KR102363975B1 - Lactobacillus plantarum strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of thereof - Google Patents

Abstract

It relates to a novel microorganism, its lysate, culture solution, extract of the culture solution, endoplasmic reticulum and their anti-inflammatory and/or antibacterial use, and according to the novel strain and the endoplasmic reticulum derived therefrom according to an aspect, inflammation-related conditions, or bacterial infections There is an effect that can be usefully used for prevention, improvement, or treatment.

Description

Lactobacillus plantarum strain, and endoplasmic reticulum derived therefrom, and anti-inflammatory and antibacterial uses 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 microbes supply nutrients that cannot be produced by the host's enzymes alone and are closely related to the host's metabolism and immune system, while preventing various diseases such as irritable bowel syndrome, obesity, atopy, depression, rheumatoid arthritis, autism spectrum disorder, and dementia. It has been reported to be associated with

Recently, due to Western dietary habits and reckless use of antibiotics, the intestinal microbiota is imbalanced and intestinal health is deteriorating, and the importance of intestinal microbes and interest are rising due to research on intestinal microbes and various diseases. .

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 is recognized as a medium for information exchange between cells, so methods for using the vesicle as a drug carrier are being 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 10-0509558

One aspect is to provide a Lactobacillus plantarum ( Lactobacillus plantarum ) strain belonging to the Lactobacillus genus ( Lactobacillus sp. ) deposited with accession number KCTC 14265BP.

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

Another aspect is to provide a pharmaceutical composition for preventing or treating inflammatory diseases 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.

Another aspect is to provide a health functional food for the prevention or improvement of inflammatory diseases 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.

Another aspect is to provide a health functional food for improving intestinal health 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.

Another aspect is to provide a pharmaceutical composition for preventing or treating a bacterial 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.

Another aspect is to provide a health functional food for preventing or improving bacterial 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.

Another aspect is to provide a cosmetic composition comprising the strain, the endoplasmic reticulum derived from the strain, a lysate of the strain, a culture solution, or a mixture thereof.

Another aspect is to provide an antibacterial composition for external application for skin comprising the strain, the endoplasmic reticulum derived from the strain, a lysate of the strain, a culture solution, or a mixture thereof.

One aspect provides a Lactobacillus genus ( Lactobacillus sp. ) belonging to the Lactobacillus plantarum ( Lactobacillus plantarum ) strain.

The Lactobacillus plantarum strain may be a strain deposited with accession number KCTC 14265BP.

The Lactobacillus plantarum 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, for example, pro-inflammatory cytokines (eg, TNF, or CCL2), or increases anti-inflammatory cytokines (IL-10) it could be

Another aspect provides an endoplasmic reticulum derived from the Lactobacillus plantarum strain, 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, 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 solution of the Lactobacillus plantarum 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 can provide nutrients so that Lactobacillus plantarum can grow and survive in vitro. It may mean the entire medium including the strain obtained by culturing the strain in a medium for a certain period of time, its metabolites, and extra nutrients. 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 lower 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 and taking the part that has sunk to the lower layer, or by removing the liquid from the upper layer after leaving it still for a certain period of time because it sinks to the lower layer of the 　 culture solution by gravity.

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 solution is obtained by culturing Lactobacillus plantarum 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. it could be

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 Lactobacillus plantarum 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 fractions obtained by fractionation thereof. can

Another aspect is Lactobacillus plantarum ( Lactobacillus plantarum ), the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture solution, or the disease improvement, prevention or treatment use of the 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.

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 Clostridium , Helicobactor , Escherichia , Salmonella , Staphylococcus , Streptococcus , Haemophilus ), Klebsiella , Moraxella , Enterobacter , Proteus , Serratia , Pseudomonas , Acinetobacter , Citrobacter ), Stenotrophomonas , Bacteroides , Prevotella , Fusobacterium ( Fusobacterium ) It may include an infection caused by bacteria belonging to the genus. More specifically, the bacterial infection is Clostridium difficile infection (CDI), or Clostridium difficile associated disease (CDAD), for example, Clostridium difficile associated diarrhea. diarrhea) may be included.

