Microbiome composition of fermentation culture supernatant of plant lactobacillus strain KM2 with anti-inflammatory effect

Abstract

The invention relates to a microbiome composition of a fermentation culture supernatant of a plant lactobacillus strain KM2 with an anti-inflammatory effect. It has been demonstrated that a fermentative culture supernatant of the strain exhibits anti-inflammatory activity, such as inhibition of nitric oxide activity and regulation of expression of inflammatory cytokines, and thus the fermentative culture supernatant can be used as a composition for preventing, treating or ameliorating inflammatory diseases.

Description

Technical Field

[0001] The present disclosure relates to a microbiome composition comprising a fermentation culture supernatant of Lactiplantibacillus plantarum strain KM2 having anti-inflammatory efficacy. Background Art

[0002] Inflammation is a normal, protective in vivo defense mechanism that occurs locally in response to tissue damage caused by physical attack, harmful chemicals, microbial infection, or irritants in in vivo metabolites. Such inflammation is triggered by a variety of chemical mediators produced by damaged tissues and migrating cells, and these chemical mediators are known to vary depending on the type of inflammatory process. Under normal circumstances, the body neutralizes or clears pathogenic factors through the inflammatory response and regenerates damaged tissues, thereby restoring normal structure and function.

[0003] However, when these inflammatory responses occur abnormally, they may develop into diseases such as chronic inflammation. Moreover, when inflammation is triggered by harmless substances such as pollen, or by inappropriate autoimmune reactions such as asthma or rheumatoid arthritis, the defense response itself may instead damage tissues, leading to various diseases.

[0004] Currently, steroid and non-steroid drugs are used as the most common therapies for preventing or treating inflammatory diseases, but most of them have side effects. Therefore, active efforts are being made to develop drugs for inflammatory diseases that overcome the above disadvantages. Summary of the Invention Technical Problem

[0005] An object of the present disclosure is to provide a pharmaceutical composition for preventing or treating inflammatory diseases.

[0006] Another object of the present disclosure is to provide a health functional food composition for preventing or improving inflammatory diseases.

[0007] Still another object of the present disclosure is to provide a method for preventing or treating inflammatory diseases. Technical Solution

[0008] To achieve the above object, the present disclosure provides a pharmaceutical composition for preventing or treating inflammatory diseases, the composition comprising a fermentation culture supernatant of a Lactiplantibacillus plantarum strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof as an active ingredient.

[0009] In addition, the present disclosure provides a health functional food composition for preventing or improving inflammatory diseases, the composition comprising the fermentation culture supernatant of the strain, its concentrate, its dried product, its fermentation metabolite or a mixture thereof as an active ingredient.

[0010] In addition, the present disclosure provides a method for preventing or treating inflammatory diseases, the method comprising treating the following patient groups with the pharmaceutical composition according to item 1: compared with the normal group, the patient group suffers from inflammatory diseases and has a reduced number of one or more strains selected from the group consisting of Weizmannia coagulans, Clostridium perfringens, Clostridium botulinum, Clostridium sporogenes, Clostridium baratii and Clostridium butyricum; or compared with the normal group, the patient group suffers from inflammatory diseases and has an increased number of Bifidobacterium pseudolongum strains. Beneficial effects

[0011] According to the present disclosure, it has been found that the fermentation culture supernatant of Lactiplantibacillus plantarum strain KM2 exhibits anti-inflammatory activity, such as inhibiting nitric oxide activity and regulating the expression of inflammatory cytokines, such that the fermentation culture supernatant can be effectively used as a composition for preventing, treating or improving inflammatory diseases. Description of the drawings

[0012] Figure 1 Shows the analysis results of the cytotoxicity of the fermentation culture supernatant of Lactiplantibacillus plantarum strain KM2 (hereinafter referred to as the sample) in macrophages.

[0013] Figure 2 Shows the analysis results of the effect of the sample on the production of nitric oxide (hereinafter referred to as NO) in macrophages.

[0014] Figure 3 Shows the analysis results of the effect of the sample on the production of prostaglandin E2 (hereinafter referred to as PGE2) in macrophages.

[0015] Figure 4 Shows the analysis results of the effect of the sample on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in macrophages.

[0016] Figure 5The analysis results showing the effects of the sample on the production of pro-inflammatory and anti-inflammatory cytokines in macrophages are presented.

[0017] Figure 6 The analysis results showing the effects of the sample on the transepithelial electrical resistance (TEER) in intestinal epithelial cells are presented.

[0018] Figure 7 The analysis results showing the effects of the sample on the paracellular permeability in intestinal epithelial cells are presented.

