WO2024085623A1 - Microbiome composition of fermentation culture supernatant of lactiplantibacillus plantarum strain km2 having anti-inflammatory effect - Google Patents

Abstract

The present invention relates to a microbiome composition of a fermentation culture supernatant of Lactiplantibacillus plantarum strain KM2 having an anti-inflammatory effect. It has been confirmed that the fermentation culture supernatant of the strain exhibits anti-inflammatory activity, such as suppressing nitric oxide activity and controlling the expression of inflammatory cytokines, and thus, the fermentation culture supernatant can be useful as a composition for preventing, treating, or ameliorating inflammatory diseases.

Description

Microbiome composition of fermentation culture supernatant of Lactiplantibacillus plantarum KM2 strain with anti-inflammatory effect

The present invention relates to a microbiome composition of the fermentation culture supernatant of Lactiplantibacillus plantarum KM2 ( Lactiplantibacillus plantarum KM2) strain, which has anti-inflammatory effects.

Inflammation is the expression of a normal, protective defense mechanism in the body that appears locally in response to tissue damage caused by physical trauma, harmful chemicals, infection by microorganisms, or irritating substances in the body's metabolites. This inflammation is triggered by various chemical mediators produced from damaged tissues and migrating cells, and these chemical mediators are known to come in many different types depending on the type of inflammatory process. In normal cases, the body neutralizes or eliminates pathogenic factors through an inflammatory response and regenerates damaged tissues to restore normal structure and function.

However, if this inflammatory reaction occurs abnormally, it may progress to a disease such as chronic inflammation, and if inflammation is triggered inappropriately by harmless substances such as pollen or an autoimmune reaction such as asthma or rheumatoid arthritis, the defense reaction itself Rather, it damages tissues and causes various diseases.

Currently, steroidal and non-steroidal therapeutic agents are largely used as the most common preventive or therapeutic agents for inflammatory diseases, but most of them have the problem of side effects, and the development of inflammatory disease therapeutic agents that overcome the above problems is actively underway. am.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases.

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

Another object of the present invention is to provide a method for preventing or treating inflammatory diseases.

In order to achieve the above object, the present invention prevents inflammatory diseases comprising the fermentation culture supernatant of Lactiplantibacillus plantarum strain, its concentrate, its dried product, its fermentation metabolite, or a mixture thereof as an active ingredient. Alternatively, a pharmaceutical composition for treatment is provided.

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

In addition, the present invention compares to the normal group, Weizmannia coagulans , Welch's bacillus ( clostridium perfringens ), Clostridium botulinum , Clostridium sporogenis, Clostridium sporogenes A group of patients with inflammatory diseases in which the number of at least one strain selected from the group consisting of clostridium baratii and clostridium butyricum is reduced; Alternatively, a method for preventing or treating an inflammatory disease is provided, comprising treating a group of patients with an inflammatory disease in which the number of Bifidobacterium pseudolongum strains is increased compared to a normal group with the pharmaceutical composition of claim 1.

According to the present invention, it was confirmed that the fermentation culture supernatant of the Lactiplantibacillus plantarum KM2 strain exhibits anti-inflammatory activity, such as suppressing nitric oxide activity and controlling the expression of inflammatory cytokines. By doing so, it can be usefully used as a composition for preventing, treating, or improving inflammatory diseases.

Figure 1 shows the results of analyzing the cytotoxicity of the fermentation culture supernatant (hereinafter referred to as sample) of the Lactiplantibacillus plantarum KM2 strain in macrophages.

Figure 2 shows the results of analyzing the effect of samples on the production of nitric oxide (hereinafter referred to as NO) in macrophages.

Figure 3 shows the results of analyzing the effect of samples on the production of prostaglandin E2 (Prostaglandin E2; hereinafter referred to as PGE2) in macrophages.

Figure 4 shows the results of analyzing the effect of samples on the expression of iNOS (Inducible nitric oxide synthase) and COX-2 (Cyclooxygenase-2) in macrophages.

Figure 5 shows the results of analyzing the effect of samples on the production of pro-inflammatory and anti-inflammatory cytokines in macrophages.

Figure 6 shows the results of analyzing the effect of samples on TEER (Trans-epithelial electrical resistance) in intestinal epithelial cells.

