KR102620189B1 - Blautia brookingsii strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of thereof - Google Patents

Abstract

Relates to novel microorganisms, their lysates, cultures, extracts of culture fluids, endoplasmic reticulum and their anti-inflammatory and/or antibacterial uses, and according to one aspect, the novel strain and endoplasmic reticulum derived therefrom are used to treat inflammation-related conditions or bacterial infections. It has effects that can be useful for prevention, improvement, or treatment.

Description

Blautia brookingsii strain, and vesicles from it and anti-inflammation and anti-bacteria uses of it}

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

Microbiome refers to the microorganisms that exist in a specific environment and their entire genetic information, and refers to a collection of genomes that represent the entire genetic information of a single organism. Therefore, the human microbiome refers to the 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 intestines are the optimal environment for microorganisms to consume nutrients and form systematic communities, so the largest number of microorganisms exist. Intestinal microorganisms supply nutrients that cannot be produced by the host's own enzymes alone and are deeply related to the host's metabolism and immune system, while also preventing various diseases such as irritable bowel syndrome, obesity, atopy, depression, rheumatoid arthritis, autism spectrum disorder, and dementia. It is reported that it is related to the outbreak.

Recently, intestinal health has been worsening due to an imbalance in the intestinal microbiota due to Western eating habits and indiscriminate use of antibiotics. Research on intestinal microorganisms and various diseases has highlighted the importance of intestinal microorganisms and is raising interest. .

Meanwhile, endoplasmic reticulum is a nano-sized substance of about 20 to 200 nm produced and excreted by cells, and can move freely between cells. In addition, the endoplasmic reticulum contains membrane lipids, membrane proteins, DNA and RNA, and it is known that these genetic materials act as a complex to transfer toxic factors between cells and play a role in regulating inflammation and immune responses. From single-celled organisms to multicellular organisms, information exchange between cells is an essential process of life. Recently, endoplasmic vesicles have been recognized as mediators of information exchange between cells, and methods of using vesicles as drug carriers are being developed.

Accordingly, there is a need to develop materials for improving, preventing, or treating diseases using new microorganisms derived from the human intestine and endoplasmic reticulum derived from them.

Korean Patent Publication No. 10-2018-0012849

One aspect is to provide a Blautia brookingsii strain belonging to the Blautia genus ( Blautia sp. ), deposited under accession number KCTC 14940BP.

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

Another aspect is inflammatory bowel diseases (IBD) comprising the Blautia brookingsi strain, endoplasmic reticulum derived from the strain, lysate of the strain, 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 an inflammatory bowel disease comprising the Blautia brookingsi strain, endoplasmic reticulum derived from the strain, lysate of the strain, culture medium, or a mixture thereof as an active ingredient; Irritable Bowel Syndrome; It provides health functional foods for preventing or improving enteritis, Crohn's disease, or ulcerative colitis.

Another aspect is Clostridioides difficile inhibition or inflammation inhibition comprising the Blautia brookingsi strain, endoplasmic reticulum derived from the strain, lysate of the strain, culture medium, or a mixture thereof as an active ingredient. To provide health functional foods for improving intestinal health.

Another aspect is the prevention or treatment of Clostridioides difficile infection (CDI) comprising the Blautia brookingsi strain, endoplasmic reticulum derived from the strain, lysate of the strain, culture medium, or a mixture thereof as an active ingredient. To provide a pharmaceutical composition for use.

Another aspect is a health function for preventing or improving Clostridioides difficile infection comprising the Blautia brookingsi strain, endoplasmic reticulum derived from the strain, lysate of the strain, culture medium, or a mixture thereof as an active ingredient. It is to provide food.

One aspect provides a strain of Blautia brookingsii belonging to the Blautia genus ( Blautia sp. ).

The Blautia Brookingsi strain may be a strain deposited under the accession number KCTC 14940BP.

The Blautia brooking strain may be a strain containing 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 inflammation-induced cells, suppresses the expression of inflammatory cytokines (e.g., TNF-α or IL-6), or inhibits bacterial proliferation (e.g., C. difficile ). It may be suppressing. In addition, the strain reduces inflammatory factors induced by C. difficile , such as pro-inflammatory cytokines (e.g., TNF, or CCL2), or reduces anti-inflammatory cytokines (e.g., IL-10). ) may increase.