[0019] Figure 8 The analysis results showing the effects of the sample on the expression of intestinal junction proteins in macrophages are presented.

[0020] Figure 9 The analysis results showing the effects of the sample on body weight and disease activity index scores in an animal model are presented.

[0021] Figure 10 The analysis results showing the effects of the sample on colon length in an animal model are presented.

[0022] Figure 11 The analysis results showing the effects of the sample on the production of inflammatory cytokines in an animal model are presented.

[0023] Figure 12 The analysis results showing the effects of the sample on intestinal tissue in an animal model are presented.

[0024] Figure 13 The analysis results showing the effects of the sample on intestinal microbiota in an animal model are presented.

[0025] This indicates that at a 95% confidence level, there are statistical differences among all the figures between the treatment groups represented by different letters (such as a, b, etc.). For example, if there is group 1 represented as a, group 2 represented as b, and group 3 represented as ab, then there are statistical differences between group 1 and group 3 and between group 1 and group 2 at the 95% level; while there is no statistical difference between group 2 and group 3. Detailed implementation mode

[0026] Hereinafter, the present disclosure will be elaborated in more detail.

[0028] The present disclosure provides a pharmaceutical composition for preventing or treating inflammatory diseases, which composition contains the fermentation culture supernatant of Lactiplantibacillus plantarum strains, its concentrate, its dried product, its fermentation metabolite, or a mixture thereof as an active ingredient.

[0029] The strain may be Lactiplantibacillus plantarum KM2 strain deposited under the deposit number KCTC 14637BP.

[0030] The inflammatory disease can be one or more selected from the group consisting of ulcerative colitis, ulcerative duodenitis, Crohn's disease, irritable bowel syndrome, intestinal Behcet's disease, hemorrhagic rectal ulcer, pouchitis, enteritis, ischemic colitis, acne, dermatitis with extraintestinal manifestations, atopic dermatitis, allergic dermatitis, seborrheic dermatitis, papular urticaria, eczema, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, pharyngitis, tonsillitis, pneumonia, pancreatitis, gastritis, hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome, and multiple sclerosis, but not limited thereto.

[0031] The pharmaceutical composition may further comprise inactivated cells or spores of Lactiplantibacillus plantarum strain KM2.

[0032] In addition, the pharmaceutical composition can modulate one or more gut microbiota selected from the group consisting of Weissella coagulans, Clostridium perfringens, Clostridium botulinum, Clostridium sporogenes, Clostridium baratii, Clostridium butyricum, and Bifidobacterium pseudolongum.

[0033] In addition, the pharmaceutical composition can inhibit the production or expression of one or more selected from the group consisting of prostaglandin E2, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), or promote the production or expression of one or more selected from the group consisting of ZO-1, Occludin, Claudin-1, and interleukin-10 (IL-10), but not limited thereto.

[0034] According to a method that can be easily implemented by those of ordinary skill in the art to which the present disclosure pertains, the pharmaceutical composition of the present disclosure can be formulated using a pharmaceutically acceptable carrier to be prepared in unit dose form, or can be prepared by encapsulating into multi-dose containers.

[0035] The pharmaceutically acceptable carrier is commonly used in formulations, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate / ester, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present disclosure may further comprise lubricants, humectants, sweeteners, flavoring agents, emulsifying agents, suspending agents, and preservatives.

[0036] In the present disclosure, the content of the additive contained in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range that can be used in conventional preparations.

[0037] The pharmaceutical composition can be formulated in the form of one or more topical skin agents selected from the group consisting of, but not limited to: injectable preparations such as aqueous solutions, suspensions and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes and cataplasms.

[0038] The pharmaceutical composition of the present disclosure may include pharmaceutically acceptable carriers and diluents that are additionally present in the preparation. The pharmaceutically acceptable carriers and diluents include, but are not limited to: excipients such as starch, sugar and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethyl cellulose and hydroxypropyl cellulose; binders such as gelatin, alginate / ester and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol; disintegrants such as polyvinylpyrrolidone and cross-linked polyvinylpyrrolidone; and surfactants such as polysorbate, cetyl alcohol and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically friendly to the subject. Examples of diluents may include, but are not limited to, saline, water-soluble buffers, solvents and / or dispersion media.

[0039] According to the desired method, the pharmaceutical composition of the present disclosure can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically). When administered orally, it can be formulated into tablets, lozenges, troches, water-soluble suspensions, oil-based suspensions, powders, granules, emulsions, hard capsules, soft capsules, syrups and elixirs. When administered parenterally, it can be formulated into injection solutions, suppositories, powders for respiratory inhalation, aerosol sprays, ointments, topical powders, oils and creams.

