KR102365420B1 - Roseburia intestinalis strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of thereof - Google Patents

Abstract

The present invention relates to a novel Roseburia intestinalis strain (Accession No: KCTC 14838BP) belonging to the genus Roseburia and a lysate thereof, a culture medium, an extract of the culture medium, an endoplasmic reticulum, and anti-inflammatory and/or antibacterial uses thereof. The novel strain and an endoplasmic reticulum derived therefrom according to an aspect of the present invention have an effect that can be effectively used for the prevention, alleviation, or treatment of inflammation-related conditions, or bacterial infections.

Description

Roseburia intestinalis strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of thereof

It relates to novel microorganisms, their lysates, cultures, extracts of cultures, endoplasmic reticulum and their anti-inflammatory and/or antibacterial uses.

Microbiome refers to microorganisms that exist in a specific environment and their entire genetic information, and refers to a collection of genomes that refer to the entire genetic information of a single organism. Therefore, the human microbiome refers to microorganisms living inside and outside the human body and their entire genetic information.

The human body lives in a symbiotic relationship with many microorganisms, and in particular, the intestine contains the most microorganisms as an optimal environment for the microorganisms to take in nutrients and form a systematic community. Intestinal microorganisms provide nutrients that cannot be produced by the host’s enzymes alone and are deeply related to the host’s metabolism and immune system. It has been reported to be associated with

Recently, due to Western eating habits and indiscriminate use of antibiotics, the intestinal microbiota is imbalanced and intestinal health is deteriorating. .

On the other hand, the endoplasmic reticulum is a nano-sized material of about 20 to 200 nm produced and discharged by cells, and it is free to move between cells. In addition, it is known that the endoplasmic reticulum contains membrane lipids, membrane proteins, DNA or RNA, and the like, and these genetic materials act as a complex to deliver toxic factors between cells and regulate inflammation and immune response. From unicellular organisms to multicellular organisms, information exchange between cells is an essential process of life phenomena, and recently, the endoplasmic reticulum has been recognized as a medium for information exchange between cells, so methods for using the vesicle as a drug carrier have been developed.

Accordingly, there is a need for the development of substances for improving, preventing, or treating diseases using novel microorganisms derived from the human intestine and endoplasmic reticulum derived therefrom.

Korean Patent Publication 10-2018-0012849

One aspect is to provide a Roseburia intestinalis strain belonging to the genus Roseburia sp. , deposited with accession number KCTC 14838BP.

Another aspect is to provide an endoplasmic reticulum derived from the strain, a lysate of the strain, or a culture solution.

Another aspect is inflammatory bowel diseases (IBD) comprising the strain, the endoplasmic reticulum derived from the strain, a lysate of the strain, a culture medium, or a mixture thereof as an active ingredient; irritable bowel syndrome; To provide a pharmaceutical composition for preventing or treating enteritis, Crohn's disease; or ulcerative colitis.

Another aspect is inflammatory bowel disease comprising the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient; irritable bowel syndrome; To provide a functional food for preventing or improving enteritis, Crohn's disease; or ulcerative colitis.

Another aspect is the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient Clostridioides difficile ) Inhibition of intestinal health by inhibition or inflammation inhibition To provide health functional food for improvement.

Another aspect is a pharmaceutical composition for preventing or treating Clostridioides difficile infection (CDI) comprising the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient will provide

Another aspect is to provide a health functional food for the prevention or improvement of Clostridioides difficile infection comprising the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient .

One aspect provides a strain of Roseburia intestinalis belonging to the genus Roseburia sp .

The Roseburia Intestinalis strain may be a strain deposited with accession number KCTC 14838BP.

The Roseburia intestinalis strain may be a strain comprising 16S rRNA of SEQ ID NO: 1.

The strain may have anti-inflammatory and/or antibacterial activity.

The strain inhibits the production of nitric oxide in the inflamed cells, inhibits the expression of inflammatory cytokines (eg, TNF-α or IL-6), or inhibits the proliferation of bacteria (eg, C. difficile ) may be inhibiting In addition, the strain reduces inflammatory factors induced by C. difficile , such as pro-inflammatory cytokines (eg, TNF, or CCL2), or reduces anti-inflammatory cytokines (eg, IL-10). ) may be increased.