Figure 7 shows the results of analyzing the effect of samples on paracellular permeability in intestinal epithelial cells.

Figure 8 shows the results of analyzing the effect of samples on intestinal junction protein expression in macrophages.

Figure 9 shows the results of analyzing the effect of samples on body weight and disease activity in animal models.

Figure 10 shows the results of analyzing the effect of samples on colon length in an animal model.

Figure 11 shows the results of analyzing the effect of samples on inflammatory cytokine production in an animal model.

Figure 12 shows the results of analyzing the effect of samples on intestinal tissue in an animal model.

Figure 13 shows the results of analyzing the effect of samples on intestinal microorganisms in an animal model.

In all figures, there is a statistical difference at the 95% confidence level between treatment groups indicated with different alphabets (ex. a, b, etc.). For example, if there is group 1 denoted by a, group 2 denoted by b, and group 3 denoted by ab, there is a statistical difference at the 95% level between groups 1 and 3, and between groups 1 and 2; And it means that there is no statistical difference between groups 2 and 3.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising as an active ingredient a fermentation culture supernatant of Lactiplantibacillus plantarum strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof. do.

The strain may be Lactiplantibacillus plantarum KM2 ( Lactiplantibacillus plantarum KM2) deposited with the deposit number KCTC 14637BP.

The inflammatory diseases include ulcerative colitis, ulcerative duodenitis, Crohn's disease, irritable bowel syndrome, intestinal Behcet's disease, and bleeding rectal ulcers ( hemorrhagic rectal ulcer, pouchitis, enteritis, ischemic colitis, acne, extra-intestinal manifestations dermatitis, 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, Sjögren's syndrome. It may be one or more selected from the group consisting of multiple sclerosis, but is not limited thereto.

The pharmaceutical composition may further include dead cells or spores of the Lactiplantibacillus plantarum KM2 strain.

In addition, the pharmaceutical composition may be used against Weizmannia coagulans , Welch's bacillus ( clostridium perfringens) , Clostridium botulinum , Clostridium sporogenes , Clostridium baratii ( It is possible to control one or more intestinal microorganisms selected from the group consisting of clostridium baratii , clostridium butyricum , and bifidobacterium pseudolongum .

In addition, the pharmaceutical composition contains prostaglandin E2 (Prostaglandin E2), iNOS (Inducible nitric oxide synthase), COX-2 (Cyclooxygenase-2), IL-1β (Interleukin-1β), TNF-α (tumor necrosis factor-α), and Inhibit the production or expression of one or more selected from the group consisting of IL-6 (Interleukin-6), or the production or expression of one or more selected from the group consisting of ZO-1, Occludin, Claudin-1 and IL-10 (Interleukin-10) can promote, but is not limited to this.

The pharmaceutical composition of the present invention is prepared in unit dose form or in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by those skilled in the art. It can be manufactured by internalizing it.

The pharmaceutically acceptable carriers are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Includes, but is not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

In the present invention, the content of additives included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

The pharmaceutical compositions include injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniment agents, paste agents, and cataplasmase agents. It may be formulated in the form of one or more external skin preparations selected from the group consisting of, but is not limited to this.

The pharmaceutical composition of the present invention may additionally contain pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include excipients such as starch, sugar and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, gelatin, alginate, polyvinyl pyrrolidone. It includes, but is not limited to, binders such as talc, calcium stearate, lubricants such as hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone and crospovidone, and surfactants such as polysorbate, cetyl alcohol, glycerol, etc. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically friendly to the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and/or dispersion media.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method. For oral administration, it can be formulated as tablets, troches, lozenges, aqueous suspensions, oily suspensions, powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it can be formulated as an injection, suppository, powder for respiratory inhalation, aerosol for spray, ointment, powder for application, oil, cream, etc.

The dosage of the pharmaceutical composition of the present invention is determined by the patient's condition, weight, age, gender, health, dietary constitution specificity, nature of the preparation, degree of disease, administration time of the composition, administration method, administration period or interval, excretion rate, and The range may vary depending on the drug form and can be appropriately selected by a person skilled in the art. For example, it may range from about 0.1 to 10,000 mg/kg, but is not limited and may be administered once or in divided doses several times a day.