[0040] The dosage range of the pharmaceutical composition of the present disclosure can vary according to the following and can be appropriately selected by those skilled in the art: the patient's condition, weight, age, gender, health status, dietary structure specificity, nature of the preparation, severity of the disease, time of administration of the composition, administration method, duration or interval of administration, excretion rate and dosage form of the drug. For example, the range can be from about 0.1 mg / kg to 10000 mg / kg, but is not limited thereto, and it can be administered once or several times a day.

[0041] According to the desired method, the pharmaceutical composition can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically). The pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present disclosure can vary depending on the method of preparation of the pharmaceutical composition, the type of administration, the time of administration, and the route of administration, and those of ordinary skill in the art can easily determine and prescribe an effective dose for the desired treatment. The administration of the pharmaceutical composition of the present disclosure can be once daily or in multiple separate doses.

[0043] In addition, the present disclosure provides a health functional food composition for preventing or improving inflammatory diseases, the composition comprising a fermentation culture supernatant of Lactiplantibacillus plantarum strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof as an active ingredient.

[0044] The strain can be Lactiplantibacillus plantarum KM2 strain deposited under the accession number KCTC 14637BP.

[0045] The present disclosure can be widely used as a conventionally used food.

[0046] The food composition of the present disclosure can be used as a health functional food. As used herein, the term "health functional food" refers to a food processed and manufactured using raw materials or ingredients having beneficial functions for the human body in accordance with the Health Functional Food Act; and the term "function" as used herein refers to consumption for the purpose of producing beneficial effects for health purposes, such as regulating nutrients or physiological effects on the human body structure and functions, etc.

[0047] The health functional food composition may contain conventional food additives, and unless otherwise specified, the applicability as a "food additive" is determined according to the general rules and general test methods of the Korean Food Additive Codex approved by the Ministry of Food and Drug Safety of Korea, and the relevant standards and guidelines for the corresponding items.

[0048] Items listed in the Korean Food Additive Codex may include, for example: chemically synthesized compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sorghum pigment, and guar gum; and mixed preparations such as L-glutamate preparation, noodle alkali agent, preservative, and tar colorant.

[0049] The food compositions of the present disclosure can be manufactured or processed into forms such as tablets, capsules, powders, granules, liquids, and pills. For example, a hard capsule preparation among health functional foods in capsule form can be prepared by mixing the composition of the present disclosure with additives such as excipients and filling them into a conventional hard capsule, while a soft capsule preparation can be manufactured by mixing the composition of the present disclosure with additives such as excipients and then filling it into a capsule matrix such as gelatin. If necessary, the soft capsule preparation may contain plasticizers such as glycerol or sorbitol, colorants, and preservatives.

[0050] The terms of excipients, binders, disintegrants, lubricants, flavor enhancers, and flavoring agents are defined in the literature known in the art and include those having the same or similar functions. The type of food is not particularly limited and includes all health functional foods in the general sense.

[0051] As used herein, the term "prevention" refers to any action of inhibiting or delaying an inflammatory disease by administering the composition described in the present disclosure. As used herein, the term "treatment" refers to any action of improving the condition of an inflammatory disease or making it better by administering the composition described in the present disclosure. As used herein, the term "improvement" refers to any action of making the adverse condition of an inflammatory disease better by administering the composition described in the present disclosure.

[0053] Furthermore, the present disclosure provides a method for preventing or treating an inflammatory disease, the method comprising treating the following patient groups with the pharmaceutical composition: compared with a normal group, the patient group suffers from an inflammatory disease and has a reduced number of one or more strains selected from the group consisting of Weissella coagulans, Clostridium perfringens, Clostridium botulinum, Clostridium sporogenes, Clostridium baratii, and Clostridium butyricum; or compared with a normal group, the patient group suffers from an inflammatory disease and has an increased number of Bifidobacterium pseudolongum strains. Examples

[0054] The present disclosure will be described in more detail below through examples to help understand the present disclosure. However, the following examples are only for illustrating the present disclosure, and the scope of the present disclosure is not limited to the following examples. The examples of the present disclosure are provided to more completely explain the present disclosure to those skilled in the art.