Another aspect provides an endoplasmic reticulum derived from a Roseburia intestinalis strain belonging to the genus Roseburia sp. , a lysate of the strain, a culture solution, an extract of the culture solution, or a mixture thereof.

The strain is as described above.

As used herein, the term “vesicle” refers to a particle secreted from a cell and released into the extracellular space, and includes an exosome, an ectosome, a microvesicle, and a microparticle. , exosome-like vesicles, and the like. The extracellular ER can reflect the state of the secreting cell of origin (donor cell), exhibit various biological activities depending on which cell it is secreted from, and play an important role in cell-to-cell interactions by transferring genetic material and proteins between cells can do. In addition, the cell-derived substances including the endoplasmic reticulum cause disease or stimulate immune cells to fight disease, and have the effect of helping to break down and absorb substances that humans cannot digest through the metabolic process of microorganisms. there is. The endoplasmic reticulum is a membrane-structured endoplasmic reticulum, which is divided into an inside and an outside, and has a plasma membrane lipid, a plasma membrane protein, a nucleic acid, and a cytoplasmic component of the cell, and is larger than the original cell. may be small.

In one embodiment, the endoplasmic reticulum may be isolated from the cell lysate of the culture medium of the Roseburia intestinalis strain.

In one embodiment, the extracellular vesicles may have a diameter of 10 nm to 400 nm. For example, it may be 10 nm to 400 nm, 10 nm to 350 nm, 10 nm to 300 nm, or 10 nm to 250 nm.

As used herein, the term "culture medium" may be used interchangeably with "culture supernatant", "conditioned culture medium" or "conditioned medium", and provides nutrients so that Roseburia intestinalis can grow and survive in vitro. It may mean the entire medium including the strain obtained by culturing the strain for a certain period of time in a medium that can be supplied, its metabolites, extra nutrients, and the like. In addition, the culture solution may refer to a culture solution obtained by culturing the strain in which the cells are removed from the culture solution. On the other hand, the liquid from which the cells have been removed from the 　 culture medium is also called a "supernatant", and the 　 culture solution is left for a certain period of time to take only the liquid from the upper layer except for the part that has sunk to the bottom layer, remove the cells through filtration, or 　 centrifuge the culture solution to the lower part It can be obtained by removing the precipitation of and taking only the liquid at the top. The "cell" of the present invention refers to the strain itself of the present invention, and includes the strain isolated and selected from a skin sample, or the strain isolated from the culture solution by culturing the strain. The cells can be obtained by centrifuging the 　 culture medium to take the part that has sunk to the lower layer, or by gravity 　 sink to the lower layer of the culture solution, leaving it still for a certain period of time and then removing the upper liquid It can be obtained.

The culture medium may include the culture medium itself obtained by culturing the strain, its concentrate, or a lyophilisate or a culture supernatant obtained by removing the strain from the culture medium, its concentrate or lyophilisate.

The culture medium may be obtained by culturing Roseburia intestinalis in an appropriate medium (eg, R2A medium or TSA medium) at any temperature above 10° C. or below 40° C. for a certain period of time, for example, 4 to 50 hours. may be obtained.

In one embodiment, the culture supernatant of the strain may be obtained by centrifuging or filtering the strain culture to remove the strain.

In another embodiment, the concentrate may be obtained by concentrating the supernatant obtained after filtering the strain culture solution itself, or the culture solution using centrifugation or a filter.

The culture medium and culture conditions for culturing the Roseburia intestinalis may be appropriately selected or modified by those of ordinary skill in the art.

As used herein, the term "lysate" may refer to a product obtained by crushing the cell wall of the strain itself by chemical or physical force.

As used herein, the term "culture solution extract" means extraction from the culture solution or its concentrate, and may include an extract, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or these prepared or purified products, and a fraction obtained by fractionation thereof. can

Another aspect provides a disease improvement, prevention or treatment use of a Roseburia intestinalis strain, an endoplasmic reticulum derived from the strain, a lysate of the strain, a culture solution, or an extract of the culture solution.

As used herein, the term “treat” may mean that inflammation or bacterial infection is cured in a shorter time compared to natural healing. The treatment may include amelioration and/or alleviation of inflammation or bacterial infection. In addition, the treatment may refer to healing and/or recovery of symptoms caused by inflammation or bacterial infection.