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 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.

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 level of inflammatory cytokines (TNF-α and IL-6) 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.
Figure 3 is a graph showing the protein expression levels of inflammatory cytokines (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: the endoplasmic reticulum of Example 2.
4 is a graph showing the culture rate according to the co-culture of the strain and C. difficile according to an embodiment.
5 is a graph showing the culture rate of C. difficile according to the treatment with C. difficile of the endoplasmic reticulum of the strain according to one embodiment.
6 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.
7 is a graph showing the cytotoxicity and nitric oxide production reduction activity results of the endoplasmic reticulum of the strain and the standard strain according to one embodiment; CdEV: Clostridium difficile ER, BBH006: ER of Example 1 strain, Type strain: ER of Lactobacillus plantarum standard strain.
8 is a graph showing the reduction activity of inflammation induced by Clostridium difficile of the endoplasmic reticulum of the strain according to one embodiment; CdEV: Clostridium difficile ER, BBH006: ER of Example 1 strain, Type strain: ER of Lactobacillus plantarum 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

Fecal Microbiota Transplantation (FMT) was performed as follows to isolate and identify strains from the composition.

First, 50 g of feces collected from healthy people and 300 ml of a physiological saline solution mixed with 10% Glycerol are placed in Stomacher (Bagmixer 400, Interscience) to remove undigested food and small particles, and homogenized to prepare a fecal mixture. did. Thereafter, the fecal mixture was filtered once more through 0.25 mm sieves to obtain a filtrate, and then 250 ml of FMT graft was prepared. The composition containing the microorganism was serially diluted in TSA medium (Tryptic soy agar, Green Tech) containing 5% sheep blood, spread on a plate, and the bacteria were selected by incubating at 37 ° C. for 2 days. 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, Lactobacillus plantarum BBH 006 having 98% homology was isolated. The selected Lactobacillus plantarum BBH 006 strain was deposited with the Korea Biological Resources Center on August 6, 2020 and was given an accession number KCTC 14265BP, and the Lactobacillus plantarum BBH 006 strain has a 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, in order to prepare the endoplasmic reticulum, the isolated strain was cultured in BHIs broth (Brain Heart Infusion-supplemented, Eco cell) at 30° C. under anaerobic conditions for 3 days. Thereafter, the culture medium was centrifuged at 3000 RPM for 20 minutes and then centrifuged again at 3000 RPM for 40 minutes to remove bacterial debris. Thereafter, after filtering with a 0.45um filter, filter with a 0.22um filter again, and use a TFF system (Tangential Flow Filtration System, Green Tech) using a filter (Millipore Pellicon XL50, Biomax-100 filter) to concentrate the material of 100 kda or more and the endoplasmic reticulum of the isolated strain was isolated.

Experimental Example 1. Anti-inflammatory activity assay

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 RPMI1640 culture medium containing 20% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) in the presence of 5% CO ₂ 37 Incubated at ℃. Thereafter, the Raw 264.7 cells were aliquoted at a concentration of 5Х10 ⁴ cells/well in a 48-well plate by 250 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) 10ug/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 0.01 to 100 ug/ml and treated, followed by incubation 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 570 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 reduced the amount of NO production of the inflamed cells to the level of the negative control.

Next, the inflammatory cytokine inhibitory activity of the endoplasmic reticulum of the strain was 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 or 10 ug/ml, and then cultured at 37° C. for 48 hours. Thereafter, the protein expression of TNF-α, IL-6, CCL2 and IL-10 of the cells was measured for absorbance at 540 nm using an ELISA kit (eBioscience, USA) according to the manufacturer's instructions, and the results are shown in each figure. 2 and 3 are shown.

