inoculated onto 5% soybean flour medium and cultured at 40℃ for 2 days with shaking, and the supernatant was centrifuged and α-glucosidase inhibitory activity was measured. Five strains with increased α-glucosidase inhibitory activity were selected, and DH-128-46Y93 was selected based on sporulation ability and α-glucosidase inhibitory activity. The DH-128-46Y93 strain was repeatedly mutated using the same method as described above, and a strain with high α-glucosidase inhibitory activity and high spore formation ability was selected. It was named Bacillus coagulans CC strain and deposited at the Korea Comprehensive Biotechnology and Technology Center (KCTC) on August 6, 2020 (accession number KCTC14267BP).

[0068] As control strains, α-glucosidase inhibitor producing natto strain Bacillus subtilis DC-15 and α-glucosidase inhibitor non-producing strain Bacillus coagulans ATCC 7050 were used, and mutant strains DH-128, DH-128-46Y93 and Bacillus coagulans CC strain (Bacillus coagulans CC) were cultured aerobically or anaerobically in 5% soybean flour medium at 40℃ for 2 days, respectively, and the α-glucosidase inhibitory activity produced is shown in Table 2 below.

[0069] [Table 2]

[0070] As shown in Table 2, the Bacillus coagulans CC strain with significantly increased α-glucosidase inhibitory activity was bred from the selected strain DH-128 by sequential mutation breeding. The control strain, Bacillus subtilis DC-15, is an aerobic bacterium used in natto production, which produces a significant amount of α-glucosidase inhibitor under aerobic conditions but does not produce any under anaerobic conditions, and Bacillus coagulans ATCC 7050 does not produce α-glucosidase inhibitor under either aerobic or anaerobic conditions. Therefore, the Bacillus coagulans CC strain has a very different characteristic from other aerobic bacteria that produce α-glucosidase inhibitors by showing the ability to produce α-glucosidase inhibitors even in anaerobic environments such as the intestine.

[0071] <4.2> Morphological and biochemical characteristics of isolated Bacillus coagulans CC strain The bacterial colonies formed by the Bacillus coagulans CC strain bred through the above-mentioned isolation process on L-broth agar plate medium were small and flat, with smooth rounded edges, and the surface was non-glossy white. The effect of temperature on the growth of the bacteria was good in the range of 25°C to 40°C, and no growth was observed above 60°C. The nitrate reducing power was positive, and the Voges-Proskauer test using citric acid was negative. In addition, it decomposed casein, gelatin, and starch, produced acid from glucose, but did not produce acid from arabinose, xylose, and mannitol, was catalase positive, and grew under aerobic and anaerobic conditions. When the logarithmic phase of the growth of the bacteria using L-broth liquid medium was observed under a microscope, it was similar to the comparative strain Bacillus coagulans in terms of its short rod-like external shape, and was gram-positive.

[0072] In addition, the biochemical characteristics of the isolated strain were investigated using an API50CHB kit (BioMerieux, France), and it was confirmed that the strain had the biochemical characteristics (API test results) shown in Table 3 below.

[0073] [Table 3]

[0074] The results of the analysis of the taxonomic characteristics of Bacillus strains according to Bergey's Manual of Systematic Bacteriology (2002) are summarized in Table 4 below, and it was confirmed that Bacillus coagulans CC has the 16s rRNA sequence of SEQ ID NO: 1 below.

[0075] [Table 4]

[0076] [SEQ ID NO: 1]

[0077] Example 5: Analysis of 1-deoxynojirimycin produced by Bacillus coagulans CC strain Bacillus coagulans CC strain was cultured in 5% soy flour medium at 37℃ for 48 hours, centrifuged, and the supernatant was developed by TLC, then scraped at 0.5 cm intervals, extracted with water, and α-glucosidase inhibitory activity was measured. α-glucosidase inhibitory activity was found in the same place as 1-deoxynojirimycin, and the main component of α-glucosidase inhibitor was tentatively determined to be 1-deoxynojirimycin. 1-deoxynojirimycin was further confirmed by HPLC analysis. Bacillus coagulans CC strain was inoculated into 5 ml of 5% soy flour medium and cultured anaerobically at 37℃ for 48 hours. One ml of the mixture was taken, and 20 μl of 0.4 M potassium borate buffer (pH 8.5) and 20 μl of 5 mM 9-fluorenylmethyl chloroformate dissolved in acetonitrile were added, and the mixture was reacted at room temperature for 30 minutes in the dark, and compared with the derivative of the standard product, 1-deoxynojirimycin. The reaction was stopped by adding 20 μl of 0.1 M glycine, and the product was used for HPLC analysis. The column used for HPLC analysis was an Agilent TC-C18 column (250 × 4.60 mm, 5 μm), and the mobile phase was acetonitrile: 0.1% acetic acid = 35:65, the flow rate was 1.2 ml / min, the column oven temperature was 40 °C, and detection was performed at 254 nm. As shown in Figure 1, the product of Bacillus coagulans CC strain and 1-deoxynojirimycin showed the same retention time, confirming that they were the same substance.