As used herein, the term “prevention” refers to partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or concomitant symptom thereof, preventing the acquisition or reacquisition of the disease or disorder, or preventing the disease or disorder from occurring. How to reduce the risk of acquisition. For example, the prevention refers to any action that suppresses or delays the occurrence of inflammation or bacterial infection by administration of the composition according to the present invention.

The use of the strain may include preventing, ameliorating, or treating an inflammatory disease (anti-inflammatory activity), preventing, ameliorating, or treating a bacterial infection (antibacterial activity), or preventing or improving intestinal health.

The inflammatory disease may include inflammation of the digestive system (gastrointestinal tract, etc.), intraocular inflammation, oral inflammation, respiratory system inflammation including lung, skin inflammation, cardiovascular inflammation, brain inflammation, and ear inflammation. there is.

More specifically, the inflammatory disease is inflammatory bowel disease (IBD); irritable bowel syndrome; Behcet's disease; enteritis, Crohn's disease; ulcerative colitis; vasculitis; mucositis; stomatitis; peri-implantitis; periodontitis; pulpitis; gingivitis; Pneumonia; dermatitis; atopic dermatitis; contact dermatitis; CREST syndrome; dermatitis herpetiformis; dermatomyositis; systemic scleroderma; erythema nodosum; Henoch-Schonlein purpura; Hidradenitis suppurativa; lichen planus; Majeed syndrome; Schnitzler syndrome; psoriasis; eczema; acne; mouth ulcers; uveitis; pharyngitis; tonsillitis; otitis, including otitis media; arthritis (psoriatic arthritis); synovitis; meningitis; encephalitis; Bickerstaff's encephalitis encephalomyelitis; spondylitis; osteomyelitis; Guillain-Barre syndrome; myelitis; neuromyelitis optica; cystitis; Acute inflammation at the site of an infection or wound; nephritis; And it may be any one selected from the group consisting of glomerulonephritis.

In addition, the improvement of intestinal health may be a help in intestinal beneficial growth and suppression of harmful bacteria, help in intestinal health by regulating immunity, or help in smooth bowel movements.

The term “antibacterial agent,” as used herein, refers to (i) inhibiting, reducing or preventing the growth of bacteria; (ii) inhibit or reduce the ability of the bacteria to cause infection in a subject; or (iii) a substance capable of inhibiting or reducing the ability of a bacterium to multiply or maintain infectivity in the environment.

Examples of the bacterial infection may include an infection caused by gram-positive bacteria or gram-negative bacteria. Specifically, the bacterial infection is Clostridioides ( Clostridioides ), Helicobactor ( Helicobactor ), Escherichia ( Escherichia ), Salmonella ( Salmonella ), Staphylococcus ), Streptococcus ( Streptococcus ), Haemophilus ( Haemophilus , Klebsiella , Moraxella , Enterobacter , Proteus , Serratia , Pseudomonas , Acinetobacter , Citrobacter Citrobacter ), Stenotrophomonas ), Bacteroides ), Prevotella ( Prevotella ), and may include infections caused by bacteria belonging to the genus Fusobacterium ( Fusobacterium ). More specifically, the bacterial infection is Clostridioides difficile infection (CDI), or Clostridioides difficile associated disease (CDAD), such as Clostridioides difficile associated disease (CDAD), for example, Clostridioides difficile associated diarrhea ( Clostridioides difficile associated diarrhea).

The composition comprises 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt% with respect to the total weight of the composition %, 0.00001% to 10% by weight, 0.00001% to 5% by weight, 0.05% to 60% by weight, 0.05% to 40% by weight, 0.05% to 30% by weight, 0.05% to 20% by weight, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1% by weight % to 10% by weight, or 0.1% to 5% by weight of the strain, a lysate thereof, a culture medium, or an extract of the culture medium thereof.

The term, "included as an active ingredient" means that the strain of the present specification, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or the extract of the culture medium is added to the extent that it can exhibit the above-mentioned effects, For drug delivery and stabilization, it is meant to include formulations in various forms by adding various components as sub-components.

In another embodiment, the composition may be a pharmaceutical composition.

