CN116209363A - Novel bacillus coagulans CC strain producing alpha-glucosidase inhibitor - Google Patents

Abstract

The novel bacillus coagulans CC (Bacillus coagulans CC) (KCTC14267BP) bacterial strain of the present invention is the novel bacterial strain that also can survive under the anaerobic condition, can produce and supply α-glucosidase inhibitor in intestinal tract, thus in α-glucose Glycosidase and its main component 1‑deoxynojirimycin (1‑deoxynojirimycin) act on the small intestine to maintain the effective concentration at a specified level, thereby maximizing the absorption of monosaccharides, thereby showing weight loss and The effect of lowering blood sugar. In addition, since the α-glucosidase inhibitor and its main component 1-deoxynojirimycin (1-deoxynojirimycin) can be produced and supplied in the intestinal tract, separate fermentation costs and processing costs other than spore production costs are not required , so the production cost can be reduced, and since there will be no problems such as fermentation smell, it can be applied to various forms of food, feed and pharmaceutical compositions.

Description

A novel Bacillus coagulans CC strain producing α-glucosidase inhibitor

Technical Field

The present invention relates to a novel strain, Bacillus coagulans CC (KCTC14267BP), which produces a large amount of α-glucosidase inhibitor in the intestinal tract and its use.

Background Art

Carbohydrates are broken down into disaccharides such as maltose during digestion in the body, and then broken down into monosaccharides such as glucose by an enzyme called α-glucosidase, which are then absorbed into the blood vessels. In this process, if the action of α-glucosidase is blocked, the decomposition of disaccharides is delayed, thereby controlling the absorption of sugars. Therefore, α-glucosidase inhibitors can significantly suppress high blood sugar levels after meals, thereby effectively preventing or improving diabetes, obesity, etc.

α-glucosidase inhibitors are distributed in some plants or microorganisms. Plant-derived α-glucosidase inhibitors include aza sugars contained in mulberry leaves, isoflavones and flavone glycosides contained in soybeans, etc. Microbial-derived α-glucosidase inhibitors include pseudooligosaccharides derived from Actinoplanes strains, 1-deoxynojirimycin and tris base produced by microorganisms such as Streptomyces or Bacillus, etc. There are many α-glucosidase inhibitors in the pharmaceutical category, which are already marketed as diabetes treatment agents.

Recently, in order to verify the effectiveness of mulberry leaf extract represented by 1-deoxynojirimycin or α-glucosidase inhibitors derived from natural substances such as natto, a lot of research has been carried out (non-patent literature 005). In addition, these primary processed products or extracts are produced in large quantities, but there are factors such as taste and color turbidity that cause objection, so there are limitations in the diversification of products. And, since this belongs to the situation of utilizing secondary metabolites generated by plants or microorganisms, the concentration of active ingredients is very low, and only by taking it multiple times a day according to the prescribed amount can the intended purpose be achieved, which becomes its shortcoming.

On the other hand, facultative anaerobic bacteria can be proliferated under anaerobic conditions such as in the intestinal tract of animals. Therefore, facultative anaerobic bacteria with the ability to generate alpha-glucosidase inhibitors can proliferate in the intestinal tract and continuously generate and supply alpha-glucosidase inhibitors in the intestinal environment. The subtilis (Bacillus subtilis) strain used in the generation of alpha-glucosidase inhibitors utilizing microorganisms belongs to aerobic bacteria, is difficult to proliferate in the intestinal tract, and is therefore difficult to supply alpha-glucosidase inhibitors in the intestinal tract. The technology surrounding alpha-glucosidase inhibitors that have been concerned about so far and the result of continuous research with safety, high intestinal reachability achieved by forming spores, and alpha-glucosidase inhibitors that can be generated in large quantities in the intestinal tract as a target, have been successfully separated and a strain that can generate a large amount of alpha-glucosidase inhibitors in the intestinal tract has been completed.

Summary of the invention

Technical issues

The inventors of the present invention actively explored microorganisms with strong α-glucosidase inhibitory activity and that can grow in the intestine from a variety of samples derived from nature. As a result, as a facultative anaerobic, spore-forming, safe bacterium, a novel Bacillus coagulans CC (KCTC14267BP) strain capable of producing a large amount of α-glucosidase inhibitors and 1-deoxynojirimycin (1-deoxynojirimycin) as its main component in the intestine of animals was isolated and identified from microorganisms that exhibit α-glucosidase inhibitory activity. It was confirmed that the nutrient cells of the fermentation products or spores containing the above-mentioned microorganisms can be effectively used in food, medicine, feed, etc., to achieve the purposes of weight loss, lowering blood sugar, eliminating diabetes and constipation. Therefore, the object of the present invention is to provide technical content related to the Bacillus coagulans CC (KCTC14267BP) strain as a new strain that produces a large amount of α-glucosidase inhibitors.

Technical Solution

In order to achieve the above technical objectives, the present invention provides a novel Bacillus coagulans CC strain (Bacillus coagulans CC, deposit number KCTC14267BP) having an improved ability to produce an α-glucosidase inhibitor.

According to one embodiment of the present invention, the strain may include the base sequence of 16s rRNA of sequence number 1, but is not limited thereto.

According to an embodiment of the present invention, the above-mentioned strain may be anaerobic, but is not limited thereto.

According to an embodiment of the present invention, the strain may be excreted from the organism within 2 to 4 weeks after administration of the strain, but the present invention is not limited thereto.

According to one embodiment of the present invention, the above-mentioned strain can produce an α-glucosidase inhibitor in the intestine, but is not limited thereto.

According to an embodiment of the present invention, the α-glucosidase inhibitor produced in the intestine can be excreted from the organism within 2 to 4 weeks after administration of the strain, but the present invention is not limited thereto.

According to an embodiment of the present invention, the α-glucosidase inhibitor may be 1-deoxynojirimycin, but is not limited thereto.

