KR20230014138A - Roseburia intestinalis strain having efficacy of butyric acid production, efficacy of dietary fiber fermentation and efficacy of strengthening intestinal epithelial barrier and uses thereof - Google Patents

Abstract

The present invention relates to a Roseburia intestinalis strain having butyric acid production, dietary fiber fermentation, and intestinal epithelial barrier reinforcement effects. The strain, a culture solution thereof, a concentrate or a dried product of the culture solution can be used as a probiotic preparation, and can be usefully used for prevention, alleviation or treatment of inflammatory bowel diseases, diabetes or colon cancer.

Description

Roseburia intestinalis strain having efficacy of butyric acid production, efficacy of dietary fiber fermentation and efficacy of strengthening intestinal epithelial barrier and uses it}

The present invention relates to a Roseburia interiorlis strain having butyric acid-producing ability, dietary fiber fermenting ability, and intestinal epithelial barrier strengthening effect, and uses thereof.

Butyric acid has been reported as a metabolite derived from intestinal microbes that is effective in alleviating and treating diseases such as inflammatory bowel disease, diabetes or colon cancer. However, it is known that Lactobacillus genus strains ( Lactobacillus ) or Bifidobacterium genus strains ( Bifidobacterium ), which are traditionally used as health functional foods to improve intestinal health, do not have butyric acid metabolism or have very weak levels.

Therefore, there is a need to develop new probiotic candidate bacteria capable of producing an excessive amount of butyric acid by overcoming the limitations of conventional probiotics preparations. In addition, at the same time, there is a demand for the discovery of probiotics with excellent fermentability for various prebiotic dietary fibers used as food for beneficial intestinal bacteria.

In order to overcome the limitations of conventional probiotics, the present inventors tested the butyric acid-producing ability and dietary fiber fermentation ability of four microorganisms isolated from human feces. By comparison, new probiotic candidate bacteria were discovered.

Korean Patent Registration No. 10-2249897

An object of the present invention is to provide a Roseburia intestinalis strain having butyric acid producing ability, dietary fiber fermenting ability and intestinal epithelial barrier strengthening effect.

Another object of the present invention is to provide a probiotics preparation containing the strain, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient.

Another object of the present invention is to provide a composition for preventing, improving or treating inflammatory bowel disease comprising the strain, its culture medium, or a concentrate or dried product of the culture medium as an active ingredient.

Another object of the present invention is to provide a composition for preventing, improving or treating diabetes comprising the strain, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient.

Another object of the present invention is to provide a composition for preventing, improving or treating colorectal cancer comprising the strain, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient.

Another object of the present invention is to provide a method for producing butyric acid comprising culturing the strain.

In order to achieve the above object, the present invention provides a Roseburia intestinalis strain having butyric acid producing ability, dietary fiber fermenting ability and intestinal epithelial barrier strengthening effect.

In addition, the present invention provides a probiotic preparation comprising the strain, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient.

In addition, the present invention provides a composition for preventing, improving or treating inflammatory bowel disease comprising the strain, its culture medium, or a concentrate or dried product of the culture medium as an active ingredient.

In addition, the present invention provides a composition for the prevention, improvement or treatment of diabetes comprising the strain, its culture medium, a concentrated or dried product of the culture medium as an active ingredient.

In addition, the present invention provides a composition for preventing, improving or treating colorectal cancer comprising the strain, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient.

In addition, the present invention provides a method for producing butyric acid comprising culturing the strain.

Roseburia intestinalis strain according to the present invention is capable of producing butyric acid and fermenting dietary fiber, so it can be used as a probiotics preparation, and in addition, a health function that can induce a human intestinal health promoting effect It can be usefully used for food or medicine.

1 is a result of evaluating the dietary fiber fermentation ability for four human intestinal butyric acid producing bacteria ( Eubacterium rectale , Faecalibacterium prausnitzii , Roseburia hominis , and Roseburia intestinalis ).
Figure 2 is a result of comparative analysis of the butyric acid-producing ability of four types of butyric acid-producing bacteria in the human intestine.
Figure 3 is a result of analyzing the mRNA expression of Claudin 3 protein in intestinal epithelial cells when the cell culture extract of the human intestinal butyric acid-producing strain was treated (***p<0.001; YCFA was the medium control).
Figure 4 is the result of analyzing the mRNA expression of Claudin 3 protein in intestinal epithelial cells when the cell culture extract of the human intestinal butyric acid-producing strain was treated (***p<0.001; YCFA was the control medium).
Figure 5 shows the results of analyzing the mRNA expression of tight junction protein-1 protein in intestinal epithelial cells when the cell culture extract of the human intestinal butyric acid-producing strain is treated (***p<0.001; YCFA is the control medium).
6 is a result of confirming the cytotoxicity to intestinal epithelial cells when the human intestinal epithelial cell line was treated with Roseburia intestinalis probiotics (NS: Not significant).

Hereinafter, the present invention will be described in detail.