The types of pharmaceutically active ingredients capable of delivering the active ingredient into an individual include anticancer agents, contrast agents (dyes), hormones, antihormones, vitamins, calcium agents, inorganic agents, saccharides, organic acid preparations, protein amino acid preparations, detoxifying agents, enzymes Agents, metabolic agents, diabetic combination agents, tissue revitalization agents, chlorophyll agents, pigment agents, tumor agents, tumor agents, radiopharmaceuticals, tissue cell diagnostic agents, tissue cell therapies, antibiotic agents, antiviral agents, combined antibiotics, chemicals Therapeutic agents, vaccines, toxins, toxoids, antitoxins, leptospirin serum, blood products, biologics, analgesics, immunogenic molecules, antihistamines, allergens, non-specific immunogenic agents, anesthetics, stimulants, psychoneurologic agents, small molecule compounds, nucleic acids, aptamers, antisense nucleic acids, oligonucleotides, peptides, siRNAs and microRNAs, and the like.

The pharmaceutical composition may further include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be carboxymethyl cellulose calcium, sodium starch glycolate, anhydrous calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

When formulating the pharmaceutical composition, it is usually prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, Witepsol, macrogol, Tween 61, cacao butter, liulinji, glycerogelatin, etc. may be used.

The pharmaceutical composition may include carbohydrates such as glucose, sucrose or dextran, antioxidants such as Ascorbic acid or Glutathione, chelating agents, and low molecular weight proteins to increase stability or absorption. Alternatively, other stabilizers may be used as medicaments.

The pharmaceutical composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or a combination thereof. The parenteral dosage form may be an injection.

The composition may be a health functional food composition.

The health functional food composition may be used alone or in combination with other foods or food ingredients of the strain or its culture, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic treatment). In general, in the production of food or beverage, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw material. There is no particular limitation on the type of the health functional food. Among the types of health functional food, the beverage composition may contain various flavoring agents or natural carbohydrates as an additional component like a conventional beverage. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumartin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The health food composition can also be added to nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages carbonation agent used, or a combination thereof. The health functional food composition may also contain natural fruit juice, fruit juice beverage, fruit flesh for the production of vegetable beverage, or a combination thereof.

The composition may be a cosmetic composition.

The cosmetic composition may have a cosmetic formulation of, for example, a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, an essence, a pack, a gel, an ampoule, or a skin adhesion type.

Components included in the cosmetic composition may include components commonly used in cosmetic compositions in addition to the composition as an active ingredient, for example, conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances. may include

In addition, the composition may be a composition for external application to the skin.

In the present specification, the external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermally delivered patch, drug-containing bandage, lotion, or a combination thereof. The external preparation for skin is a component usually used in external preparations for skin such as cosmetics or pharmaceuticals, for example, an aqueous component, an oily component, a powder component, alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, a preservative, an antioxidant, a surfactant, a fragrance , colorant, various skin nutrients, or a combination thereof may be appropriately formulated as needed. The external preparation for skin includes metal-blocking agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belapamil, licorice extract, glablidine, and kaline. Fruit hot water extracts, various herbal medicines, tocopherol acetate, glitylittic acid, tranexamic acid and derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can be mix|blended suitably.

Another aspect also provides a method of preventing, ameliorating, or treating a condition in a subject comprising treating or administering to the subject in need thereof an effective amount of the composition described above.

The subject's condition may be a condition associated with inflammation or a condition associated with a bacterial infection.

Administration may be administered by methods known in the art. Administration may be administered directly to a subject by any means, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. can The administration may be systemically or locally.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be a subject in need of improvement of a condition associated with inflammation or a condition associated with bacterial infection.

The administration is 0.00001 mg to 1,000 mg per subject per day, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg may be administered. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art The dosage may be appropriately adjusted in consideration of these factors. The number of administration may be once a day or twice or more within the range of clinically acceptable side effects, and may be administered to one or two or more places for the site of administration, and total daily or at intervals of 2 to 5 days The number of days of administration may range from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period. For animals other than humans, the dose is the same as that of a human per kg, or the above dose is converted, for example, by the volume ratio (for example, average value) of the target animal and the organ (heart, etc.) of the human One dose can be administered.

According to the novel strain and its derived endoplasmic reticulum according to an aspect, there is an effect that can be usefully used for the prevention, improvement, or treatment of inflammation-related conditions, or bacterial infections.