Furthermore, the present invention provides a food composition for preventing or improving obesity, comprising a novel Bacillus coagulans CC strain (Bacillus coagulans CC, deposit number KCTC14267BP), a culture solution, a culture filtrate or a fermentation product thereof as an effective ingredient.

Furthermore, the present invention provides a feed composition for preventing or improving obesity, comprising a novel Bacillus coagulans CC strain (Bacillus coagulans CC, deposit number KCTC14267BP), a culture solution, a culture filtrate or a fermentation product thereof as an effective ingredient.

Furthermore, the present invention provides a pharmaceutical composition for preventing or treating obesity, comprising a novel Bacillus coagulans CC strain (Bacillus coagulans CC, deposit number KCTC14267BP), a culture solution, a culture filtrate or a fermentation product thereof as an effective ingredient.

Furthermore, the present invention provides a food composition for preventing and improving hyperglycemia or diabetes, comprising a novel Bacillus coagulans CC strain (Bacillus coagulans CC, deposit number KCTC14267BP), a culture solution, a culture filtrate or a fermentation product thereof as an effective ingredient.

Furthermore, the present invention provides a feed composition for preventing and improving hyperglycemia or diabetes, comprising a novel Bacillus coagulans CC strain (Bacillus coagulans CC, deposit number KCTC14267BP), a culture solution, a culture filtrate or a fermentation product thereof as an effective ingredient.

Furthermore, the present invention provides a pharmaceutical composition for preventing and treating hyperglycemia or diabetes, comprising a novel Bacillus coagulans CC strain (Bacillus coagulans CC, deposit number KCTC14267BP), a culture solution, a culture filtrate or a fermentation product thereof as an effective ingredient.

Finally, the present invention provides a method for preventing or treating obesity, comprising the step of administering the above-mentioned pharmaceutical composition for preventing or treating obesity.

Effects of the Invention

The novel Bacillus coagulans CC (KCTC14267BP) strain of the present invention is a novel strain that can survive even under anaerobic conditions. Since it produces and supplies an α-glucosidase inhibitor in the intestine, it can maintain the effective concentration at a predetermined level in the small intestine where α-glucosidase and its active ingredient 1-deoxynojirimycin act, thereby maximizing the absorption inhibition of monosaccharides, thereby showing the effects of reducing body weight and lowering blood sugar. The Bacillus coagulans CC strain (KCTC14267BP) of the present invention has the characteristic of temporarily surviving in the intestine, rather than being fixed in the intestine, so that the administered strain or the α-glucosidase inhibitor and its active ingredient 1-deoxynojirimycin produced by the strain can be excreted after a predetermined time as needed, so that it can be used safely.

In the case of using microorganisms to produce α-glucosidase inhibitors, the production costs such as fermentation costs and refining costs are heavy and require a lot of labor. However, by using the Bacillus coagulans CC strain (KCTC14267BP), α-glucosidase inhibitors and their active ingredient 1-deoxynojirimycin can be produced and supplied in the intestines, so there is no need for separate fermentation costs and processing costs in addition to the spore production costs, thereby reducing production costs. Since there is no problem of fermented smell, etc., it can be applied to various forms of food, feed and pharmaceutical compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of high performance liquid chromatography (HPLC) analysis to identify 1-deoxynojirimycin, a major component of the α-glucosidase inhibitor produced by Bacillus coagulans CC strain.

2 is a graph showing the effect of administration of Bacillus coagulans CC strain spores on the body weight of mice when a high-carbohydrate diet was provided, including a standard diet group (○), a high-carbohydrate diet group (□), and a high-carbohydrate diet + CC spore administration group (■).

Figure 3 shows the results of observing the fat distribution in the chest and abdomen of mice in the standard 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 in Figure 2 by electronic computed tomography (CT).

4 is a graph showing the effect of administration of Bacillus coagulans CC strain spores on the body weight of mice fed a high-fat diet, including a standard diet group (○), a high-fat diet group (□), and a high-fat diet + CC spore administration group (■).

Figure 5 shows the results of observing the fat distribution in the chest and abdomen of mice in the standard diet group (Normal-Diet), high-fat diet group (HF-Diet) and high-fat diet + CC spore administration group (HF-Diet + CC Spore) at the 11th week in Figure 4 by electronic computed tomography (CT).

FIG6 is a graph showing the inhibitory activity of α-glucosidase contained in 1 g of mouse feces, including a high-carbohydrate feed + CC spore administration group (gray) and a high-fat feed + CC spore administration group (black). CC spores were administered for 7 weeks, and then a high-carbohydrate feed or a high-fat feed without spores was provided. The arrow indicates the time when the administration of CC spores was stopped.

FIG7 is a graph showing the distribution of Bacillus coagulans CC strains excreted through feces of mice, including a high-carbohydrate diet + CC spore administration group (gray) and a high-fat diet + CC spore administration group (black). CC spores were administered for 7 weeks, followed by a high-carbohydrate diet or a high-fat diet without spores. The arrow indicates the time when the administration of CC spores was stopped.

FIG. 8 is a diagram showing the taxonomic position of Bacillus coagulans CC strains.

Best Mode for Carrying Out the Invention

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

According to one embodiment of the present invention, the strain can be obtained by isolating heat-resistant spore-forming bacteria using hay as a sample, and screening a strain that can achieve facultative anaerobic cultivation and high production of an α-glucosidase inhibitor.

According to one embodiment of the present invention, the strain may include 16s rRNA represented by the base sequence of SEQ ID NO: 1.

According to one embodiment of the present invention, the strain can have both the anaerobic property of producing α-glucosidase inhibitors in the intestine with high yield and the aerobic property of producing α-glucosidase inhibitors in vitro.