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

As shown in Figures 2 and 3, the endoplasmic reticulum of the strain according to one embodiment significantly reduced inflammatory cytokines compared to the untreated control group, and it was found that the anti-inflammatory cytokines significantly increased 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 Clostridium difficile of the strain of Example 1, the strain of Example 1 and C.difficile in 5ml BHI broth (Brain Heart Infusion, Eco cell) It was pre-cultured and adjusted to OD (600nm) 0.5 using a spectrophotometer. After that, the strain culture medium was inoculated in 30ml BHI broth at a rate of 10% and then cultured anaerobically at 37°C for 48 hours. The cultured strain was centrifuged at 4000RPM for 30 minutes to separate the pellet and the supernatant, and then the pellet was washed 3 times with PBS and then resuspended.

C. difficile and the strain of the above example were inoculated into BHI broth at the same ratio in the same amount, and then co-cultured in BHI broth medium under anaerobic conditions for 2 days. Thereafter, the culture rate of C. difficile was measured by the method of colony forming unit calculation, and the results are shown in FIG. 4 .

In addition, in order to analyze the antimicrobial activity against C. difficile of the endoplasmic reticulum of the strain of the above example, the culture supernatant of the strain was centrifuged at 4000 for 30 minutes to separate the endoplasmic reticulum. The separated supernatant was concentrated using centrifuge tubes (Amicon centrifuge tubes), and inoculated to 3 X 10 ⁶ cfu/ml C. difficile at a ratio of 90%, anaerobic conditions for 1 day in BHI broth medium cultured in Thereafter, the culture rate of C. difficile was measured by the method of colony forming unit calculation, and the results are shown in FIG. 5 .

4 is a graph showing the culture rate according to the co-culture of the strain and C. difficile according to one embodiment.

5 is a graph showing the culture rate of C. difficile according to the treatment with C. difficile of the endoplasmic reticulum of the strain according to one embodiment.

As shown in Figures 4 and 5, the strain and its derived endoplasmic reticulum according to one embodiment was found to significantly reduce the culture rate of C. difficile .

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

Experimental Example 3. Cytotoxicity test

The PreMix 　 WST-1 Cell Proliferation Assay System was used for the cytotoxicity test of the endoplasmic reticulum of the strain isolated in Example 2.

Specifically, Raw264.7 cells were aliquoted at a concentration of 5Х10 ⁴ cells/well in a 48-well plate by 250 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) 10ug/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 0.01 to 100 ug/ml and treated, followed by incubation at 37° C. for 16 hours. Thereafter, 25 μl of WST-1 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 mitochondria of living cells was measured at 560 nm to measure cell viability, and the results are shown in FIG. indicated.

6 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 FIG. 6 , it was found that no cytotoxicity was observed in the endoplasmic reticulum of the strain according to one embodiment at a concentration of 100 ug/ml.

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

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

First, Clostridium difficile endoplasmic reticulum (CdEV) was treated in an amount of 100gu/ml in mouse macrophage Raw264.7 cells in the same manner as in Experimental Example 1, and then cultured at 37°C for 4 hours. After that, the standard strain of Lactobacillus plantarum (ATCC14917) and the endoplasmic reticulum of the strain of Example 1 were treated in an amount of 0.1ug/ml or 1ug/ml, and then cultured at 37°C for 16 hours, cells The survival rate was measured in the same manner as in Experimental Example 2, and the results are shown in FIG. 7 .

In addition, the relative concentrations of cytokines TNF, CCL2 and IL-6 in the cells were measured for absorbance at 540 nm using an ELISA kit (BD bioscience, USA) according to the manufacturer's instructions, and the results are shown in FIG. 8 .

7 is a graph showing the cytotoxicity results of the endoplasmic reticulum of the strain according to one embodiment; CdEV: Clostridium difficile ER, BBH006: ER of Example 1 strain, Type strain: ER of Lactobacillus plantarum standard strain.

8 is a graph showing the activity of reducing inflammation induced by the endoplasmic reticulum Clostridium difficile of the strain according to one embodiment; CdEV: Clostridium difficile ER, BBH006: ER of Example 1 strain, Type strain: ER of Lactobacillus plantarum standard strain.