1 is a graph showing the production amount of nitric oxide according to the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
Figure 2 is a graph showing the protein expression levels of pro-inflammatory cytokines (IL-6 and CCL2) and anti-inflammatory cytokines (IL-10) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
3 is a graph showing the culture rate according to the co-culture of the strain and C. difficile according to one embodiment; C. difficile : Clostridioides difficile, BBH031: Example 1 strain, Type: Roseburia intestinalis standard strain.
4 is a graph showing the culture rate of C. difficile in the endoplasmic reticulum of the strain according to one embodiment; C. difficile : Clostridioides difficile, BBH031: endoplasmic reticulum of Example 1 strain, Type: endoplasmic reticulum of Roseburia intestinalis standard strain.
5 is a graph showing the cytotoxicity results of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
6 is a graph showing the cytotoxicity results when the endoplasmic reticulum of the strain according to one embodiment and Clostridioides difficile toxin B are treated together; C. difficile toxin B: Clostridioides difficile toxin B, BBH031: endoplasmic reticulum of Example 1 strain, Type strain: endoplasmic reticulum of Roseburia intestinalis standard strain.
7 is a graph showing the activity of reducing inflammation induced by Clostridioides difficile of the endoplasmic reticulum of the strain according to one embodiment; C. difficile toxin B: Clostridioides difficile toxin B, BBH031: endoplasmic reticulum of Example 1 strain, Type strain: endoplasmic reticulum of Roseburia intestinalis standard strain.

Hereinafter, it will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1. Isolation and identification of strains

In order to isolate and identify strains from feces of healthy people, it was performed as follows.

First, 1 g of feces collected from a healthy person and 10 ml of 1 x PBS (Phosphate buffer saline) were mixed and then vortexed to suspend the feces. Then, it was filtered with a cell strainer to remove undigested food and small particles. The filtered fecal suspension was serially diluted and spread at 10 ^-6 to 10 ^-8 on a BHIs (Brain Heart Infusion + Supplement) Plate, and the bacteria were selected after culturing at 37°C for 3 days or more. PCR amplification was performed on the cultured colonies, and the nucleotide sequence of the 16S rRNA region determined among the isolated and cultured microbial colonies was transferred to another registered BLAST program provided on the website of the National Center for Biotechnology Information (NCBI). The strains were compared and analyzed. As a result of comparative analysis, Roseburia intestinalis BBH 031 with 99% homology was isolated. The selected Roseburia intestinalis BBH 031 strain was deposited with the Korea Biological Resources Center on December 20, 2021 and was given accession number KCTC 14838BP, and the Roseburia intestinalis BBH 031 strain has the 16S rRNA sequence of SEQ ID NO: 1 (complementary DNA).

Example 2. Isolation of the endoplasmic reticulum

The endoplasmic reticulum of the strain isolated in the above example was isolated.

Specifically, to prepare the endoplasmic reticulum, the isolated strain was cultured in PYG broth (DSMZ 104) at 37° C. under anaerobic conditions for 2 days. Thereafter, the culture medium was centrifuged at 5000 x g for 20 minutes to remove bacterial debris. Thereafter, after filtering with a 0.45 μm filter and again with a 0.22 μm filter, a centrifuge tube (Amicon® Ultra-15 Centrifugal Filter Unit) was used to concentrate the material of 100 kda or more, and the endoplasmic reticulum of the isolated strain was isolated.

Experimental Example 1. Analysis of anti-inflammatory activity

The anti-inflammatory activity of the endoplasmic reticulum of the strain isolated in Example 2. was analyzed.

First, in order to evaluate the anti-inflammatory activity, a decrease in nitric oxide (NO) production was measured. Mouse macrophage Raw264.7 cells were treated with DMEM (Dulbecco Modified Eagle Medium) containing 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) for 5 Incubated at 37° C. in the presence of % CO ₂ . Thereafter, the Raw 264.7 cells were aliquoted at a concentration of 5Х10 ⁴ cells/well in a 48-well plate by 300 μL, and incubated at 37° C. and 24 hours in a CO ₂ incubator. The well supernatant was discarded, and a medium supplemented with lipopolysaccharide (LPS) 10 μg/ml was dispensed to induce inflammation, followed by further incubation for 4 hours. The supernatant containing LPS was discarded, and the endoplasmic reticulum was added to the medium at a concentration of 1 X 10 ³ or 1 X 10 ⁴ particles/ml to treat and incubate at 37° C. for 16 hours. Thereafter, 50 μL of the well supernatant and 50 μL of Griess reagent were mixed and reacted at room temperature for 10 minutes, and then absorbance was measured at 540 nm with a plate reader to measure the amount of nitric oxide produced, and the results are shown in FIG. 1 .