[0078] <Example 6> Dietary experiment of Bacillus coagulans CC strain In order to confirm whether Bacillus coagulans CC strain efficiently produces α-glucosidase inhibitors in the intestinal tract of animals, spores of Bacillus coagulans CC strain were orally administered to mice while they were being raised, and the growth of the strain in the intestine and its excretion from the body, the production of α-glucosidase inhibitors in the intestine, and changes in body weight were examined. For this purpose, male mice (3 weeks old) were used after being pre-raised for one week with commercial standard feed and water ad libitum. The light / dark cycle was set to 08:00 to 20:00 h for the light period and 20:00 to 08:00 h for the dark period, and the temperature was maintained at 22.5 ± 0.5 °C and humidity was maintained at 50 to 60%. The diets were prepared according to the compositions in Table 5 below. Mice were divided into five groups (8 mice per group): standard diet group, high-carbohydrate diet group, high-carbohydrate diet + CC spore group, high-fat diet group, and high-fat diet + CC spore group, and were kept for 12 weeks. Bacillus coagulans CC strain spores were administered at 100 mg / kg for the high-carbohydrate diet + CC spore group and the high-fat diet + CC spore group. 9 The remaining groups were fed a standard diet, a high-carbohydrate diet, and a high-fat diet, and were given 100 μl of saline instead of the spore dilution every other day. As the animals were fed, feces were collected periodically and used as samples to measure α-glucosidase inhibitory activity in the feces and the excreted Bacillus coagulans CC strain.

[0079] [Table 5]

[0080] <6.1>Analysis of the effect of administration of Bacillus coagulans CC strain spores on the body weight of mice fed a high-carbohydrate diet The effect of administration of Bacillus coagulans CC strain spores on the body weight of mice fed a high-carbohydrate diet was analyzed. As shown in Figure 2, the high-carbohydrate diet group showed a significant weight gain compared to the standard diet group, but the high-carbohydrate diet + CC spore administration group showed a significantly reduced weight gain compared to the high-carbohydrate diet group. At the 11th week of the experiment, CT scans were taken of the mice in each group to observe the distribution of fat in the body. As a result, Figure 3 shows that the bright areas indicated by the arrows in the photographs indicate the fat layer, and it can be seen that the high-carbohydrate diet group has a large amount of fat accumulated in the chest and abdomen compared to the standard diet. On the other hand, the high-carbohydrate diet group administered Bacillus coagulans CC strain spores showed a significant decrease in the accumulation of body fat, indicating that the administration of Bacillus coagulans CC strain spores, which shows the same tendency as the standard diet group, efficiently inhibits weight gain and the accumulation of body fat.

[0081] <6.2>Effect of administration of Bacillus coagulans CC strain spores on the body weight of mice fed a high-fat diet The effect of administration of Bacillus coagulans CC strain spores on the body weight of mice fed a high-fat diet was analyzed. As shown in Figure 4, the high-fat diet group showed a significant weight gain compared to the standard diet group, but it was confirmed that the weight gain was significantly reduced compared to the high-fat diet group, similar to the high-carbohydrate diet + CC spore administration group. Similarly, CT scans were taken of the mice in each group in the high-fat diet group at the 11th week of the experiment to observe the fat distribution in the body. Figure 5 shows that fat is less likely to accumulate in the high-fat diet than in the high-carbohydrate diet, but a large amount of fat accumulates in the chest and abdomen in the high-fat diet compared to the standard diet, and administration of Bacillus coagulans CC strain spores efficiently inhibits weight gain and body fat accumulation.

[0082] Example 7: Fecal analysis During the feeding experiment of Bacillus coagulans CC strain, feces were periodically collected and the amount of α-glucosidase inhibitor and Bacillus coagulans CC strain excreted in the feces were measured. The α-glucosidase inhibitor was measured for α-glucosidase inhibitor activity, and the number of Bacillus coagulans CC strain bacteria was measured by adding sterilized water to a certain amount of feces, suspending it well, smearing the supernatant on a BCP plate medium, and incubating it anaerobically at 55℃ for 2 days, and then counting the number of colonies that appeared.