The pharmaceutical composition may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally, or topically applied) depending on the desired method. The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time, administration route, etc. of the pharmaceutical composition, and those skilled in the art will know that it is effective for the desired treatment. Dosage can be easily determined and prescribed. The pharmaceutical composition of the present invention may be administered once a day, or may be administered in several divided doses.

In addition, the present invention provides a health function for preventing or improving inflammatory diseases comprising the fermentation culture supernatant of Lactiplantibacillus plantarum strain, its concentrate, its dried product, its fermentation metabolite, or a mixture thereof as an active ingredient. A food composition is provided.

The strain may be Lactiplantibacillus plantarum KM2 ( Lactiplantibacillus plantarum KM2) deposited with the deposit number KCTC 14637BP.

The present invention can be generally used with commonly used foods.

The food composition of the present invention can be used as a health functional food. The term “health functional food” refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with the Health Functional Food Act, and “functionality” refers to food that is related to the structure and function of the human body. It means ingestion for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects.

The health functional food composition may contain common food additives, and its suitability as a “food additive” is determined in accordance with the general provisions and general test methods of the food additive code approved by the Ministry of Food and Drug Safety, unless otherwise specified. The decision is made based on the specifications and standards for the item.

Items listed in the “Food Additives Code” include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as subchromic pigment, licorice extract, crystalline cellulose, high-liquid pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations.

The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among health functional foods in the form of capsules, hard capsules can be manufactured by mixing and filling the composition according to the present invention with additives such as excipients in a regular hard capsule, and soft capsules can be manufactured by mixing and filling the composition according to the present invention. It can be manufactured by mixing with additives such as excipients and filling it with a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary.

Definitions of terms such as excipients, binders, disintegrants, lubricants, coagulants, flavoring agents, etc. are described in literature known in the art and include those with the same or similar functions. There is no particular limitation on the type of food, and it includes all health functional foods in the conventional sense.

In the present invention, the term “prevention” refers to all actions that suppress or delay inflammatory diseases by administering the composition according to the present invention. In the present invention, the term “treatment” refers to any action that improves or beneficially changes the symptoms of an inflammatory disease by administering the composition according to the present invention. In the present invention, the term “improvement” refers to all actions that improve the bad state of an inflammatory disease by administering the composition according to the present invention.

In addition, the present invention compares to the normal group, Weizmannia coagulans , Welch's bacillus ( clostridium perfringens) , Clostridium botulinum , Clostridium sporogenes , Clos. A group of patients with inflammatory diseases in which the number of at least one strain selected from the group consisting of clostridium baratii and clostridium butyricum is reduced; Alternatively, a method for preventing or treating an inflammatory disease is provided, comprising treating the pharmaceutical composition to a group of patients with an inflammatory disease in which the number of Bifidobacterium pseudolongum strains is increased compared to a normal group.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

[Experimental Example 1] Sample preparation

To prepare the fermentation culture supernatant of the KM2 strain ( Lactiplantibacillus plantarum KM2), which is a sample of this experiment, the KM2 stock strain stored at -70°C was activated and subjected to primary seed culture in a test tube and flask, followed by 20L in a 50L fermenter. 2% (v/v) was inoculated into the working volume and secondary seed culture was performed for 6 hours. This culture was incubated for 12 hours by inoculating 2% (v/v) in a 350L working volume in a 500L fermenter, and glucose was additionally fed once at 6 hours of culture. After completion of the culture, the cell slurry was removed by first centrifugation at 7200 rpm and 2 L/min in a disk centrifuge, and the supernatant was second centrifuged at 15000 rpm and 1.5 L/min in a tubular centrifuge to form a cell cake. It was removed and the supernatant was recovered. The recovered supernatant was filtered through a 0.2 μm sterilization filter to obtain a fermentation culture supernatant of the KM2 strain from which the bacterial cells were finally removed. 3% (w/v) of trehalose was added as an excipient to the sterilized fermentation culture supernatant and freeze-dried for 96 to 120 hours to obtain a powdered fermentation culture supernatant.