According to one embodiment of the present invention, the strain can form spores and exhibit the characteristics of temporarily surviving in the intestines instead of being fixed in the intestines, so the administered strain can be discharged from the intestines as needed, so that it can be used safely. Preferably, the strain can be discharged from the body of the organism after 2 to 4 weeks from the time of administration to the organism, and more preferably, it can be discharged after 1 week.

According to one embodiment of the present invention, the strain as described above can produce the above-mentioned α-glucosidase inhibitor in the intestine, can maintain the effective concentration of the α-glucosidase inhibitor at a specified level in the intestine, can excrete the above-mentioned α-glucosidase inhibitor produced in the intestine, and can be used safely. Preferably, the above-mentioned α-glucosidase inhibitor can be excreted from the body of the organism from 2 to 4 weeks after the strain is administered to the organism.

Therefore, the composition containing the above-mentioned strain can be administered by taking it to generate an α-glucosidase inhibitor in the intestine at a high yield, and can be discharged to the outside of the organism after a specified period, so it can be effectively used for the purpose of reducing body weight and lowering blood sugar. Due to the action of the α-glucosidase inhibitor generated in large quantities in the intestine, the undecomposed oligocarbohydrates will reach the large intestine and become food for microorganisms in the intestine, playing the role of prebiotics, thereby eliminating constipation, and can be used for the purpose of relieving constipation.

According to an embodiment of the present invention, the strain may be contained in the form of spores, and may contain 10 ⁶ to 10 ¹⁴ spores per 1 gram, but is not limited thereto.

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

The term "fermentation product" as used herein may include the fermentation product itself cultured in a liquid/solid medium, a filtrate obtained by filtering or centrifuging the fermentation product to remove strains (supernatant obtained by centrifugation), or a processed product derived from the fermentation product itself such as a precipitate containing bacteria.

The term "culture" in this specification means growing microorganisms under appropriate environmental conditions artificially adjusted.

The above-mentioned Bacillus coagulans CC strain can be cultured in a conventional culture medium, for example, in a nutrient broth medium. The above-mentioned culture medium contains nutrients that need to be supplied to the cultured microorganisms as the culture object in order to culture specific microorganisms, and substances for achieving special purposes can be added and mixed. The above-mentioned culture medium can also be called a culture vessel or a culture solution, including all concepts such as a natural culture medium, a synthetic culture medium or a selective culture medium. The above-mentioned strain can be cultured according to conventional culture methods.

The culture medium used in the culture should meet the culture elements of the specific strain in a proper manner by adjusting the temperature, pH, etc. in a conventional culture medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc. The carbon sources that can be used include glucose as the main carbon source, in addition to sugars such as sucrose, lactose, fructose, maltose, starch, and cellulose; carbohydrates; oils such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as fats, palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; organic acids such as acetic acid. These substances can be used alone or in the form of a mixture. The nitrogen sources that can be used 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, as well as organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean meal (meal) or its decomposition products. These nitrogen sources can be used alone or in combination. In the above culture medium, potassium phosphate, dipotassium phosphate and corresponding sodium salts may be included as phosphate sources. Available phosphate sources include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium salts. In addition, inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate and calcium sulfate. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins may be used.

In addition, appropriate precursors may be used in the culture medium. The above raw materials may be added to the culture in a batch, valence or sequential manner during the culture process by an appropriate method, but are not limited thereto. The pH of the culture may be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide, ammonia gas, or an acid compound such as phosphoric acid or sulfuric acid in an appropriate manner.

Furthermore, the present invention provides a food composition, a feed composition and a pharmaceutical composition for preventing or improving obesity, comprising a novel Bacillus coagulans CC strain (KCTC14267BP), a culture solution, a culture filtrate or a fermentation product thereof as an effective ingredient.

Since the above-mentioned food composition, feed composition and pharmaceutical composition for preventing or improving obesity contain the above-mentioned novel Bacillus coagulans CC strain (Bacillus coagulans CC) (KCTC14267BP), in order to avoid repeated content making this specification too complicated, the content repeated with the strain of the present invention in the above content will be omitted.

In addition to containing effective ingredients, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, just like conventional food compositions.

For example, the above-mentioned natural carbohydrates include: monosaccharides, i.e. glucose, fructose, etc.; disaccharides, i.e. maltose, sucrose, etc.; and polysaccharides, i.e. dextrin, cyclodextrin, etc., in addition to this conventional sugar, there are also sugar alcohols of xylitol, sorbitol, and erythritol. The above-mentioned flavoring agent can effectively use natural flavoring agents (thaumatin), stevia extracts (such as rebaudioside A, glycyrrhizic acid glycosides, etc.) and synthetic flavoring agents (saccharin, aspartame sugar substitutes, etc.). The food composition of the present invention can be formulated in the same manner as the above-mentioned pharmaceutical composition to be used as a functional food, or can be added to various foods. For example, the food to which the composition of the present invention can be added includes beverages, meats, chocolates, foods, biscuits, pizzas, instant noodles, other noodles, chewing gums, sugars, ice creams, alcoholic beverages, vitamin complexes, and health supplements, etc.

In addition to the extract as an effective ingredient, the food composition may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers (cheese, chocolate, etc.), pectin acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerol, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp used to make natural fruit juices, fruit juice beverages, and vegetable beverages.