Another aspect provides an endoplasmic reticulum derived from a Blautia brookingsii strain belonging to the Blautia genus (Blautia sp. ), a lysate of the strain, a culture medium, an extract of the culture medium, 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, including exosome, ectosome, microvesicle, and microparticle. , exosome-like vesicles, etc. Extracellular vesicles can reflect the state of the secreting cell of origin (donor cell), exhibit various biological activities depending on which cell they are secreted from, and play an important role in intercellular interactions by transferring genetic material and proteins between cells. can do. In addition, cell-derived substances containing the endoplasmic reticulum cause disease or stimulate immune cells to fight against disease, and have the effect of helping humans break down and absorb substances that cannot be digested through the metabolic process of microorganisms. there is. The endoplasmic reticulum is a membrane-structured endoplasmic reticulum that is divided into an inside and an outside, and contains cell membrane lipids, plasma membrane proteins, nucleic acids, and cytoplasmic components, and is larger than the original cell. may be small.

In one embodiment, the endoplasmic reticulum may be isolated from cell lysate of the culture medium of the Blautia brooking 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,” which provides nutrients for Blautia brookingsi to grow and survive in vitro. It may refer to the entire medium containing the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain in a suitable medium for a certain period of time. Additionally, the culture medium may refer to a culture medium obtained by removing the bacterial cells from the bacterial culture medium obtained by culturing the strain. On the other hand, the liquid from which the bacteria have been removed from the culture medium is also called "supernatant", and the culture medium is left alone for a certain period of time and only the liquid in the upper layer excluding the part that has settled in the lower layer is taken, the bacteria are removed through filtration, or the culture medium is centrifuged and the lower layer is removed. It can be obtained by removing the precipitation and taking only the upper liquid. The "bacteria" refers to the strain itself of the present invention, and includes the strain itself isolated and selected from a skin sample, etc., or a strain isolated from the culture medium by culturing the strain. The bacterial cells can be obtained by centrifuging the culture medium and taking the part that sinks to the lower layer. Alternatively, since they sink to the lower layer of the culture medium by gravity, they can be obtained by leaving them still for a certain period of time and then removing the upper liquid.

The culture medium may include the culture medium itself, its concentrate, or freeze-dried product obtained by cultivating the strain, or the culture supernatant obtained by removing the strain from the culture medium, its concentrate, or freeze-dried product.

The culture medium is obtained by culturing Blautia brookingsi in an appropriate medium (e.g., 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. It may have happened.

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

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

The culture medium and culture conditions for culturing the Blautia brookingsi can be appropriately selected or modified by those skilled in the art.

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

As used herein, the term "culture extract" refers to an extract from the culture medium or its concentrate, and may include extracts, diluted or concentrated extracts, dried products obtained by drying the extracts, crude or purified products thereof, and fractions thereof. You can.

Another aspect provides a use of the Blautia brookingsi strain, endoplasmic reticulum derived from the strain, lysate, culture fluid, or extract of the culture fluid of the strain for disease improvement, prevention, or treatment.

As used herein, the term “treat” may mean curing inflammation or bacterial infection in a shorter time compared to natural healing. The treatment may include amelioration and/or alleviation of inflammation or bacterial infection. Additionally, the treatment may mean 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 its secondary symptoms, preventing the acquisition or re-acquisition of a disease or disorder, or preventing the development or recurrence of a disease or disorder. It refers to a method of reducing the risk of acquisition. For example, the prevention refers to all actions that suppress or delay the occurrence of inflammation or bacterial infection by administering the composition according to the present invention.

Uses of the strain may include preventing, ameliorating, or treating inflammatory diseases (anti-inflammatory activity), preventing, ameliorating, or treating bacterial infections (antibacterial activity), or preventing or improving intestinal health.

The inflammatory disease may include inflammation of the digestive system (gastrointestinal tract, etc.), inflammation within the eye, inflammation within the oral cavity, inflammation of the respiratory system including the lungs, inflammation of the skin, inflammation within the cardiovascular system, inflammation of the brain, inflammation within the ear, etc. there is.

More specifically, the inflammatory diseases include inflammatory bowel diseases (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; 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 glomerulonephritis.