The present invention provides a Roseburia intestinalis strain having butyric acid producing ability, dietary fiber fermenting ability and intestinal epithelial barrier strengthening effect.

In one embodiment of the present invention, the strain is a microbial strain distributed from the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center (KCTC), accession number KCTC 15746 Roseburia internalis ( Roseburia intestinalis ) strain can

In addition, the present invention provides a probiotic preparation comprising the strain, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient.

The probiotics preparation may be prepared and administered in various formulations and methods according to methods known in the art. For example, the Roseburia intestinalis strain of the present invention, its culture, the concentrate of the culture or its dried is mixed with a carrier commonly used in the pharmaceutical field to obtain a powder, liquid ( liquids and solutions), tablets, capsules, syrups, suspensions, or granules. The carrier may be, for example, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant and a flavoring agent, but is not limited thereto. In addition, the administration dose can be appropriately selected according to the degree of absorption of the active ingredient in the body, inactivity rate, excretion rate, age, sex, animal species, condition and severity of the disease of the recipient to be administered.

The method of culturing the strain of the present invention may be cultured according to a method commonly used in the art, and is not limited to a particular method.

When the strain or the culture of the strain or the concentrate of the culture of the strain obtained in the step of culturing the strain of the present invention is used as an additive, the strain or the culture of the strain or the concentrate of the culture of the strain is added as it is, or Other additives may be used together, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient can be suitably determined depending on the purpose of its use.

In addition, the present invention provides a health functional food composition for preventing or improving inflammatory bowel disease comprising the strain, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving diabetes comprising the strain, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving colorectal cancer comprising the strain, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient.

The health functional food composition of the present invention may include all foods in a conventional sense, and may be used interchangeably with terms known in the art, such as food composition and functional food.

The term of the present invention, "health functional food" refers to a food manufactured and processed by methods such as extracting, concentrating, refining, mixing, etc., using specific ingredients as raw materials or specific ingredients contained in food raw materials for the purpose of health supplementation. It refers to a food designed and processed to sufficiently exert biological control functions such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc. It can perform related functions, etc.

There are no restrictions on the types of foods in which the composition of the present invention can be used. In addition, the composition of the present invention may be prepared by mixing suitable other auxiliary ingredients that may be included in food and known additives according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and Vitamin complexes, etc., can be prepared by adding the extract and its fractions according to the present invention to juices, teas, jellies, juices, etc. prepared as main components.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (e.g., formula milk, infant, baby food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), health supplement food , seasonings (eg soy sauce, soybean paste, gochujang, mixed paste, etc.), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickled vegetables, etc.) ), beverages (eg fruit, vegetable drinks, soy milk, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweeteners, flavoring agents, or natural carbohydrates as additional components, like conventional beverages. In addition to the above, the health functional food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, and glycerin. , alcohol, a carbonating agent used in carbonated beverages, and the like. In addition, it may contain fruit flesh for the manufacture of natural fruit juice, fruit juice beverages and vegetable beverages.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a strain, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes comprising the strain, its culture medium, a concentrated or dried product of the culture medium as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating colorectal cancer comprising the strain, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means that it exhibits non-toxic properties to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art such as a buffer, a preservative, a pain reliever, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, a lubricant, and the like.

In addition, the pharmaceutical composition of the present invention is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions. can Furthermore, it may be used in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or skin external preparations in the form of gels. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient, for example, starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance into a subject by an appropriate method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, but not limited thereto.

The term "individual" refers to all animals such as rats, mice, livestock, and the like, including humans. Preferably, it may be a mammal including a human.

The term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's gender, age, Factors including body weight, health condition, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and rate of excretion, duration of treatment, drugs used in combination or concurrently, and other medical disciplines. It can be easily determined by a person skilled in the art according to well-known factors. Administration may be administered once a day at the recommended dose, or may be administered in several divided doses.

In addition, the present invention provides a method for producing butyric acid comprising culturing the strain. The method of culturing the strain of the present invention may be cultured according to a method commonly used in the art, and is not limited by a particular method.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. Analysis of dietary fiber fermentability and butyric acid production ability of intestinal microbial strains

Four strains of human intestinal microbes, namely Eubacterium rectale , Faecalibacterium prausnitzii , Roseburia hominis and Roseburia intestinalis ) was performed to confirm the dietary fiber fermentation ability. Briefly, in a 96-well plate, 5 monosaccharides (Arabinose, Fructose, Galactose, Glucose, and Xylose) and 6 dietary fibers (Amylopectin, Arabinoxylan, Galactan, Inulin, Pullulan, and Xylan) were supplied as nutrients, respectively, and absorbance was measured. The growth ability of the strain was evaluated through the measurement.

The Eubacterium rectale , Roseburia hominis , and Roseburia intestinalis strains are microbial strains distributed from the Korea Research Institute of Bioscience and Biotechnology (KCTC). , The deposit numbers are KCTC 5835, KCTC 5845, and KCTC 15746, respectively, in order. Faecalibacterium prausnitzii strain is a strain distributed from the German Center for Microorganisms and Biological Resources (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ), and the accession number is DSM 17677.