As shown in Figure 7, cytotoxicity was not confirmed in both the endoplasmic reticulum of the strain and the standard strain according to one embodiment.

In addition, as shown in FIG. 8, the strain according to one embodiment significantly reduced the amount of cytokines increased by the endoplasmic reticulum of Clostridium difficile compared to the standard strain for pro-inflammatory cytokines TNF, CCL2 and IL-6. And it was found.

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

Korea Biological Resources Center (Overseas) KCTC14265BP 20200806

<110> BioBankHealing INC. <120> Lactobacilus plantarum strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of its <130> PN210940 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1489 <212> DNA <213> Artificial Sequence <220> <223> sequence of Lactiplantibacillus plantarum <400> 1 ctaatcatct gtcccacctt aggcggctgg ttcctaaaag gttaccccac cgactttggg 60 tgttacaaac tctcatggtg tgaggggcgg tgtgtacaag gcccgggaac gtattcaccg 120 cggcatgctg atccgcgatt actagcgatt ccgacttcat gtaggcgagt tgcagcctac 180 aatccgaact gagaatggct ttaagagatt agcttactct cgcgagttcg caactcgttg 240 taccatccat tgtagcacgt gtgtagccca ggtcataagg ggcatgatga tttgacgtca 300 tccccacctt cctccggttt gtcaccggca gtctcaccag agtgcccaac ttaatgctgg 360 caactgataa taagggttgc gctcgttgcg ggacttaacc caacatctca cgacacgagc 420 tgacgacaac catgcaccac ctgtatccat gtccccgaag ggaacgtcta atctcttaga 480 tttgcatagt atgtcaagac ctggtaaggt tcttcgcgta gcttcgaatt aaaccacatg 540 ctccaccgct tgtgcgggcc cccgtcaatt cctttgagtt tcagccttgc ggccgtactc 600 cccaggcgga atgcttaatg cgttagctgc agcactgaag ggcggaaacc ctccaacact 660 tagcattcat cgtttacggt atggactacc agggtatcta atcctgtttg ctacccatac 720 tttcgagcct cagcgtcagt tacagaccag acagccgcct tcgccactgg tgttcttcca 780 tatatctacg catttcaccg ctacacatgg agttccactg tcctcttctg cactcaagtt 840 tcccagtttc cgatgcactt cttcggttga gccgaaggct ttcacatcag acttaaaaaa 900 ccgcctgcgc tcgctttacg cccaataaat ccggacaacg cttgccacct acgtattacc 960 gcggctgctg gcacgtagtt agccgtggct ttctggttaa ataccgtcaa tacctgaaca 1020 gttactctca gatatgttct tctttaacaa cagagtttta cgagccgaaa cccttcttca 1080 ctcacgcggc gttgctccat cagactttcg tccattgtgg aagattccct actgctgcct 1140 cccgtaggag tttgggccgt gtctcagtcc caatgtggcc gattaccctc tcaggtcggc 1200 tacgtatcat tgccatggtg agccgttacc ccaccatcta gctaatacgc cgcgggacca 1260 tccaaaagtg atagccgaag ccatctttca agctcggacc atgcggtcca agttgttatg 1320 cggtattagc atctgtttcc aggtgttatc ccccgcttct gggcaggttt cccacgtgtt 1380 actcaccagt tcgccactca ctcaaatgta aatcatgatg caagcaccaa tcaataccag 1440 agttcgttcg acttgcatgt attaggcacg ccgccagcgt tcgtctgac 1489

Claims (10)

Deposited with accession number KCTC 14265BP, Lactobacillus genus ( Lactobacillus sp. ) and Lactobacillus plantarum containing 16S rRNA of SEQ ID NO: 1 ( Lactobacillus plantarum ) strain. delete 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, a lysate of the strain, a 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, a lysate of the strain, a 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 Clostridium difficile comprising a mixture thereof as an active ingredient. Health for improving intestinal health by inhibition or inflammation inhibition functional food. A pharmaceutical composition for preventing or treating Clostridium 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 Clostridium 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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