In addition, the improvement in intestinal health may be helpful in beneficial proliferation and suppression of harmful bacteria in the intestines, helpful in intestinal health by regulating immunity, or helpful in facilitating bowel movements.

The term “antibacterial agent,” as used herein, refers to an agent that (i) inhibits, reduces, or prevents the growth of bacteria; (ii) inhibits or reduces the ability of bacteria to cause infection in a subject; or (iii) refers to a substance capable of inhibiting or reducing the ability of bacteria to multiply or remain infective in the environment.

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

The composition is 0.00001% by weight to 80% by weight, for example, 0.00001% by weight to 60% by weight, 0.00001% by weight to 40% by weight, 0.00001% by weight to 30% by weight, 0.00001% by weight to 20% by weight, based on 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 It may include % to 10% by weight, or 0.1% to 5% by weight of the strain, its lysate, culture medium, or extract of the culture medium.

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 are added to the extent that the above-mentioned effects can be exhibited, This means that it is formulated into various forms by adding various ingredients as sub-ingredients for drug delivery and stabilization.

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

Types of pharmaceutically active ingredients that can deliver the active ingredient into the subject include anticancer agents, contrast agents (dyes), hormones, antihormones, vitamins, calcium agents, mineral agents, saccharides, organic acid agents, protein amino acid agents, detoxifiers, and enzymes. Preparations, metabolic preparations, diabetic preparations, tissue revitalization preparations, chlorophyll preparations, dye preparations, tumor preparations, tumor treatment drugs, radiopharmaceuticals, tissue cell diagnostic agents, tissue cell therapeutic agents, antibiotic preparations, antivirals, combination antibiotic preparations, chemistry Therapeutics, vaccines, toxins, toxoids, antitoxins, leptospira serum, blood products, biological agents, analgesics, immunogenic molecules, antihistamines, allergy medications, non-specific immunogenic agents, anesthetics, stimulants, psychotropic agents, small molecule compounds, nucleic acids, It may include aptamers, antisense nucleic acids, oligonucleotides, peptides, siRNA, and micro RNA.

The pharmaceutical composition may additionally 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, disintegrant, binder, lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium monohydrogen phosphate anhydride, 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 prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, Witepsol, Macrogol, Tween 61, cacao, Liurinji, glycerogeratin, etc. can be used.

The pharmaceutical composition contains carbohydrates such as glucose, sucrose or dextran, antioxidants such as Ascorbic acid or Glutathione, chelating agents, and small molecules to increase stability or absorption. Proteins or other stabilizers can be used as agents.

The pharmaceutical composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrup, or combinations thereof. Parenteral dosage forms may be injectable.

The composition may be a health functional food composition.

The health functional food composition can be used alone or with other foods or food ingredients, such as the strain or its culture medium, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). In general, when manufacturing food or beverages, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw materials. There are no particular restrictions on the types of health functional foods. Among the types of health functional foods, beverage compositions may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary beverages. 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 a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used. The health food composition also contains nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonated beverages. It may contain the carbonating agent used, or a combination thereof. The health functional food composition may also contain pulp for the production of natural fruit juice, fruit juice beverage, vegetable beverage, or a combination thereof.

The composition may be a cosmetic composition.

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

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

Additionally, the composition may be a composition for external skin application.

In this specification, the external skin agent may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof. The skin external preparations include ingredients commonly used in external skin preparations such as cosmetics and medicines, such as aqueous ingredients, oil-based ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , colorants, various skin nutrients, or a combination thereof may be appropriately mixed according to need. The skin external preparations include metal sequestrants such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belafamil, licorice extract, glablidin, and calin. Hot water extract of fruit, various herbal medicines, drugs such as tocopherol acetate, glytylitinic acid, tranexamic acid and its derivatives or salts, vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, Sugars such as trehalose can also be appropriately mixed.

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

The subject's condition may be a condition related to inflammation, or a condition related to bacterial infection.

Administration can be done by methods known in the art. Administration may be administered directly to the subject by any means, such as, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. You can. The administration may be administered 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 an individual in need of an improvement in a condition related to inflammation or a condition related to bacterial infection.