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, "health functional food composition" refers to food prepared and processed by using raw materials or ingredients with effective human functions as specified in Law No. 6727 on Health Functional Foods, and refers to intake for the purpose of obtaining effective results in health care such as regulating nutrients or playing physiological roles in the structure and function of the human body. The health functional food of the present invention may contain ordinary food additives. Unless otherwise specified, whether it is suitable for use as a food additive will be judged according to the specifications and standards of the relevant categories based on the general provisions and general experimental methods in the food additive specifications approved by the Korean Food and Drug Safety Administration. For example, the categories included in the above-mentioned "Food Additive Specifications" may include: chemical synthetics such as ketones, glycine, calcium citrate, niacin, cinnamic acid, etc.; natural additives such as persimmon pigment, hay extract, microcrystalline cellulose, sorghum color, guar gum, etc.; mixed preparations such as L-glutamine sodium preparations, flour-added alkali agents, preservative preparations, and tar pigment preparations. For example, the health functional food in tablet form can be formed in the following manner, that is, after the mixture of the active ingredients, excipients, binders, disintegrants and other additives of the present invention is granulated by conventional methods, a lubricant is added for compression molding, or the above mixture is directly compressed. In addition, the health functional food in the form of tablets may also contain bittering agents, etc. as needed. The hard capsules in the health functional food in the form of capsules can be made by filling ordinary hard capsules with a mixture of additives such as the active ingredients, excipients, etc. of the present invention, and the soft capsules can be made by filling a mixture of additives such as the active ingredients, excipients, etc. of the present invention into a gelling matrix such as gelatin. As needed, the soft capsules may contain plasticizers such as glycerol or sorbitol, colorants, preservatives, etc. The health functional food in the form of pills can be made by molding a mixture of the active ingredients, excipients, binders, disintegrants, etc. of the present invention by existing known methods, and can be skinned by white sugar or other skinning agents as needed, or can be coated with starch, talcum powder, etc. on the surface. The health functional food in the form of granules can be prepared by mixing the active ingredient of the present invention, excipients, binders, disintegrants, etc. into granules by a conventionally known method, and can contain flavoring ingredients, bittering agents, etc. as needed.

The feed composition of the present invention has the effects of suppressing obesity, lowering blood sugar, etc., and can be expected to significantly improve the health of animals. The feed composition of the present invention can be prepared in the form of fermented feed, formulated feed, pellet form, and silage. The above-mentioned fermented feed can be prepared by adding the Bacillus coagulans CC of the present invention to ferment organic matter, and the formulated feed can be prepared by mixing a variety of ordinary feeds, spores of the Bacillus coagulans CC strain of the present invention, and fermented products. Feed in the form of pellets can be prepared by applying heat and pressure to the above-mentioned formulated feed in a pellet machine, and silage can be prepared by fermenting green feed with the microorganisms of the present invention. Wet fermented feed can be prepared in the following manner, that is, after organic matter such as kitchen waste is collected and transported, it undergoes a sterilization process and is mixed with an excipient for adjusting the moisture content in a prescribed ratio, and then fermented at an appropriate fermentation temperature for more than 24 hours, and adjusted to a moisture content of about 70%. Fermented dry feed can be prepared by adjusting the moisture content of wet fermented feed to 30% to 40% by subjecting the wet fermented feed to an additional drying step.

The feed composition of the present invention may also contain ingredients that have been added to feed in the past. For example, such ingredients added to feed include cereal powder, meat powder, and beans. The cereal powder may be one or more selected from rice flour, flour, barley flour, and corn flour. The meat powder may be one or more selected from chicken, beef, pork, and ostrich meat. The beans may be one or more selected from soybeans, green beans, peas, and black beans.

In order to improve the nutrition of feed, in addition to the cereal powder, meat powder and beans mentioned above as ingredients added to existing feed, the feed composition of the present invention may also be added with one or more selected from nutrients and inorganic substances. In order to prevent the quality of feed from deteriorating, it may contain one or more selected from antifungal agents, antioxidants, anticoagulants, emulsifiers and binding agents.

The pharmaceutical composition of the present invention can be prepared in the form of mixing an effective ingredient into a pharmaceutically acceptable carrier. Wherein, the pharmaceutically acceptable carrier includes a carrier, an excipient and a diluent commonly used in the pharmaceutical field. The pharmaceutically acceptable carrier that can be used for the pharmaceutical composition of the present invention is not limited thereto, and can include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated and used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods.

In terms of formulation, it can be made by using a diluent or excipient such as a commonly used filler, extender, binder, wetting agent, disintegrant, surfactant, etc. Oral solid preparations include tablets, pills, powders, granules, capsules, etc., and this solid preparation can be made by mixing more than one excipient with the active ingredient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talcum powder can also be used. Oral liquid preparations are suspensions, liquid preparations, oils, syrups, etc., and in addition to water and liquid paraffin as commonly used diluents, they can also include a variety of excipients, such as wetting agents, sweeteners, aromatics, preservatives, etc.

The above-mentioned pharmaceutical composition can be formulated into various oral dosage forms.

For example, oral dosage forms include tablets, pills, hard capsules, soft capsules, liquids, suspensions, emulsions, syrups, granules, etc. In addition to the active ingredients, these dosage forms may also contain diluents (e.g., lactose, glucose, sucrose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g., silicon dioxide, talc, stearic acid and its magnesium or calcium salt and/or polyethylene glycol). In addition, the above-mentioned tablets may contain binders such as magnesium aluminum silicate, starch ointment, gelatin, tragacanth gum, methylcellulose, sodium carboxymethyl cellulose and/or polyvinyl pyrrolidone, and may contain disintegrants such as starch, agar, alginic acid or its sodium salt or similar mixtures and/or absorbents, colorants, flavoring agents and sweeteners, etc., depending on the circumstances. The above-mentioned dosage forms can be prepared by ordinary mixing, granulation or coating methods.

Furthermore, the present invention provides a food composition, a feed composition and a pharmaceutical composition for preventing or improving hyperglycemia and diabetes, comprising a novel Bacillus coagulans CC strain (KCTC14267BP), a culture solution, a culture filtrate or a fermentation product thereof as an effective ingredient.

Since the above-mentioned food composition, feed composition and pharmaceutical composition for preventing or improving hyperglycemia and diabetes contain the above-mentioned novel Bacillus coagulans CC strain (Bacillus coagulans CC) (KCTC14267BP), in order to avoid repeated content making this specification too complicated, the content repeated with the strain of the present invention in the above content will be omitted.