1 is a graph showing the production amount of nitric oxide according to the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

As shown in Figure 1, it was found that the endoplasmic reticulum of the strain according to one embodiment significantly reduced the amount of NO production of the inflammation-induced cells compared to the untreated control.

Next, the pro-inflammatory cytokine inhibitory activity and anti-inflammatory cytokine promoting activity of the endoplasmic reticulum of the strain were measured. Specifically, the LPS-treated Raw264.7 cells in the same manner as above were treated with the endoplasmic reticulum at a concentration of 1 X 10 ⁴ to 1 X 10 ⁸ particles/ml, and then cultured at 37° C. for 16 hours. Thereafter, the protein expression of the pro-inflammatory cytokines IL-6 and CCL2 and the protein expression of the anti-inflammatory cytokine IL-10 of the cells were measured at 450 nm using an ELISA kit (BD bioscience, USA) according to the manufacturer's instructions. absorbance was measured, and the results are shown in FIG. 2 , respectively.

Figure 2 is a graph showing the protein expression levels of pro-inflammatory cytokines (IL-6 and CCL2) and anti-inflammatory cytokines (IL-10) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

As shown in Figure 2, it was found that the endoplasmic reticulum of the strain according to one embodiment significantly reduced pro-inflammatory cytokines compared to the untreated control group, and significantly increased the anti-inflammatory cytokines compared to the untreated control group.

The above results mean that the strain according to one embodiment can be usefully used for prevention, improvement, or treatment of inflammatory diseases, particularly inflammatory bowel disease or irritable bowel syndrome.

Experimental Example 2. Antibacterial activity analysis

The antibacterial activity of the strain isolated in Example 1. was analyzed.

Specifically, in order to analyze the antibacterial activity against Clostridioides difficile of the strain of Example 1, the strain of Example 1 was pre-cultured in PYG broth, adjusted to OD 0.1, and then added to PYG broth. After inoculation at 1% ratio, it was cultured anaerobically at 37°C for 48 hours, and C. difficile strain was cultured in BHI (Brain Heart Infusion) broth. The cultured strain of Example 1 was centrifuged at 5000 x g for 20 minutes to separate the pellet and the supernatant, and then the pellet was washed twice with PBS, resuspended, and adjusted to an OD of 0.5, respectively. C. difficile and the strain of Example 1 were inoculated in 30ml BHI broth at the same ratio, and then the culture rate of C. difficile was measured by the method of colony forming unit calculation. In order to compare the antibacterial effect with the strain of Example 1, the Roseburia intestinalis standard strain (KCTC15746) was also analyzed for antibacterial activity against Clostridioides difficile in the same manner as above. The analysis result is shown in FIG. 3 .

In addition, in order to analyze the antimicrobial activity of the ER of the strain of Example 2 against C. difficile , the ER was isolated by the method of Example 2. After culturing C. difficile in PYG broth for 48 hours, the OD was adjusted to 0.5, and C. difficile was inoculated into the endoplasmic reticulum of the strain of Example above at a ratio of 10%, and then cultured in anaerobic conditions for 1 day. Thereafter, the culture rate of C. difficile was measured spectrophotometrically. The analysis results are shown in FIG. 4 .

3 is a graph showing the culture rate according to the co-culture of the strain and C. difficile according to one embodiment; C. difficile : Clostridioides difficile, BBH031: Example 1 strain, Type: Roseburia intestinalis standard strain.

Figure 4 is a graph showing the culture rate of C. difficile in the endoplasmic reticulum of the strain according to one embodiment; C. difficile : Clostridioides difficile, BBH031: endoplasmic reticulum of Example 1 strain, Type: endoplasmic reticulum of Roseburia intestinalis standard strain.

As shown in Figure 3, the strain according to one embodiment was found to significantly reduce the growth rate of C. difficile to a level of 64%. On the other hand, in the case of the Roseburia intestinalis standard strain, it was found that the growth rate of C. difficile was not inhibited.