The administration of the composition according to one embodiment is 0.00001 mg to 1,000 mg, 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 mg per day. 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, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will Taking these factors into consideration, the dosage can be adjusted appropriately. The frequency of administration can be once a day or more than twice within the range of clinically acceptable side effects, and can be administered at one or two or more locations, daily or at intervals of 2 to 5 days. The number of days of administration can be from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after an appropriate period. For animals other than humans, the dosage per kg is the same as for humans, or the above dosage is converted into, for example, the volume ratio (e.g., average value) of the organs (heart, etc.) between the target animal and human. One dose can be administered.

According to one aspect, the new strain and the endoplasmic reticulum derived therefrom have effects that can be useful in preventing, improving, or treating inflammation-related conditions or bacterial infections.

Figure 1 is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF, IL-6, and CCL2) and anti-inflammatory cytokines (IL-10) upon cellular processing of the endoplasmic reticulum of a strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
Figure 2 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.
Figure 3 is a graph showing the cytotoxicity results when the endoplasmic reticulum of the strain and Clostridioides difficile toxin B were treated together; C. difficile toxin B: Clostridioides difficile toxin B, BBH033: Endoplasmic reticulum of Example 1 strain, Type strain: Endoplasmic reticulum of Blautia Brookingsi standard strain.
Figure 4 is a graph showing the activity of reducing inflammation induced by Clostridioides difficile of the endoplasmic reticulum of a strain according to one embodiment; C. difficile toxin B: Clostridioides difficile toxin B, BBH033: Endoplasmic reticulum of Example 1 strain, Type strain: Endoplasmic reticulum of Blautia Brookingsi standard strain.

This will be described in more detail through examples below. However, these examples are intended to illustrate one or more embodiments and the scope of the present invention is not limited to these examples.

Example 1. Isolation and identification of strains

To isolate and identify strains from feces of healthy people, the following procedures were performed.

First, 1 g of feces collected from a healthy person was mixed with 10 ml of 1 x PBS (Phosphate buffer saline) and then vortexed to suspend the feces. It was then filtered through a cell strainer to remove undigested food and small particle substances. 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 more than 3 days. PCR amplification was performed on the colonies for which culture was completed, and the nucleotide sequence of the 16S rRNA region determined among the isolated and cultured microbial colonies was compared to other colonies registered with the BLAST program provided on the U.S. National Center for Biotechnology Information (NCBI) website. Strains were compared and analyzed. As a result of comparative analysis , Blautia brookingsii BBH 033 with 99% homology was isolated. The selected Blautia brookingsii BBH 033 strain was deposited at the Korea Center for Biological Resources on April 13, 2022 and received accession number KCTC 14940BP. The Blautia brookingsii BBH 033 strain has the 16S rRNA sequence of SEQ ID NO: 1 (complementary DNA).

Example 2. Isolation of endoplasmic reticulum

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

Specifically, to prepare endoplasmic reticulum, the isolated strain was cultured in PYG broth at 37°C under anaerobic conditions for 2 days. Afterwards, the culture was centrifuged at 5000 x g for 20 minutes to remove bacterial debris. Afterwards, it was filtered through a 0.45 μm filter and again through a 0.22 μm filter, and then substances over 100 kda were concentrated using a centrifugal tube (Amicon® Ultra-15 Centrifugal Filter Unit), and the endoplasmic reticulum of the isolated strain was isolated.

Experimental Example 1. Anti-inflammatory activity analysis

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

The pro-inflammatory cytokine inhibitory activity and anti-inflammatory cytokine promoting activity of the endoplasmic reticulum of the above strain were measured. Specifically, mouse macrophage Raw264.7 cells were cultured in DMEM (Dulbecco Modified Eagle Medium) containing 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). ) and cultured at 37°C in the presence of 5% CO ₂ . Afterwards, 300 μL of the Raw 264.7 cells were dispensed into a 48-well plate at a concentration of 5Х10 ⁴ cells/well, and cultured in a CO ₂ incubator at 37°C for 24 hours. The well supernatant was discarded, medium containing 10 μg/ml lipopolysaccharide (LPS) was added to induce inflammation, and the cells were further cultured for 4 hours. The ^{supernatant containing} LPS was discarded, the endoplasmic reticulum was treated at a concentration of 1 Afterwards, the protein expression of the pro-inflammatory cytokines TNF, IL-6, and CCL2 and the protein expression of the anti-inflammatory cytokine IL-10 of the cells were measured using an ELISA kit (BD bioscience, USA) according to the manufacturer's instructions. Absorbance was measured at 450 nm, and the results are shown in Figure 1.