The present invention provides a method for preventing or treating obesity, comprising the step of administering the above-mentioned pharmaceutical composition for preventing or treating obesity.

The pharmaceutical composition of the present invention can be administered in a therapeutically effective amount or a pharmaceutically effective amount.

The term "pharmaceutically effective amount" as used in the present invention refers to an amount that can fully treat the disease at a reasonable benefit/risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors such as individual type and severity, age, gender, drug activity, sensitivity to the drug, administration time, administration route and excretion rate, treatment time, concurrent drugs used, and other factors well known in the medical field.

Furthermore, the administration method of the pharmaceutical composition is not particularly limited, and for example, the pharmaceutical composition may be applied to the skin, injected intradermally, injected subcutaneously, or the like.

Furthermore, the "individual" of the present invention means a subject for whom disease prevention and regulation or treatment methods are required, and more specifically, means a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc. mammals. Depending on the circumstances, humans may be excluded.

DETAILED DESCRIPTION

The present invention will be described in more detail below by way of examples. These examples are provided to more specifically illustrate the present invention, but the scope of the present invention is not limited to these examples.

<Example 1> Exploration of strains

About 800 hays collected from various places in South Korea were used as samples to isolate microorganisms. After adding a small amount of sterilized saline to each sample and suspending it, it was heat-treated for 20 minutes in a constant temperature water tank at a temperature of 80°C, and the spore liquid obtained was smeared on a plate medium containing 1.5% agar of bromocresol purple plate count agar (BCP Plate Count Agar) and anaerobically cultured for 2 days in an incubator at a temperature of 55°C to isolate the bacteria that form the bacterial colony. In order to investigate the production of α-glucosidase inhibitors, the isolated strains were inoculated into 5 ml of a medium suspended with 5% soybean powder, and shaken culture was performed at a temperature of 37°C for 24 hours to obtain a culture solution. The α-glucosidase inhibitory activity was determined from the upper layer by centrifuging the above culture solution.

<Example 2> α-glucosidase inhibition activity measurement

α-Glucosidase was extracted from pig intestine and partially tableted as an enzyme source. α-Glucosidase enzyme solution was appropriately diluted with 0.5M phosphate buffer solution (pH 7.0) to reach 24unit/ml, and p-nitrophenylα-D-glucopyranoside (pNPG) was used as a substrate. After 30μl of culture supernatant and 50μl of enzyme were mixed in the test tube and pre-incubated at 37°C for 10 minutes, 50μl of 3mM pNPG was added and the reaction was carried out at 37°C for 20 minutes. 0.1M Na ₂ CO ₃ was added to the reaction solution to stop the reaction and the absorbance was measured at 405nm with a spectrophotometer to determine the α-glucosidase inhibitory activity. In this case, the inhibitor was replaced with water as a control group to calculate the inhibitory rate according to Mathematical Formula 1.

Mathematical formula 1

Obstruction rate (%) = [(A-B)/A] × 100

In the above mathematical formula, A represents the absorbance of the control group, and B represents the absorbance of the sample containing the inhibitor.

In this case, the α-glucosidase inhibition unit (AGI unit) is determined based on the amount of the inhibitor that reduces the α-glucosidase used in the activity assay by 1%.

<Example 3> Screening of strains

After about 50 kinds of bacteria were screened as α-glucosidase inhibitor-producing bacteria for the first time through the process of Example 2, the culture solution of the strain that can be anaerobically cultured at 55°C and cannot be cultured in a medium containing propionic acid was centrifuged to measure the α-glucosidase inhibitory activity from the supernatant, thereby screening for three kinds of strains with high α-glucosidase inhibitory activity for the second time. Among the above three strains, they were classified as Bacillus coagulans according to the taxonomic characteristics analysis method based on Bergey's Manual of Systematic Bacteriology (Bergey's Manual of Systematic Bacteriology, 2002), and finally DH-128 bacteria with high α-glucosidase inhibitor production rate and easy spore formation were screened (see Table 1).

Table 1

Strains AGI (unit/ml) The simplicity of spore formation DH-128 2986 +++ DH-204 1824 + HS-061 2075 +++

<Example 4> Inducing mutation of DH-128 strain

<4.1> Screening of novel Bacillus coagulans CC and confirmation of its characteristics

After inducing mutations by treating the DH-128 strain with nitrosoguanidine (100 μg/ml) to achieve a killing rate of 99.9%, the strains were smeared on BCP Plate Count Agar plates at a rate of 200 to 300 per plate, and then cultured at 40°C for 2 days. The resulting population was randomly inoculated into a 5% soybean powder medium, shaken and cultured at 40°C for 2 days, and the supernatant was centrifuged to measure the α-glucosidase inhibitory activity. Five strains with increased α-glucosidase inhibitory activity were screened to screen DH-128-46Y93 considering spore formation ability and α-glucosidase inhibitory activity. The DH-128-46Y93 strain was repeatedly mutated by the method as described above, and a strain with high α-glucosidase inhibition activity and good spore formation ability was selected and named Bacillus coagulans CC strain (Bacillus coagulans CC), which was entrusted to the Bioresource Center (KCTC) of the Korea Institute of Biotechnology (Deposit No. KCTC14267BP) for preservation on August 6, 2020.

As control strains, Bacillus subtilis DC-15, which is a natto strain producing an α-glucosidase inhibitor, and Bacillus coagulans ATCC 7050, which is a strain not producing an α-glucosidase inhibitor, were used. Table 2 shows the α-glucosidase inhibitory activity produced by aerobic or anaerobic cultivation of mutant strains DH-128, DH-128-46Y93, and Bacillus coagulans CC at 40°C in a 5% soybean powder medium for 2 days.