[0056] [Experimental Example 1] Sample Preparation

[0057] To prepare the sample for this experiment, the fermentation culture supernatant of the KM2 strain (Lactiplantibacillus plantarum KM2), the KM2 storage strain stored at -70 °C was activated, followed by primary seed culture in test tubes and flasks. 2% (v / v) of it was inoculated into a 50 L fermenter with a working volume of 20 L and secondary seed culture was carried out for 6 hours. This culture was carried out in a 500 L fermenter with a working volume of 350 L at an inoculation amount of 2% (v / v) for 12 hours, and glucose was additionally supplemented once after 6 hours of this culture. After completion of the culture, the cell slurry was removed by primary centrifugation in a disk centrifuge at 7200 rpm and 2 L / min; and the supernatant was subjected to secondary centrifugation in a tubular centrifuge at 15000 rpm and 1.5 L / min to remove cell pellet blocks and recover the supernatant. The recovered supernatant was filtered through a 0.2 μm sterile filter to obtain the fermentation culture supernatant of the KM2 strain, and the bacterial cells were finally removed from the supernatant. 3% (w / v) trehalose was added to the sterile fermentation culture supernatant as an excipient, and then freeze-dried for 96 - 120 hours to obtain the powdered fermentation culture supernatant.

[0059] [Experimental Example 2] Cell Culture

[0060] Raw 264.7 cells, a macrophage cell line derived from mice, were obtained from the Korean Cell Line Bank (KCLB). Using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (hereinafter referred to as P / S), the cells were passaged every two days in an incubator at 37 °C with 5% CO2.

[0061] IPEC-J2 cells, porcine intestinal epithelial cells, were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). Using Dulbecco's Modified Eagle Medium - Nutrient Mixture F-12 (DMEM / F-12) medium supplemented with 10% FBS and 1% P / S, the cells were passaged every two days in an incubator at 37 °C with 5% CO2.

[0063] [Experimental Example 3] Preparation of Animal Model

[0064] C57BL / 6 mice were purchased from Orient Bio. Healthy animals were used in the experiments by checking the number of mice, observing general symptoms, measuring body weight, and verifying the test results provided by the animal supplier. All animals were checked for abnormalities and given a 7-day acclimation period to adapt to the animal housing environment. During the acclimation period, the general symptoms of all animals were observed once a day, and the body weight change was checked on the last day of the acclimation period to evaluate the animal health status. The animals were divided into four groups of nine animals each to ensure equal average body weight in each experimental group. Individual markings were made on the tails of the animals using a five-color permanent marker, and individual identification cards were pasted on the breeding cages. The animal models were raised under the following conditions and provided with food and drinking water: temperature 20°C to 26°C, relative humidity 30% to 70%, and a 12-hour light-dark cycle (9:00 am to 9:00 pm). The animals were fed ad libitum with solid feed for laboratory animals (Lab DFiet #5053 PMI Nutrition International, U.S.A). The animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of the Korea Food Industry Cluster (IACUC-22-014).

[0066] [Experimental Example 4] Statistical Analysis

[0067] All experimental results were expressed as mean ± standard error of the mean (mean ± SEM), and all statistical analyses were performed using the SAS program (version 9.4 SAS Institute Inc., Cary, NC, USA). The significant differences between experimental groups (p < 0.05) were analyzed by one-way analysis of variance and Duncan's multiple range test.

[0069] [Example 1] Physiological Activity Analysis of Samples (In Vitro)

[0070] 1-1. Cytotoxicity Assay

[0071] To identify the cytotoxicity of the samples in macrophages and intestinal epithelial cells, the EZ-Cytox kit (DAEIL lab, Korea) was used to determine the sample concentration affecting cell viability based on the decrease in absorbance according to the sample treatment concentration compared to the control group (the group not treated with the sample; control). Raw 264.7 and IPEC-J2 cells were seeded at 1 × 10 cells per well respectively 4Cells were seeded at a density of [number of cells] per well in a 96-well plate and cultured at 37 °C in an incubator with 5% CO2 for 24 hours. Subsequently, they were treated with different concentrations of the sample and then cultured for another 24 hours. Then, the medium was removed, 90 μL of DMEM and 10 μL of EZ-Cytox reagent were mixed and dispensed, and the reaction was carried out at 37 °C in an incubator with 5% CO2 in the dark for 1 hour. After that, the absorbance was measured at 450 nm using a microplate reader, and the cell viability was calculated using the following formula 1.

[0072] [Formula 1]

[0073] Cell viability = (Absorbance of control group / Absorbance of sample-treated group) × 100

[0074] The results were as Figure 1 shown. In the case of Raw 264.7 cells, no significant cytotoxicity was observed in the groups treated with 0.1 μL / mL to 50 μL / mL of the sample; while in the case of IPEC-J2 cells, no significant cytotoxicity was observed in the groups treated with 0.1 μL / mL to 10 μL / mL of the sample. Based on the above results, the biological activity of the sample was experimented within the concentration range where no cytotoxicity was observed.