Figure 1 is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF, IL-6, and CCL2) and anti-inflammatory cytokines (IL-10) upon cellular processing of the endoplasmic reticulum of a 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 pro-inflammatory cytokines compared to the untreated control group and significantly increased 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 preventing, improving, or treating inflammatory diseases, especially inflammatory bowel disease or irritable bowel syndrome.

Experimental Example 2. Cytotoxicity experiment

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

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

Figure 2 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 2, it was found that cytotoxicity was not observed in the endoplasmic reticulum of the strain according to one embodiment at a concentration of 1 × 10 ³ to 1 × 10 ⁸ particles/ml.

Experimental Example 3. Analysis of activity to reduce inflammation induced by Clostridioides difficile toxin B

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

First, mouse macrophage Raw264.7 cells were treated with Clostridioides difficile toxin B ( C. difficile toxin B) in an amount of 10 ng/ml in the same manner as in Experimental Example 1, and then incubated at 37°C for 4 days. It was cultured for some time. ^Afterwards , the endoplasmic reticulum of the Blautia Brookings standard strain (DSM107275) and the strain of Example ¹ was treated at a concentration of 1 The cells were cultured, and the cell viability was measured in the same manner as in Experimental Example 2, and the results are shown in Figure 3.

In addition, the relative concentrations of pro-inflammatory cytokines TNF and CCL2 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 Figure 4.

Figure 3 is a graph showing the cytotoxicity results when the endoplasmic reticulum of the strain and Clostridioides difficile toxin B were treated together; C. difficile toxin B: Clostridioides difficile toxin B, BBH033: Endoplasmic reticulum of Example 1 strain, Type strain: Endoplasmic reticulum of Blautia Brookingsi standard strain.

Figure 4 is a graph showing the activity of reducing inflammation induced by Clostridioides difficile of the endoplasmic reticulum of a strain according to one embodiment; C. difficile toxin B: Clostridioides difficile toxin B, BBH033: Endoplasmic reticulum of Example 1 strain, Type strain: Endoplasmic reticulum of Blautia Brookingsi standard strain.

As shown in Figure 3, 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 4, the endoplasmic reticulum of the strain according to one embodiment was found to significantly reduce the pro-inflammatory cytokines TNF and CCL2 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, preventing, improving, and preventing Clostridioides difficile infection (CDI) or irritable bowel syndrome resulting therefrom. Or it means that it can be usefully used for treatment.

Korea Biological Resources Center (overseas) KCTC14940BP 20220413

Claims (10)

A Blautia brookingsii strain belonging to the Blautia genus and containing the 16S rRNA of SEQ ID NO: 1, deposited with accession number KCTC 14940BP. delete Endoplasmic reticulum derived from the strain of claim 1. Crushed liquid of the strain of claim 1. Culture medium of the strain of claim 1. Inflammatory bowel diseases (IBD) comprising the strain of claim 1, endoplasmic reticulum derived from the strain, lysate of the strain, culture medium, or a mixture thereof as an active ingredient; irritable bowel syndrome; A pharmaceutical composition for preventing or treating enteritis, Crohn's disease, or ulcerative colitis. Inflammatory bowel diseases (IBD) comprising the strain of claim 1, endoplasmic reticulum derived from the strain, lysate of the strain, culture medium, or a mixture thereof as an active ingredient; irritable bowel syndrome; Health functional food for preventing or improving enteritis, Crohn's disease, or ulcerative colitis. For improving intestinal health by inhibiting Clostridioides difficile or inhibiting inflammation, comprising the strain of claim 1, endoplasmic reticulum derived from the strain, lysate of the strain, culture medium, or a mixture thereof as an active ingredient. Health functional food. A pharmaceutical composition for preventing or treating Clostridioides difficile infection (CDI), comprising the strain of claim 1, endoplasmic reticulum derived from the strain, lysate of the strain, culture medium, or a mixture thereof as an active ingredient. A health functional food for preventing or improving Clostridioides difficile infection (CDI), comprising the strain of claim 1, endoplasmic reticulum derived from the strain, lysate of the strain, culture medium, or a mixture thereof as an active ingredient.