Table 2

As shown in Table 2 above, the α-glucosidase inhibitory activity of Bacillus coagulans CC strain was greatly increased by sequentially cultivating mutations from DH-128 as the screening strain. Bacillus subtilis DC-15 as the control strain is an aerobic bacterium used for natto production. It produces a considerable amount of α-glucosidase inhibitors under aerobic conditions, but it cannot be produced at all under anaerobic conditions. Bacillus coagulans ATCC 7050 does not produce α-glucosidase inhibitors in both aerobic and anaerobic environments. Therefore, the Bacillus coagulans CC strain has the ability to produce α-glucosidase inhibitors in anaerobic environments such as the intestines, which is very different from other aerobic bacteria that produce α-glucosidase inhibitors.

<4.2> Determine the morphological and biochemical characteristics of the isolated Bacillus coagulans CC strain

The colonies formed on the L-broth agar plate medium of the Bacillus coagulans CC strain cultivated through the above separation process are very small and flat, with rounded edges and a matte white surface. The temperature that affects the growth of the bacteria is in the range of 25°C to 40°C, and good growth is shown in this range. No bacterial growth is confirmed above 60°C. The reducing power of nitrate is positive, and the Voges-Proskauer test is negative using citric acid. In addition, it decomposes sodium caseinate, gelatin, and starch, generates acid from glucose, does not generate acid from arabinose, xylose, and mannitol, is catalase positive, and will grow under aerobic and anaerobic conditions. The results of microscopic observation during the logarithmic growth phase of bacterial growth using L-broth liquid medium showed that it was similar to the comparative strain Bacillus coagulans with a short rod-shaped appearance and was Gram-positive.

Furthermore, when the biochemical characteristics of the isolated strain were investigated using the API50CHB kit (BioMerieux, France), it was confirmed that the strain possessed the biochemical characteristics as shown in Table 3 (API test results).

Table 3

The results of the taxonomic characteristics analysis of Bacillus strains based on Bergey's Manual of Systematic Bacteriology (2002) are summarized in Table 4. Bacillus coagulans CC was confirmed to have the following 16s rRNA sequence of SEQ ID NO: 1.

Table 4

[Serial No. 1]

CTCACCAAGGCAACGATGCGTAGCCGACCCGAGAAGGGGAGCGGCCACATTGGGACTGAGACACGGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCCACCGCCGCGTGAGTGAAGAAGGCCTTCGGGGCGTAAAAACTCCGTTGCCGGGGGAAGAACAAGGGCCGTTCCAACAGGGCGGGGCCTTTGGCGGTACCGGGCCAAAAAGGCCCGGGTCACCTCC TGTCCCCACAAGCCGCGCGTAATACTGAGGGGGGAAAGGGTTTCTCGCGAAAATTTGGGGGTGAAAAAGCGGCCCGCGGCGGGGTTTTTAAAATTTGTGTGGGTTAAAATTTTTGGCGCCCCCCCCCGGCGGGCGTTTTTTAAAACGGGGGGGGGCTGTGTGGAAAAAAGAGAAAGGGTATAAATCTCCCGTATTGTTGTGGAAAAAGTATATAAAATGTGGGAAAAACACAAGGGCGGAGGGGGCCTCGGTGGAAA

<Example 5> Analysis of 1-deoxynojirimycin produced by Bacillus coagulans CC strain

The culture solution of Bacillus coagulans CC strain cultured at 37°C for 48 hours in a 5% soy flour medium was centrifuged, and after developing the upper layer by thin layer chromatography (TLC), the upper layer was scraped out at intervals of 0.5 cm and extracted with water to determine the α-glucosidase inhibitory activity. The α-glucosidase inhibitory activity was present at the same position as 1-deoxynojirimycin, and the main component of the α-glucosidase inhibitor was provisionally determined to be 1-deoxynojirimycin. In order to further confirm 1-deoxynojirimycin, it was analyzed by high performance liquid chromatography (HPLC). After inoculating Bacillus coagulans CC strain into 5 ml of 5% soy flour medium, anaerobically cultured at 37°C for 48 hours. 1 ml of the sample was added with 20 μl of 0.4 M potassium borate buffer (pH 8.5) and 20 μl of 5 mM 9-fluorophenylmethyl chloroformate dissolved in acetonitrile, and the reaction was performed at room temperature for 30 minutes under light shielding, and the sample was compared with the standard 1-deoxynojirimycin derivative. 20 μl of 0.1 M glycine was added to stop the reaction, and the sample was used as a high performance liquid chromatography (HPLC) analysis sample. The column used for high performance liquid chromatography (HPLC) analysis was an Agilent TC-C18 column (250 x 4.60 mm, 5 μm), with a mobile phase of acetonitrile:0.1% acetic acid = 35:65, a flow rate of 1.2 ml/min, an oven temperature of 40°C, and detection at 254 nm. As shown in FIG1 , the product of the Bacillus coagulans CC strain and 1-deoxynojirimycin showed the same retention time, confirming that they were the same substance.

<Example 6> Feed experiment of Bacillus coagulans CC strain

In order to confirm whether the Bacillus coagulans CC strain effectively produces α-glucosidase inhibitors in the intestines of animals, spores of the Bacillus coagulans CC strain were orally administered during the feeding of mice, and the proliferation of the strain in the intestines and whether it was excreted to the body, whether α-glucosidase inhibitors were produced in the intestines, and changes in body weight were examined. For this purpose, male mice (3 weeks old) that were fed 1 week in advance were provided with standard feed and water sold on the market freely and used. In terms of light and dark cycles, the time period from 08:00 to 20:00 was used as the light period, and the time period from 20:00 to 08:00 was used as the dark period, and the temperature was maintained at 22.5±0.5℃ and the humidity was maintained at 50% to 60%. The feed was prepared according to the composition in Table 5. The mice were divided into 5 groups (8 each) including a standard feed group, a high carbohydrate feed group, a high carbohydrate feed + CC spore group, a high fat feed group, and a high fat feed + CC spore group and were fed for 12 weeks. For the high carbohydrate diet + CC spore group and the high fat diet + CC spore group, Bacillus coagulans CC spores were diluted in 100 μl of saline solution to give a body weight of 10 ⁹ cells/kg and administered every other day. For the standard diet group, the high carbohydrate diet group, and the high fat diet group, 100 μl of saline solution was used instead of the spore dilution solution and administered every other day. During the feeding process, feces were collected regularly to serve as samples for measuring the α-glucosidase inhibitory activity in the feces and the excreted Bacillus coagulans CC strain.