[0076] 1-2. Analysis of NO production

[0077] To determine the effect of the sample on the production of nitric oxide (NO), the NO activity was measured using a nitric oxide (NO) detection kit (Promega Corp, USA, catalog number: G2930). Raw 264.7 cells were seeded at a density of 2.5×10 5 cells per well in a 24-well plate and cultured at 37 °C in an incubator with 5% CO2 for 24 hours. Then, 1 μg / mL of lipopolysaccharide (LPS, Sigma-Aldrich, USA, derived from Escherichia coli 055:B5) and different concentrations of the sample were diluted with DMEM medium supplemented with 1% FBS and 1% P / S, and the culture was carried out for 24 hours. Subsequently, the amount of NO produced during cell culture was measured. For the amount of NO produced, according to the kit instructions, 50 μL of cell culture medium was mixed with 100 μL of Griess reagent (Sigma-Aldrich) and reacted at room temperature for 10 minutes. The absorbance of the colored reaction solution was measured at 520 nm using a microplate reader, and the nitrite concentration was calculated through a standard curve.

[0078] The results were as Figure 2As shown, compared with the control group (the group not treated with LPS and the sample), the production of NO in the group treated only with LPS increased significantly; while compared with the group treated only with LPS, the production of NO in the sample-treated group decreased significantly in a concentration-dependent manner.

[0080] 1-3. Analysis of the inhibitory activity on PGE2 production

[0081] To identify the effect of the sample on the production of prostaglandin E2 (PGE2), Raw 264.7 cells were seeded in 24-well plates at a density of 2.5×10 5 cells per well and cultured in an incubator with 5% CO2 at 37°C for 24 hours. Then, 1 μg / mL lipopolysaccharide (LPS, Sigma-Aldrich, USA, derived from Escherichia coli 055:B5) and different concentrations of the sample were diluted with DMEM medium supplemented with 1% FBS and 1% P / S, and the culture was carried out for 24 hours. After that, each cell culture medium was taken, centrifuged at 13,000 rpm for 10 minutes, and the PGE2 content in the supernatant obtained by centrifugation was measured. All samples were stored frozen at -20°C until quantification. PGE2 was quantified using a mouse enzyme-linked immunosorbent assay (ELISA) kit (Catalog No.: KGE004B, R&D Systems Inc., Minneapolis, MN, USA), and the R2 value of the standard curve of the standard was above 0.99.

[0082] The results are as Figure 3 shown. Compared with the control group (the group not treated with LPS and the sample), the production of PGE2 in the group treated only with LPS increased significantly; while compared with the group treated only with LPS, the production of PGE2 in the sample-treated group decreased significantly.

[0084] 1-4. Analysis of the inhibitory activity on iNOS and COX-2 expression

[0085] To determine the effects of the sample on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), qPCR was performed. Total RNA was isolated from Raw 264.7 cells using the Trizol reagent kit (Invitrogen, USA), and the RNA was precipitated with 100% isopropanol and then washed with 75% ethanol. The extracted RNA was dissolved in nuclease-free distilled water, and its concentration was measured using a micro-spectrophotometer. cDNA was synthesized using the Maxime RT premix (Oligo dt 15 primer, iNtRON Biotechnology, Korea) according to the method provided by the manufacturer. To analyze the gene expression of iNOS (NCBI gene ID: 396859) and COX-2 (NCBI gene ID: 808504), the KAPA SYBR fast qPCR kit (KAPA biosystems, USA) was used. The qPCR conditions were set as follows: 1 cycle at 95 °C for 5 minutes; 35 cycles of 20 seconds at 96 °C, 20 seconds at 60 °C, and 20 seconds at 72 °C; and 1 cycle at 72 °C for 5 minutes. Data analysis was performed using the Light Cycler 96 software provided by the manufacturer (Roche Applied Science). The quantitative results were represented by comparing with the reference mRNA (GAPDH) using the 2 -ΔΔCT -ΔΔCt method (Livak & Schmittgen, 2001).

[0086] The results are as Figure 4 shown. Compared with the control group (the group untreated with LPS and the sample), the expression of iNOS and COX-2 in the group treated with LPS alone was significantly increased; while compared with the group treated with LPS alone, the expression of iNOS and COX-2 in the sample-treated group was significantly decreased in a concentration-dependent manner.