As shown in Figure 4, the endoplasmic reticulum of the strain according to one embodiment was found to significantly reduce the growth rate of C. difficile to a level of 20%. On the other hand, in the case of the endoplasmic reticulum of Roseburia intestinalis standard strain, it was shown that the growth rate of C. difficile was reduced to a level of 34%.

The above result means that the strain and/or the endoplasmic reticulum derived therefrom according to one embodiment has antibacterial activity against bacteria, for example, Gram-negative bacteria, and the activity is higher than that of the standard strain. In particular, these results show that the strain and/or endoplasmic reticulum derived therefrom according to one embodiment are Clostridioides difficile infection (CDI), or the prevention, improvement, or treatment of irritable bowel syndrome resulting therefrom. This means that it can be used

Experimental Example 3. Cytotoxicity test

Cell Counting Kit-8 (CCK-8, Abbkine, China) was used for the cytotoxicity test of the endoplasmic reticulum of the strain isolated in Example 2.

Specifically, 300 μL of Raw264.7 cells were dispensed in a 48-well plate at a concentration of 5Х10 ⁴ cells/well, and incubated at 37° C. and 24 hours in a CO ₂ incubator. The well supernatant was discarded, and a medium supplemented with lipopolysaccharide (LPS) 10 μg/ml was dispensed to induce inflammation, followed by further incubation for 4 hours. The supernatant containing LPS was discarded, and the endoplasmic reticulum was added to the medium at a concentration of 1 X 10 ⁴ to 1 X 10 ⁸ particles/ml and treated, and then incubated at 37° C. for 16 hours. Thereafter, the supernatant was removed, and 300 μl of a medium containing 10% CCK-8 solution was treated in each well and reacted for 4 hours. After 4 hours, the concentration of water-soluble formazan produced by succinate dehydrogenase present in the mitochondria of living cells was measured at 450 nm to measure the cell viability, and the results are shown in FIG. indicated.

5 is a graph showing the cytotoxicity results of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

As shown in Figure 5, the endoplasmic reticulum of the strain according to one embodiment was found to have no cytotoxicity observed at a concentration of 1 X 10 ⁴ to 1 X 10 ⁸ particles/ml.

Experimental Example 4. Analysis of the activity of reducing inflammation induced by Clostridioides difficile

The effect of reducing the pro-inflammatory cytokines induced by Clostridioides difficile of the endoplasmic reticulum of the strain isolated in Example 1 was confirmed.

First, in the same manner as in Experimental Example 1, mouse macrophage Raw264.7 cells were treated with Clostridioides difficile toxin B ( C. difficile toxin B) in an amount of 10 ng/ml, and then 4 incubated for hours. Thereafter, after treating the endoplasmic reticulum of the Roseburia Intestinalis standard strain (KCTC15746) and the strain of Example 1 at a concentration of 1 X 10 ⁴ to 1 X 10 ⁸ particles/ml, at 37° C. for 16 hours was cultured for a while, and the cell viability was measured in the same manner as in Experimental Example 2, and the results are shown in FIG. 6 .

In addition, the relative concentrations of pro-inflammatory cytokines TNF and IL-6 in the cells were measured at 450 nm using an ELISA kit (BD bioscience, USA) according to the manufacturer's instructions, and the results are shown in FIG. it was

6 is a graph showing the cytotoxicity results when the endoplasmic reticulum of the strain according to one embodiment and Clostridioides difficile toxin B are treated together; C. difficile toxin B: Clostridioides difficile toxin B, BBH031: endoplasmic reticulum of Example 1 strain, Type strain: endoplasmic reticulum of Roseburia intestinalis standard strain.

7 is a graph showing the activity of reducing inflammation induced by Clostridioides difficile of the endoplasmic reticulum of the strain according to one embodiment; C. difficile toxin B: Clostridioides difficile toxin B, BBH031: endoplasmic reticulum of Example 1 strain, Type strain: endoplasmic reticulum of Roseburia intestinalis standard strain.

As shown in Figure 6, cytotoxicity was not confirmed in both the endoplasmic reticulum of the strain according to one embodiment and the standard strain.

In addition, as shown in Figure 7, it can be seen that the endoplasmic reticulum of the strain according to one embodiment significantly reduced the pro-inflammatory cytokines TNF and IL-6 increased by Clostridioides difficile toxin B compared to the standard strain. .