[0083] <7.1> Changes in Bacillus coagulans CC strains excreted in feces The number of spores in the feces of mice administered Bacillus coagulans CC strain spores was measured, and the change in the strains excreted in the feces was analyzed. As a result, as shown in Figure 6, the amount of bacteria excreted steadily increased until the 5th week, and thereafter, the number of spores excreted per 1 g of feces increased to 10 7 It can be concluded that the administered Bacillus coagulans CC strain grows in the intestine and some of it is excreted from the body in order to maintain the individuality. On the other hand, the number of Bacillus coagulans CC strains gradually decreases from the 8th week when the administration of the bacteria was stopped, indicating that Bacillus coagulans CC strains do not adhere to the intestine and do not remain there for very long. Therefore, it was confirmed that there is an advantage that this strain can be removed from the intestine if necessary, since it is expected that the intestinal flora will change if the administration of this strain is discontinued. Since no colony formation is observed in the feces of mice fed the standard diet, the colonies that appeared can be identified as Bacillus coagulans CC strain.

[0084] <7.2> Changes in α-glucosidase inhibitory activity excreted in feces The inhibitory activity of α-glucosidase excreted in the feces of mice administered spores of Bacillus coagulans CC strain was analyzed. As shown in Figure 7, Bacillus coagulans CC strain (KCTC14267BP) proliferated vigorously in the intestine to the extent that it was excreted in the feces, and it was confirmed that α-glucosidase inhibitors were produced. As shown in Figure 6, it was confirmed that the amount of α-glucosidase inhibitors excreted during the period from the 5th to the 7th week when the amount of excreted Bacillus coagulans CC strain stabilized was also constant. In other words, it can be seen that α-glucosidase inhibitors are always produced in the intestine, so that the concentration in the intestine is maintained at a constant level, which has the advantage of being highly efficient, and that a constant amount of the produced inhibitors is excreted.

[0085] The above results indicate that the Bacillus coagulans CC strain of the present invention can continuously supply bioactive substances to the body, instead of administering bioactive substances produced by industrial fermentation into the body. This is because there is no need for separate fermentation and processing costs to produce α-glucosidase inhibitors, which reduces production costs, and it has been confirmed that there is an advantage in that the effective concentration of α-glucosidase inhibitors can be maintained at a constant level in the small intestine, which is the site of action of α-glucosidase inhibitors, by continuously producing them in the body. In addition, the use of spores of Bacillus coagulans CC strains has the advantage that problems such as fermentation odor, volume, and color do not occur during product processing, making it applicable to various types of foods.

[0086] Depository institution name: Korea Institute of Bioscience and Biotechnology Accession number: KCTC14267BP Date of acceptance: 20200806

[0087] JPEG0007634910000007.jpg210153

Claims

1. A novel Bacillus coagulans CC strain having improved ability to produce an α-glucosidase inhibitor (Bacillus coagulans CC, accession number KCTC14267BP).

2. The Bacillus coagulans CC strain according to claim 1 , wherein the strain comprises the 16s rRNA base sequence of SEQ ID NO:

1.

3. The Bacillus coagulans CC strain according to claim 1 , wherein the strain is anaerobic.

4. The Bacillus coagulans CC strain according to claim 1, wherein the strain is excreted from the organism within 2 to 4 weeks after administration.

5. The Bacillus coagulans CC strain according to claim 1, which produces an α-glucosidase inhibitor in the intestine.

6. The Bacillus coagulans CC strain according to claim 5, wherein the α-glucosidase inhibitor produced in the intestine is excreted from the organism within 2 to 4 weeks after administration of the strain.

7. The Bacillus coagulans CC strain according to claim 1, wherein the α-glucosidase inhibitor is 1-deoxynojirimycin.

8. A food composition for preventing or improving obesity, comprising a novel Bacillus coagulans CC strain (Bacillus coagulans CC, accession number KCTC14267BP), its culture medium, culture filtrate or fermentation product as an active ingredient.

9. A feed composition for preventing or improving obesity, comprising a novel Bacillus coagulans CC strain (Bacillus coagulans CC, accession number KCTC14267BP), a culture broth, a culture filtrate or a fermentation product thereof as an active ingredient.

10. A pharmaceutical composition for preventing or treating obesity, comprising a novel Bacillus coagulans CC strain (Accession No. KCTC14267BP), a culture medium, a culture filtrate or a fermentation product thereof as an active ingredient.

11. A food composition for preventing and improving hyperglycemia or diabetes, comprising a novel Bacillus coagulans CC strain (Bacillus coagulans CC, accession number KCTC14267BP), its culture broth, culture filtrate or fermentation product as an active ingredient.

12. A feed composition for preventing and improving hyperglycemia or diabetes, comprising a novel Bacillus coagulans CC strain (Bacillus coagulans CC, accession number KCTC14267BP), a culture broth, a culture filtrate or a fermentation product thereof as an active ingredient.

13. A pharmaceutical composition for preventing and treating hyperglycemia or diabetes, comprising a novel Bacillus coagulans CC strain (Bacillus coagulans CC, Accession No. KCTC14267BP), its culture medium, culture filtrate or fermentation product as an active ingredient.

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