As a result, by confirming that Roseburia intestinalis can digest all five types of monosaccharides and digest three types of dietary fiber (Amylopectin, Arabinoxylan and Pullulan), four types of It was confirmed that it could digest the most monosaccharide and dietary fiber among microbial strains (FIG. 1).

In addition, an experiment was performed in which dietary fiber (Amylopectin, Arabinoxylan, Inulin, and Pullulan) was supplied to four human intestinal microbial strains, respectively, and the production of butyric acid at 48 hours after culture was measured.

As a result, among the four microbial strains, it was confirmed that Roseburia intestinalis had a significantly higher butyric acid production ability than other strains (Fig. 2).

Example 2. Intestinal epithelial barrier strengthening efficacy of intestinal microbial strains

The present inventors performed an experiment to confirm the intestinal epithelial barrier strengthening effect on four types of human intestinal microbial strains. Briefly, four human intestinal microbial strains were each cultured for 48 hours, and their cell culture supernatants were extracted using ethyl acetate. The extraction method is as follows. First, 30 mL of each cell culture medium is centrifuged at 3000 rpm for 10 minutes, and 20 mL of the culture supernatant is separated. Add 20 mL of ethyl acetate to the culture supernatant and stir for 5 minutes. Then centrifuged at 3000 rpm for 10 minutes to obtain 10 mL of supernatant. After evaporating the solvent in a centrifugal vacuum concentrator, it is dissolved in 10 mL of DMEM medium (Dulbecco's Modified Eagle's Medium). Then, caco-2 cell, an intestinal epithelial cell line, was cultured for 72 hours by treating 20 μL of the culture supernatant extract with 2 mL of DMEM medium, and then using RT-PCR to detect the cell junction proteins CLDN3 (Claudin 3) and OCLN (Occludin) and TJP1 (tight junction protein-1) mRNA expression changes were measured. Butyrate, used as a positive control, was diluted to a concentration of 1 mM in 2 mL of DMEM. The sequence of the primer used was from PrimerBank in the US, and the synthesis of the primer was carried out through Macrogen in Korea.

As a result, among the four microbial strains, the group treated with the cell culture extract of Roseburia intestinalis CLDN3 (Claudin 3), OCLN ( Occludin) and TJP1 (Tight junction protein-1) mRNA expression levels were significantly high (Figs. 3 to 5).

From the above results, the cell culture extract of Roseburia intestinalis increases mRNA expression of cell junction proteins CLDN3 (Claudin 3), OCLN (Occludin) and TJP1 (Tight junction protein-1), It was confirmed that there is an effect of strengthening the intestinal barrier.

Example 3. Roseburia Intestinalis ( Roseburia intestinalis ) Cytotoxicity analysis of strains

The present inventors performed an experiment confirming the cytotoxicity to Roseburia intestinalis live bacteria. Briefly, Roseburia intestinalis was treated with live bacteria in caco-2 cells and cultured for 12 hours, and then cell viability (cell viablitiy) was measured.

As a result, it was confirmed that the human intestinal flora, Roseburia intestinalis strain, did not affect the cell viability of human intestinal host cells (FIG. 6).

From the above results, it was confirmed that the Roseburia intestinalis strain was not cytotoxic to human intestinal host cells.

Thus, in the present invention, Roseburia intestinalis strain is a strain capable of overproducing butyric acid, and butyric acid is effective in preventing, improving or treating diseases such as inflammatory bowel disease, diabetes, or colorectal cancer Known, it was confirmed that Roseburia intestinalis strain is effective in preventing, improving or treating diseases such as inflammatory bowel disease, diabetes, or colorectal cancer.

Claims (10)

Roseburia intestinalis strain having butyric acid producing ability, dietary fiber fermenting ability and intestinal epithelial barrier strengthening effect. According to claim 1,
The strain is Accession No. KCTC 15746, which is Roseburia Intestinalis Roseburia intestinalis strain. A probiotic preparation comprising the strain of claim 1, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient. A health functional food composition for preventing or improving inflammatory bowel disease, comprising the strain of claim 1, its culture medium, or a concentrate or dried product of the culture medium as an active ingredient. A health functional food composition for preventing or improving diabetes comprising the strain of claim 1, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient. A health functional food composition for preventing or improving colorectal cancer comprising the strain of claim 1, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient. A pharmaceutical composition for preventing or treating inflammatory bowel disease comprising the strain of claim 1, its culture medium, or a concentrate or dried product of the culture medium as an active ingredient. A pharmaceutical composition for the prevention or treatment of diabetes comprising the strain of claim 1, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient. A pharmaceutical composition for preventing or treating colorectal cancer comprising the strain of claim 1, its culture medium, or a concentrated or dried product of the culture medium as an active ingredient. A method for producing butyric acid comprising culturing the strain of claim 1.

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