These results show that the strain and / or the endoplasmic reticulum derived therefrom according to one embodiment has significant anti-inflammatory activity, Clostridioides difficile infection (CDI), or prevention of irritable bowel syndrome resulting therefrom, improvement, Or it means that it can be usefully used for treatment.

Korea Center for Biological Resources (Overseas) KCTC14838BP 20211220

<110> BioBankHealing <120> Roseburia intestinalis strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of its <130> PN211310KR <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1434 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA for Roseburia intestinalis <400> 1 cgatcctgcc ttcggcagct ccctccttgc ggttgggtca ctgacttcgg gcattaccaa 60 ctcccatggt gtgacgggcg gtgtgtacaa gacccgggaa cgtattcacc gcgacattct 120 gattcgcgat tactagcgat tccagcttcg tgcagtcgag ttgcagactg cagtccgaac 180 tgagacgtta tttttgagat ttgctccccc tcgcaggctc gcttcccttt gtttacgcca 240 ttgtagcacg tgtgtagccc aagtcataag gggcatgatg atttgacgtc atccccacct 300 tcctccaggt tatccctggc agtctcccta gagtgcccgg cttacccgct ggctactaag 360 aataggggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca 420 accatgcacc acctgtcacc gatgctccga agagaaaaca cattacatgt tctgtcatcg 480 ggatgtcaag acttggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg 540 cttgtgcggg tccccgtcaa ttcctttgag tttcattctt gcgaacgtac tccccaggtg 600 gaatacttat tgcgtttgct gcggcaccga agagcaatgc tccccgacac ctagtattca 660 tcgtttacgg cgtggactac cagggtatct aatcctgttt gctccccacg ctttcgagcc 720 tcagcgtcag taatcgtcca gtaagccgcc ttcgccactg gtgttcctcc taatatctac 780 gcatttcacc gctacactag gaattccact tacccctccg acactctagt ccgacagttt 840 ccaatgcagt accggggttg agccccgggc tttcacatca gacttgccgt accgcctgcg 900 ctccctttac acccagtaaa tccggataac gcttgcacca tacgtattac cgcggctgct 960 ggcacgtatt tagccggtgc ttcttagtca ggtaccgtca tttcttcttc cctgctgata 1020 gagctttaca taccgaaata cttcttcgct cacgcggcgt cgctgcatca gggtttcccc 1080 cattgtgcaa tattccccac tgctgcctcc cgtaggagtt tgggccgtgt ctcagtccca 1140 atgtggccgg tcaccctctc aggtcggcta ctgatcgtcg ctttggtagg ccgttacccc 1200 accaactggc taatcagacg cgggtccatc tcataccacc ggagtttttc acaccaggtc 1260 atgcgaccct gtgcgcttat gcggtattag cagtcgtttc caactgttat ccccctgtat 1320 gaggcaggtt acccacgcgt tactcacccg tccgccactc agtcacaaaa tcttcattcc 1380 gaagaaatca aataaagtgc ttcgttcgac ttgcatgtgt taagcacgcc gcca 1434

Claims (10)

Roseburia intestinalis ( Roseburia intestinalis ) strain belonging to the genus Roseburia sp. , deposited with accession number KCTC 14838BP. The strain according to claim 1, wherein the strain comprises 16S rRNA of SEQ ID NO: 1. The endoplasmic reticulum derived from the strain of claim 1. The lysate of the strain of claim 1 The culture solution of the strain of claim 1 The strain of claim 1, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient; inflammatory bowel diseases (IBD); irritable bowel syndrome; Enteritis (enteritis), Crohn's disease; or a pharmaceutical composition for preventing or treating ulcerative colitis (ulcerative colitis). The strain of claim 1, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient; inflammatory bowel diseases (IBD); irritable bowel syndrome; A health functional food for preventing or improving enteritis, Crohn's disease; or ulcerative colitis. The strain of claim 1, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or Clostridioides difficile comprising a mixture thereof as an active ingredient For improvement of intestinal health by inhibition or inflammation inhibition health functional food. A pharmaceutical composition for preventing or treating Clostridioides difficile infection (CDI) comprising the strain of claim 1, the endoplasmic reticulum derived from the strain, a lysate of the strain, a culture medium, or a mixture thereof as an active ingredient. The health functional food for preventing or improving Clostridioides difficile infection (CDI) comprising the strain of claim 1, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient.

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