[Experimental Example 2] Cell culture

Raw 264.7 cells, a mouse-derived macrophage cell line, were purchased from the Korean cell line bank (KCLB) and used. Cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (fetal bovine serum) and 1% penicillin/streptomycin (hereinafter referred to as P/S) at 37°C and 5% _CO2 . Subculture was carried out once every two days in a conditioned incubator.

IPEC-J2 cells, porcine intestinal epithelial cells, were purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). Cells were cultured in DMEM/F-12 (Dulbecco's Modified Eagle's Medium-Nutrient-Mixture F-12 medium) medium supplemented with 10% FBS and 1% P/S for 2 days in an incubator under 5% CO ₂ and 37°C conditions. Subcultured once a year.

[Experimental Example 3] Animal model preparation

C57BL/6 mice were purchased from Orient Bio. Healthy animals were used in the experiment by confirming the mouse population, observing general symptoms, measuring body weight, and confirming the test report provided by the animal supplier. All animals were checked for any abnormalities and were given an acclimatization period of 7 days to adapt to the animal room environment. During the acclimation period, all animals were observed for general symptoms once a day, and the health status of the animals was evaluated by checking body weight changes on the end of acclimatization. To ensure that the average weight of each experimental group was equal, they were separated into 4 groups with 9 animals per group. The animal's tail was marked as an individual using five-color permanent marker, and an individual identification card was attached to the breeding box. The animal model was raised at a temperature of 20-26°C, relative humidity of 30-70%, and light/dark cycle of 12 hours/day (9:00 AM to 9:00 PM), and was supplied with food and drinking water. Solid feed for laboratory animals (Lab DFiet #5053 PMI Nutrition International, U.S.A.) was fed ad libitum. Animal testing was performed with approval from the Korea Food Industry Cluster Promotion Agency Animal Experiment Ethics Committee (IACUC-22-014).

[Experimental Example 4] Statistical analysis

All experimental results were expressed as mean ± standard error (mean ± SEM), and all statistical analyzes were performed using the SAS (release 9.4 SAS Institute Inc., Cary, NC, USA) program. Significant differences (p<0.05) between experimental groups were analyzed using one-way ANOVA and Duncan's multiple range test.

[Example 1] Analysis of physiological activity of samples ( in vitro )

1-1. Cytotoxicity assay

In order to confirm the cytotoxicity of the sample on macrophages and intestinal epithelial cells, the EZ-Cytox kit (DAEIL lab, Korea) was used to determine the cell absorbance based on the decrease in absorbance at each sample treatment concentration compared to the control group (sample untreated group; control). The concentration of samples that affected survival rates was determined. Raw 264.7 and IPEC-J2 cells were each seeded in a 96 well plate at a density of 1×10 ⁴ cells/well, cultured for 24 hours in an incubator under 5% CO ₂ and 37°C, and then samples were processed by concentration. Cultured for 24 hours. Afterwards, the culture medium was removed, 90 μL of DMEM and 10 μL of EZ-Cytox reagent were mixed and dispensed, and reacted for 1 hour in an incubator with 5% CO ₂ and 37°C in the dark. Afterwards, absorbance was measured at 450 nm using a microplate reader, and cell viability was measured using Equation 1 below.

[Equation 1]

Cell viability = (absorbance of control group) / (absorbance of sample treatment group) × 100

As a result, as shown in Figure 1, for Raw 264.7 cells, no significant cytotoxicity was observed in the 0.1~50㎕/㎖ sample treatment group, and for IPEC-J2 cells, significant cytotoxicity was observed in the 0.1~10㎕/㎖ sample treatment group. No cytotoxicity was observed. Based on the above results, an experiment on the physiological activity of the sample was performed in a concentration range where cytotoxicity was not observed.

1-2. NO production assay

To determine the effect of the sample on NO (nitric oxide) production, NO activity was measured using a NO (nitric oxide) assay kit (Promega Corp, USA, Cat.#G2930). Raw 264.7 cells were seeded in _a ²⁴ -well plate at a density of 2.5 Using the added DMEM medium, 1 μg/ml LPS (Lipopolysaccharides, Sigma-Aldrich, USA) (derived from Escherichia coli 055:B5) and samples were diluted by concentration and cultured for 24 hours. Afterwards, the amount of NO produced during cell culture was measured. The amount of NO produced was determined by mixing 50 μL of cell culture medium and 100 μL of Griess reagent (Sigma-Aldrich Co.) according to the kit protocol and reacting for 10 minutes at room temperature. The absorbance of the colored reaction solution was measured at 520 nm using a microplate reader. Nitrite concentration was calculated using the standard curve.