[0088] 1-5. Analysis of Pro-inflammatory and Anti-inflammatory Cytokine Production

[0089] To identify the effects of the sample on the production of pro-inflammatory cytokines and anti-inflammatory cytokines, ELISA kits were used to detect the production of these cytokines against the following: IL-1β (NCBI Gene ID: 16176) (Interleukin-1β, Catalog number: MLB00C), TNF-α (NCBI Gene ID: 21926) (Tumor necrosis factor-α, Catalog number: MTA00B), IL-6 (NCBI Gene ID: 16193) (Interleukin-6, Catalog number: DY406-05), and IL-10 (NCBI Gene ID: 16153) (Interleukin-10, Catalog number: M1000B-1). According to the kit instructions, the experiment was conducted using the Raw 264.7 cell culture supernatant. The absorbance of the colored reaction solution was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The concentration of each cytokine was calculated using a standard curve.

[0090] The results are as Figure 5 shown. Compared with the control group (the group untreated with LPS and the sample), the production of IL-1β, IL-6, and TNF-α in the group treated with only LPS was significantly increased; while compared with the group treated with only LPS, the production of these three cytokines in the sample-treated group was significantly decreased. In addition, the production of IL-10 in both the group treated with only LPS and the sample-treated group was significantly increased, and the increase in the production of IL-10 in the sample-treated group was more significant compared with the group treated with only LPS.

[0092] 1-6. Analysis of Trans-epithelial Electrical Resistance (TEER)

[0093] To identify the effects of the sample on Trans-epithelial Electrical Resistance (TEER), a Millicell-ERS-2 instrument (Millipore, MA, USA) was used to analyze the TEER of the IPEC-J2 cell monolayer cultured on a 0.4 μm Transwell membrane (Corning, NY, USA). The IPEC-J2 cells were seeded into 24 Transwell chambers at a density of 2 × 10 5 cells per well and cultured at 37 °C in the presence of 5% CO2 for 24 hours. Subsequently, the upper chamber was replaced with 500 μL of a specific concentration of the sample, the lower chamber was replaced with 1.5 mL of medium, and the culture plate was incubated for an additional 24 hours (37 °C, 5% CO2, and 95% humidity). Then, it was treated with 1 μg / mL lipopolysaccharide (LPS, Sigma-Aldrich, USA, derived from Escherichia coli 055:B5) dissolved in Hank's balanced salt solution (HBSS), and the TEER values were measured regularly using a Miller-ERS instrument (Millipore, MA, USA) immediately after sample treatment. The TEER value was calculated using the following formula 2.

[0094] [Formula 2]

[0095] TEER = Resistance value (Ohm) × Filter area (cm 2 ) = Ω × cm 2

[0096] The results were as Figure 6 shown. The TEER values in all experimental groups decreased over time, and the TEER value in the group treated with only LPS decreased most significantly, indicating that the decrease in the TEER value in the sample-treated group was inhibited compared to the group treated with only LPS.

[0098] 1-7. Paracellular permeability assay

[0099] To identify the effect of the sample on paracellular permeability, IPEC-J2 cells were treated with 1 μg / mL LPS for 48 hours, and then paracellular permeability was measured using 4 kDa FITC-dextran (Sigma-Aldrich, USA). For the LPS-treated cells, FITC-dextran was dissolved in HBSS and aliquoted into the upper chamber at a concentration of 1 mg / mL, followed by incubation for 4 hours, and then paracellular permeability was measured using a Tecan microplate reader (excitation wavelength 492 nm; emission wavelength 520 nm, Tecan Group Ltd., Switzerland).

[0100] The results were as Figure 7 shown. Compared with the control group (the group untreated with LPS and the sample), the flux of FITC-dextran in the group treated with only LPS increased significantly; while compared with the group treated with only LPS, the flux of FITC-dextran in the sample-treated group decreased significantly in a concentration-dependent manner.

[0102] 1-8. Analysis of intestinal junction protein expression

[0103] To identify the effect of the sample on the expression of intestinal junction proteins, IPEC-J2 cells cultured with LPS for 48 hours were washed once with Dulbecco's phosphate-buffered saline (DPBS). PRO-PREP reagent was dispensed at 100 μL to 150 μL per well, and a cell scraper was used to collect the cells into a test tube. The collected cells were placed on ice for 30 minutes and then centrifuged at 4 °C and 13,000 rpm for 5 minutes to obtain the supernatant. Subsequently, the protein in the supernatant was quantified using the Bradford assay, and 50 μg of protein was separated by 8% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Then, it was blocked with a blocking buffer (4% skim milk powder, 10 mM Tris, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 hour, followed by washing 3 times with 1% Tween 20-PBS for 15 minutes each. Antibodies (primary antibodies) against ZO-1 (NCBI gene ID: 100736682), occludin (NCBI gene ID: 397236), claudin-1 (NCBI gene ID: 100625166), and β-actin were treated at room temperature for 3 hours and then washed with 1% Tween 20-PBS. Subsequently, the secondary antibody was treated for 1 to 2 hours, followed by washing, and the protein expression levels of zonula occludens protein 1 (ZO-1), occludin, and claudin-1 were measured using an enhanced chemiluminescence (ECL) kit with a LAS 4000 instrument for detection.