Table 5

<6.1> Analysis of the effect of administration of Bacillus coagulans CC spores on body weight in mice provided with a high carbohydrate diet

The results of analyzing the effect of administration of CC strain spores of Bacillus coagulans on the body weight of mice fed a high-carbohydrate diet, as shown in FIG2, showed that the high-carbohydrate diet group showed a significant increase in weight compared to the standard diet group, but in the high-carbohydrate diet + CC spore administration group, the increase in weight was significantly less than that of the high-carbohydrate diet group. In the 11th week of the experiment, CT images were taken of the mice in each group and the fat distribution in the body was observed. In the results of FIG3, the bright part indicated by the scissors in the photo represents the fat layer, and it can be seen that a large amount of fat is accumulated in the chest and abdomen when a high-carbohydrate diet is provided compared to the standard diet. On the other hand, in the high-carbohydrate diet group administered with CC strain spores of Bacillus coagulans, the accumulation of body fat was significantly reduced, showing a trend similar to that of the standard diet, indicating that the administration of CC strain spores of Bacillus coagulans effectively inhibited the increase in weight and the accumulation of body fat.

<6.2> Effect of administration of Bacillus coagulans CC spores on body weight in mice fed a high-fat diet

The results of analyzing the effect of administration of CC strain spores of Bacillus coagulans on the body weight of mice fed a high-fat diet are shown in FIG4. Compared with the standard diet group, the high-fat diet group showed a significant increase in weight, but showed the same trend as the high-carbohydrate diet + CC spore administration group, and the increase in weight was significantly less than that of the high-fat diet group. In the 11th week of the experiment, CT scans were taken of mice in each group of the high-fat diet group and the distribution of body fat was observed. In the results of FIG5, the accumulation of fat was lighter than that of the high-carbohydrate diet, but compared with the standard diet, the high-fat diet showed a large amount of fat accumulated in the chest and abdomen, but it was shown that the administration of CC strain spores of Bacillus coagulans effectively suppressed the increase in weight and the accumulation of body fat.

<Example 7> Analysis of feces

During the above-mentioned feeding experiment of Bacillus coagulans CC strain, feces were collected regularly to measure the amount of α-glucosidase inhibitor and Bacillus coagulans CC strain excreted through feces. The α-glucosidase inhibitor was analyzed by measuring the α-glucosidase inhibitory activity, and the number of Bacillus coagulans CC strains was measured as follows, that is, sterile water was added to a predetermined amount of feces to make a good suspension, the supernatant was spread on a bromocresol purple (BCP) plate medium, and then anaerobically cultured at a temperature of 55°C for 2 days, and then the number of colonies present was measured.

<7.1> Changes in Bacillus coagulans CC strains excreted in feces

The results of analyzing the changes in the strains excreted through feces by measuring the number of spores contained in the feces of mice administered with spores of the Bacillus coagulans CC strain are as shown in FIG6 . The amount of bacteria excreted continued to increase until the 5th week, and then maintained at 107 per 1g of feces. The administered Bacillus coagulans CC strain proliferated in the intestines, and it was judged that a part of it was excreted from the body. On the other hand, it gradually decreased from the 8th week when the administration of the bacteria was stopped, showing that the Bacillus coagulans CC strain did not adhere to the intestines and did not stay for a long time. Therefore, if the administration of this strain is interrupted, it is expected that the bacterial colonies in the intestines will change, and it can be confirmed that there is an advantage that this strain can be removed from the intestines as needed. In the feces of mice provided with standard feed, no bacterial colonies were observed, and the bacterial colonies that appeared were identified as Bacillus coagulans CC strains.

<7.2> Changes in α-glucosidase inhibitory activity excreted in feces

The inhibitory activity of α-glucosidase excreted by the feces of mice administered with spores of Bacillus coagulans CC strain. As shown in FIG7 , it can be confirmed that Bacillus coagulans CC strain (KCTC14267BP) proliferates vigorously in the intestine to the extent that it can be excreted through feces, and produces α-glucosidase inhibitors. As shown in FIG6 , it is confirmed that the amount of Bacillus coagulans CC strain excreted from the 5th week to the 7th week is stable, and the amount of α-glucosidase inhibitor excreted during this period is also constant. That is, since the α-glucosidase inhibitor is always produced in the intestine, the concentration is maintained at a constant amount in the intestine, which has the advantage of high efficiency of action, and it can be known that the constant amount of the inhibitor produced is excreted.

From the above results, it is shown that the Bacillus coagulans CC strain of the present invention can continuously produce and supply physiologically active substances in vivo, rather than administering physiologically active substances produced by industrial fermentation into the body. This will not require separate fermentation costs and processing costs for producing α-glucosidase inhibitors, thereby reducing production costs, and can be continuously produced in vivo, and it can be confirmed that the effective concentration can be maintained in the small intestine where the α-glucosidase inhibitor takes effect. In addition, since spores of the Bacillus coagulans CC strain are used, there will be no problems with fermented smell, volume, color, etc. during product processing, and there is an advantage that it can be used in various forms of food.