As a result, as shown in Figure 2, NO production significantly increased in the LPS-only treated group compared to the control group (LPS and sample untreated group), and NO production significantly decreased in a concentration-dependent manner in the sample-treated group compared to the LPS-only treated group.

1-3. PGE2 production inhibition activity analysis

To determine the effect of the sample on PGE2 (Prostaglandin E2) production, Raw 264.7 cells were seeded in a 24-well plate at a density of 2.5 × 10 ⁵ cells/well and incubated in an incubator under 5% CO ₂ and 37°C conditions. After culturing for 24 hours, 1 μg/ml of LPS (Lipopolysaccharides, Sigma-Aldrich, USA) (from Escherichia coli 055:B5) was added to the sample using DMEM medium supplemented with 1% FBS and 1% P/S. It was diluted and cultured for 24 hours. Afterwards, each cell culture was taken and centrifuged at 13000 rpm for 10 minutes, and the PGE2 content of the supernatant obtained through centrifugation was measured. All samples were stored frozen at -20°C until quantification, and PGE2 was quantified using a mouse enzyme-linked immunosorbent assay (ELISA) kit (Cat.# KGE004B, R&D Systems Inc., Minneapolis, MN, USA). The R2 value of the standard curve was over 0.99.

As a result, as shown in Figure 3, PGE2 production significantly increased in the LPS-only treated group compared to the control group (LPS and sample untreated group), and PGE2 production significantly decreased in the sample-treated group compared to the LPS-only treated group.

1-4. Analysis of iNOS and COX-2 expression inhibition activity

To determine the effect of the sample on the expression of iNOS (Inducible nitric oxide synthase) and COX-2 (Cyclooxygenase-2), qPCR was performed. To isolate total RNA from Raw 264.7 cells, a Trizol reagent kit (Invitrogen, USA) was used, and RNA was precipitated using 100% isopropanol and washed with 75% ethanol. The extracted RNA was dissolved in distilled water from which nucleic acid degrading enzymes were removed, and then the concentration was measured using a nanospectrophotometer. cDNA was synthesized using Maxime RT pre-Mix (Oligo dt 15 Primer) (iNtRON Biotechnology, Korea) according to the method provided by the manufacturer. To analyze gene expression of iNOS (NCBI Gene ID: 396859) and COX-2 (NCBI Gene ID: 808504), KAPA SYBR fast qPCR Kit (KAPA biosystems, USA) was used. qPCR conditions were set to 1 cycle at 95°C for 5 minutes, 35 cycles at 96°C for 20 seconds, 60°C for 20 seconds, 72°C for 20 seconds, and 1 cycle at 72°C for 5 minutes. Light Cycler 96 software provided by the manufacturer (Roche Applied Science) was used for data analysis. Quantitative results were compared with reference mRNA (GAPDH) using the 2 ^-ΔΔCT method (Livak & Schmittgen, 2001).

As a result, as shown in Figure 4, iNOS and COX-2 expression was significantly increased in the LPS-only treated group compared to the control group (LPS and sample untreated group), and iNOS and COX-2 expression was significantly increased in the sample-treated group compared to the LPS-only treated group. It decreased significantly in a concentration-dependent manner.

1-5. Pro-inflammatory and anti-inflammatory cytokine production analysis

To determine the effect of the sample on the production of pro-inflammatory cytokines and anti-inflammatory cytokines, IL-1β (NCBI Gene ID: 16176) (Interleukin-1β, Cat.# MLB00C), TNF-α (NCBI Gene ID: 21926) (tumor necrosis factor-α, Cat.# MTA00B), IL-6 (NCBI Gene ID: 16193) (Interleukin-6, Cat.# DY406-05) and IL -10 (NCBI Gene ID: 16153) (Interleukin-10, Cat.# M1000B-1) The amount of cytokine production was measured using an ELISA kit. The experiment was conducted using Raw 264.7 cell culture supernatant according to the kit protocol. The absorbance of the colored reaction solution was measured at 450 nm using a microplate reader. The concentration of each cytokine was calculated using the standard curve.