[0104] The results are as Figure 8 shown. Compared with the control group (the group not treated with LPS and the sample), the protein expressions of ZO-1, occludin, and claudin-1 in the group treated with only LPS decreased; while compared with the group treated with only LPS, the expressions of these three proteins in the sample-treated group increased in a concentration-dependent manner.

[0106] [Example 2] Analysis of the Physiological Activity of the Sample (In Vivo)

[0107] 2-1. Construction of an Animal Model Induced with Enteritis

[0108] To construct an animal model induced with enteritis, 3% dextran sulfate sodium (DSS) salt was provided as drinking water to the animal model in Experimental Example 3 to induce enteritis. 50 mg / kg of 5-aminosalicylic acid (5-ASA) was used as a positive control, and starting from the intake of 3% DSS, the sample and 5-ASA were orally administered to the animal model's stomach once a day using a disposable syringe (1 mL) connected to an oral administration probe for 5 weeks. The experimental groups were specifically set as follows:

[0109] 1) Normal group (CON)

[0110] 2) Negative control group (DSS): Administered 3% DSS

[0111] 3) Positive control group (DSS + ASA): Administered 3% DSS + orally administered 50 mg / kg 5-ASA

[0112] 4) Sample treatment group (DSS + KM2): Administered 3% DSS + orally administered 2 g / kg sample

[0114] 2-2. Analysis of body weight and disease activity index score

[0115] To identify the effect of the sample on body weight and disease activity index (DAI) score, the body weight of the animal model was measured once a day for a total of 5 weeks (DSS administration period), and the fecal condition (fecal form and blood stool color) was examined using a blood stool kit to measure the disease activity index score (fecal form, blood stool color, and weight loss).

[0116] The results were as Figure 9 shown. Compared with the normal group (CON), the body weight decreased in the negative control group (DSS); while compared with the negative control group, the body weight increased in the positive control group (DSS + ASA) and the sample treatment group (DSS + KM2). In addition, since the 5th day of induced colitis (DSS administration), compared with the normal group, the weight loss and blood in the stool significantly increased in the negative control group, positive control group, and sample treatment group, resulting in an increase in the disease activity index score, while compared with the negative control group, the disease activity index score decreased in the positive control group and the sample treatment group.

[0118] 2-3. Analysis of colon length

[0119] To identify the effect of the sample on colon length, the animal model was fasted for 24 hours, sacrificed, and dissected to collect blood from the heart, and then its appearance, thoracic cavity, and abdominal cavity were observed macroscopically. After that, the colon was removed and its length was measured.

[0120] The results were as Figure 10 shown. Compared with the normal group (CON), the colon length significantly shortened in the negative control group (DSS); while compared with the negative control group, the colon length increased in the positive control group (DSS + ASA) and the sample treatment group (DSS + KM2).

[0122] 2-4. Blood biochemical analysis (analysis of inflammatory cytokine production)

[0123] To identify the effect of the sample on the production of inflammatory cytokines, the animal model was sacrificed and blood was then collected from the heart, and immediately centrifuged to isolate the serum, which was then stored in an ultra-low temperature freezer at -80 °C until analysis. Subsequently, a spectrophotometer and an analysis kit were used to measure the cytokine content.

[0124] The results are as Figure 11 shown. Compared with the normal group (CON), the production of IL-6, IL-1β, and TNF-α in the negative control group (DSS) was significantly increased, while the production of IL-10 was significantly decreased. In contrast, compared with the negative control group, the production of IL-6, IL-1β, and TNF-α in the positive control group (DSS+ASA) and the sample treatment group (DSS+KM2) was significantly decreased, while the production of IL-10 was significantly increased.

[0126] 2-5. Histological analysis

[0127] To identify the effect of the sample on the intestinal tissue, the colon tissue of the animal model was fixed with 10% formaldehyde, made into paraffin blocks, and stained with hematoxylin & eosin (H&E). After photographing the stained tissue sections, quantitative image analysis was performed. In addition, the degree of intestinal mucus secretion was analyzed by performing Alcian blue staining.