[Accession number]

Name of depository institution: Korea Institute of Biotechnology

Accession number: KCTC14267BP

Date of preservation: 2020.08.06

Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure International Form

Recipient: Li Haiyi Confirm at the bottom of the page

Chuncheon, Gangwon Province, South Korea

Sheting Road, 26 According to the international authorization,

Deposit recognition permitted under Article 7.1 of the Treaty

SEQUENCE LISTING

<110> Li Haiyi

<120> A novel Bacillus coagulans CC strain producing alpha-glucosidase inhibitor

<130> P23111855WP

<150> KR 10-2020-0128609

<151> 2020-10-06

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1534

<212> DNA

<213> Artificial Sequence

<220>

<223> 16s rRNA of Bacillus coagulans CC

<400> 1

ccgttggggt ctcccagcgg agtgcttaat gcgttagctg cagcactaaa gggcggaaac 60

cctctaacac ttagcactca tcgtttacgg cgtggactac cagggtatct aatcctgttt 120

gctccccacg ctttcgcgcc tcagcgtcag ttacagacca gagagccgcc ttcgccactg 180

gtgttcctcc acatctctac gcatttcacc gctacacgtg gaattccact ctcctcttct 240

gcactcaagc ctcccagttt ccaatgaccg cttgcggttg agccgcaaga tttcacatca 300

gacttaagaa gccgcctgcg cgcgctttac gcccaataat tccggacaac gcttgccacc 360

tacgtattac cgcggctgct ggcacgtagt tagccgtggc tttctggccg ggtaccgtca 420

aggcgccgcc ctgttcgaac ggcacttgtt cttccccggc aacagagttt tacgacccga 480

aggccttctt cactcacgcg gcgttgctcc gtcagacttt cgtccattgc ggaagattcc 540

ctactgctgc ctcccgtagg agtttgggcc gtgtctcagt cccaatgtgg ccgatcaccc 600

tctcaggtcg gctacgcatc gttgccttgg tgagccgtta ccccaccaac tagctaatgc 660

gccgcgggcc catctgtaag tgacagccga agccgtcttt cctttttcct ccatgcggag 720

gaaaaaacta tccggtatta gccccggttt cccggcgtta tcccgatctt acaggcaggt 780

tgcccacgtg ttactcaccc gtccgccgct aaccttttaa aagcaagctt ttaaaaggtc 840

cgcacgactt gcatgtatta ggcacgccgc cagcgttcgt cctgagcaga gaacaaaacc 900

caaaaaaagg ggccaatacc gggaaagttt attcctccgc ctgggaggag aaaggaaaga 960

cggcatcggc gggcccctac agaagggccc gcggcgcaat agccagttgg gcggggaacg 1020

gctcaccaag gcaacgatgc gtagccgacc cgagaagggg agcggccaca ttgggactga 1080

gacacgggcc caaactccta cgggaggcag cagtagggga atcttccgca atggacgaaa 1140

gtctgacgga gccaccgccg cgtgagtgaa gaaggccttc ggggcgtaaa aactccgttg 1200

ccgggggaag aacaagggcc gttccaacag ggcggggcct ttggcggtac cgggccaaaa 1260

aggcccgggt cacctcctgt ccccacaagc cgcgcgtaat actgaggggg gaaagggttt 1320

ctcgcgaaaa tttgggggtg aaaaagcggc cccgcggcgg ggtttttaaa tatgtgtggg 1380

ttaaaatttt tggcgccccc ccccggcggg cgttttttaa aacggggggg ggctgtgtgg 1440

aaaaaagaga aagggtataa atctcccgta ttgttgtgga aaaagtatat aaaatgtggg 1500

aaaaacacaa gggcggaggg ggcctcggtg gaaa 1534

Claims (14)

1. A Bacillus coagulans CC strain, characterized in that it belongs to a novel Bacillus coagulans CC strain with the preservation number KCTC14267BP whose production ability of α-glucosidase inhibitors has been improved. 2. The Bacillus coagulans CC strain according to claim 1, characterized in that said strain comprises the base sequence of the 16s rRNA of SEQ ID NO: 1. 3. The Bacillus coagulans CC bacterial strain according to claim 1, wherein said bacterial strain is anaerobic. 4. The Bacillus coagulans CC strain according to claim 1, wherein said strain is excreted from the organism within 2 to 4 weeks after the strain is administered. 5. The Bacillus coagulans CC strain according to claim 1, wherein said strain produces α-glucosidase inhibitors in the intestinal tract. 6. The Bacillus coagulans CC strain according to claim 5, wherein the α-glucosidase inhibitor produced in the intestinal tract is excreted from the living body within 2 to 4 weeks after administration of the strain. 7. The Bacillus coagulans CC strain according to claim 1, characterized in that the α-glucosidase inhibitor is 1-deoxynojirimycin. 8. A food composition for preventing or improving obesity, characterized in that it contains the novel CC strain of Bacillus coagulans with the preservation number KCTC14267BP, its culture solution, culture filtrate or fermented product as an active ingredient. 9. A feed composition for preventing or improving obesity, characterized in that it contains the novel Bacillus coagulans CC strain with the preservation number KCTC14267BP, its culture solution, culture filtrate or fermented product as an active ingredient. 10. A pharmaceutical composition for preventing or treating obesity, characterized in that it contains the novel CC strain of Bacillus coagulans with the deposit number KCTC14267BP, its culture solution, culture filtrate or fermented product as an active ingredient. 11. A food composition for preventing and improving hyperglycemia or diabetes, characterized in that it contains the novel Bacillus coagulans CC strain with the preservation number KCTC14267BP, its culture solution, culture filtrate or fermented product as an active ingredient. 12. A feed composition for preventing and improving hyperglycemia or diabetes, characterized in that it contains the novel Bacillus coagulans CC strain with the preservation number KCTC14267BP, its culture solution, culture filtrate or fermented product as an active ingredient. 13. A pharmaceutical composition for preventing and treating hyperglycemia or diabetes, characterized in that it contains the novel Bacillus coagulans CC strain with the deposit number KCTC14267BP, its culture solution, culture filtrate or fermented product as an active ingredient. 14. A method for preventing or treating obesity, characterized by comprising the step of administering the pharmaceutical composition according to claim 10.

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