As a result, as shown in Figure 5, the production of IL-1β, IL-6, and TNF-α was significantly increased in the LPS-only treatment group compared to the control group (LPS and sample untreated group), and the above-described production in the sample-treated group compared to the LPS-only treatment group. The production of three types of cytokines was significantly reduced. In addition, IL-10 production significantly increased in the LPS-only treatment group and the sample-treatment group, and the increase in IL-10 production was more significant in the sample-treatment group compared to the LPS-only treatment group.

1-6. Trans-epithelial electrical resistance (TEER) analysis

To determine the effect of samples on trans-epithelial electrical resistance (TEER), samples were analyzed on 0.4 μm transwell membranes (Corning, NY, USA) using a Millicell-ERS-2 instrument (Millipore, MA, USA). TEER of cultured IPEC-J2 cell monolayer was analyzed. IPEC-J2 cells were seeded in 24 transwells at a density of 2×10 ⁵ cells/well and cultured at 37°C and 5% CO ₂ for 24 hours. Afterwards, the top wells were replaced with 500 μL of samples of each concentration, the bottom wells were replaced with 1.5 mL medium, and the plates were further incubated for 24 hours (37°C, 5% CO ₂ and 95% humidity). Afterwards, it was treated with LPS (Lipopolysaccharides, Sigma-Aldrich, USA) (from Escherichia coli 055:B5) dissolved in HBSS (Hank's Balanced Salt Solution) at a concentration of 1㎍/mL, and TEER was performed using Miller-ERS equipment (Millipore, MA, USA) was used to measure each TEER value over time starting immediately after sample processing. The TEER value was calculated using Equation 2 below.

[Equation 2]

TEER = resistance(Ohm) × filter area(㎠) = Ω × ㎠

As a result, as shown in Figure 6, the TEER value decreased over time in all experimental groups, and the decrease in TEER value was most significant in the LPS-only treatment group, and the TEER value decreased in the sample treatment group compared to the LPS-only treatment group. It was confirmed that it was suppressed.

1-7. Paracellular permeability analysis

To determine the effect of the sample on paracellular permeability, IPEC-J2 cells were treated with 1 μg/mL of LPS for 48 hours, and then cells were permeated using 4 kDa FITC-dextran (Sigma-Aldrich, USA). Ambient permeability was measured. FITC-dextran dissolved in HBSS was dispensed to the LPS-treated cells into the upper well at a concentration of 1 mg/ml, and incubated for 4 hours, followed by Tecan Reader (excitation, 492 nm; emission, 520 nm, Tecan Group Ltd., Switzerland). ) was used to measure paracellular permeability.

As a result, as shown in Figure 7, the flux of FITC-dextran significantly increased in the LPS-only treated group compared to the control group (LPS and sample untreated group), and the flux of FITC-dextran in the sample-treated group compared to the LPS-only treated group was concentration dependent. decreased significantly.

1-8. Intestinal junction protein expression analysis

To determine the effect of the sample on intestinal junction protein expression, IPEC-J2 cells cultured with LPS for 48 hours were washed once with DPBS (Dulbecco's phosphate-buffered saline), and 100-150 ㎕ of PRO-PREP reagent was added to each well. After dispensing, the cells were recovered in a tube using a scraper. The recovered cells were left on ice for 30 minutes and then centrifuged at 13000 rpm and 4°C for 5 minutes to obtain a supernatant. Afterwards, the supernatant was quantified for protein using a Bradford assay, and 50 μg of protein was separated using 8% SDS-PAGE and then transferred to a PVDF membrane (Polyvinylidene Fluoride membrane). Afterwards, block for 1 hour using blocking buffer (4% nonfat dry milk, 10mM Tris, 100mM NaCl, and 0.1% Tween 20, pH 7.5) and wash with 1% Tween 20-PBS three times for 15 minutes each. did. Treated with ZO-1 (NCBI Gene ID: 100736682), Occludin (NCBI Gene ID: 397236), Claudin-1 (NCBI Gene ID: 100625166) and β-actin antibodies (primary antibodies) at room temperature for 3 hours, 1 Washed with % Tween 20-PBS. Afterwards, it was treated with a secondary antibody for 1 to 2 hours, and after washing, the proteins of Zonula occludens-1 (ZO-1), Occludin, and Claudin-1 were detected with a LAS 4000 instrument using an enhanced chemiluminescence (ECL) kit for detection. The expression level was measured.