[0128] The results are as Figure 12 shown. Compared with the normal group (CON), the structure and size of the epithelial tissue forming the colon surface in the negative control group (DSS) changed irregularly and the infiltration of inflammatory cells increased. On the other hand, it was found that compared with the negative control group, the symptoms that appeared in the negative control group were improved in the positive control group (DSS+ASA) and the sample treatment group (DSS+KM2). In addition, compared with the normal group in which the mucosal layer was stained blue, the degree of blue staining in the negative control group decreased, while compared with the negative control group, the degree of blue staining in the positive control group and the sample treatment group increased.

[0130] 2-6. Intestinal microbiota analysis

[0131] To identify the effect of the sample on the intestinal microbiota, the animal model was sacrificed, the cecum was removed, and shotgun metagenomic sequencing was used to analyze the changes in the intestinal microbiota.

[0132] The results are as Figure 13As shown, in the results of alpha diversity analysis that allows the identification of the microbial diversity present in a single sample, no significant differences in the Shannon index were found between the experimental groups. In addition, there were no significant differences in the Firmicutes / Bacteroidetes (F / B) ratio, which is an inflammatory indicator, between the experimental groups. The results of beta diversity analysis based on unweighted UniFrac principal coordinate analysis (PCA), which allows the identification of microbial colony patterns, showed significant differences between the experimental groups. To identify the significantly different microbiomes between the experimental groups, as a result of performing linear discriminant analysis (LDA) and KEGG pathway analysis, as Figure 13 shown, compared with the negative control group (DSS), the numbers of Weissella coagulans, Clostridium perfringens, Clostridium botulinum, Clostridium sporogenes, Clostridium baratii, and Clostridium butyricum strains in the sample treatment group (DSS+KM2) were significantly increased. In addition, compared with the sample treatment group, the number of Bifidobacterium pseudolongum strains in the negative control group was significantly increased.

[0134] Although specific parts of the present disclosure have been described in detail above, it will be apparent to those skilled in the art that the specific description is only a preferred exemplary embodiment, but the scope of the present disclosure is not limited thereto. In other words, the substantial scope of the present disclosure is defined by the appended claims and their equivalents.

[0135] (Translation) Receipt Certificate in the Original Deposit Situation Recipient: Cheng Wenxi Gukmin University, 77 Jeongneung-ro, Seongbuk-gu, Seoul, Korea

Claims

1. A pharmaceutical composition for preventing or treating inflammatory diseases, said composition comprising a fermentation culture supernatant of Lactiplantibacillus plantarum strain, its concentrate, its dried product, its fermentation metabolite or a mixture thereof as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein, The strain is Lactiplantibacillus plantarum KM2 strain deposited under the accession number KCTC 14637BP.

3. The pharmaceutical composition according to claim 1, wherein, The inflammatory diseases are one or more selected from the group consisting of ulcerative colitis, ulcerative duodenitis, Crohn's disease, irritable bowel syndrome, intestinal Behcet's disease, hemorrhagic rectal ulcer, pouchitis, enteritis, ischemic colitis, acne, dermatitis with extraintestinal manifestations, atopic dermatitis, allergic dermatitis, seborrheic dermatitis, papular urticaria, eczema, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iridocyclitis, pharyngitis, tonsillitis, pneumonia, pancreatitis, gastritis, hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome and multiple sclerosis.

4. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition further comprises inactivated cells or spores of the Lactiplantibacillus plantarum KM2 strain.

5. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition modulates one or more intestinal microorganisms selected from the group consisting of Weissella coagulans, Clostridium perfringens, Clostridium botulinum, Clostridium sporogenes, Clostridium baratii, Clostridium butyricum and Bifidobacterium pseudolongum.

6. A health functional food composition for preventing or improving inflammatory diseases, said composition comprising a fermentation culture supernatant of Lactiplantibacillus plantarum strain, its concentrate, its dried product, its fermentation metabolite or a mixture thereof as an active ingredient.

7. The health functional food composition according to claim 1, wherein, The strain is Lactiplantibacillus plantarum KM2 strain deposited under the accession number KCTC14637BP.

8. A method for preventing or treating inflammatory diseases, said method comprising treating the following patient groups with the pharmaceutical composition according to claim 1: compared with the normal group, the patient group suffers from inflammatory diseases and has a reduced number of one or more strains selected from the group consisting of Weissella coagulans, Clostridium perfringens, Clostridium botulinum, Clostridium sporogenes, Clostridium baratii and Clostridium butyricum; or compared with the normal group, the patient group suffers from inflammatory diseases and has an increased number of Bifidobacterium pseudolongum strains.

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