As a result, as shown in Figure 8, the protein expression of ZO-1, Occludin, and Claudin-1 was decreased in the LPS-only treated group compared to the control group (LPS and sample untreated group), and the above three types were decreased in the sample-treated group compared to the LPS-only treated group. Protein expression increased in a concentration-dependent manner.

[Example 2] Analysis of physiological activity of samples ( in vivo )

2-1. Production of enteritis-induced animal model

In order to create an enteritis-inducing animal model, enteritis was induced by providing 3% DSS (Dextran sulfate sodium salt) as a negative water to the animal model of Experimental Example 3 above. 50mg/kg of 5-ASA (5-aminosalicyclic acid) was used as a positive control group, and samples and 5-ASA were administered once daily from the start date of 3% DSS intake using a disposable syringe (1 ml) attached to a sonde for oral administration. It was orally administered into the stomach of an animal model for 5 weeks. The experimental group was specifically set as follows.

1) Normal group (CON)

2) Negative control group (DSS): 3% DSS intake

3) Positive control group (DSS+ASA): 3% DSS intake + 50mg/kg oral administration of 5-ASA

4) Sample treatment group (DSS+KM2): 3% DSS intake + oral administration of 2g/kg sample

2-2. Body weight and disease activity analysis

In order to determine the effect of the sample on body weight and disease activity index score (DAI score), the body weight of the animal model was measured once daily for 5 weeks (DSS intake period), and stool was measured using a hematochezia kit. The condition (shape of stool and color of bloody stool) was confirmed and disease activity (shape of stool, color of bloody stool, and weight loss) was measured.

As a result, as shown in Figure 9, body weight decreased in the negative control group (DSS) compared to the control group (CON), and increased body weight in the positive control group (DSS+ASA) and sample treatment group (DSS+KM2) compared to the negative control group. In addition, from the 5th day of enteritis induction (DSS intake), weight loss and occurrence of bloody stools significantly increased in the negative control group, positive control group, and sample treatment group, and disease activity increased compared to the control group, and in the positive control group and sample treatment group compared to the negative control group. Disease activity decreased.

2-3. Colon length analysis

To confirm the effect of the sample on colon length, the animal model was sacrificed by fasting for 24 hours, dissected, blood was collected from the heart, and the appearance, abdominal cavity, and thoracic cavity were visually observed. Afterwards, the large intestine was removed and the length of the large intestine was measured.

As a result, as shown in Figure 10, the colon length significantly decreased in the negative control group (DSS) compared to the control group (CON), and the colon length decreased in the positive control group (DSS+ASA) and sample treatment group (DSS+KM2) compared to the negative control group. increased.

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

To determine the effect of the sample on inflammatory cytokine production, the animal model was sacrificed, blood was collected from the heart, centrifuged immediately, and serum was separated. The separated serum was stored in an ultra-low temperature freezer at -80°C until analysis. did. Afterwards, the cytokine content was measured using a spectrophotometer and analysis kit.

As a result, as shown in Figure 11, the production of IL-6, IL-1β, and TNF-α significantly increased, and the production of IL-10 significantly decreased in the negative control group (DSS) compared to the control group (CON). On the other hand, IL-6, IL-1β, and TNF-α production significantly decreased, and IL-10 production significantly increased in the positive control group (DSS+ASA) and sample treatment group (DSS+KM2) compared to the negative control group.

2-5. Histological analysis

To confirm the effect of the sample on intestinal tissue, the colonic tissue of the animal model was fixed with 10% formaldehyde, a paraffin block was prepared, and H&E (Hematoxylin & Eosin) staining was performed. Quantitative image analysis was performed after photographing the stained tissue slides. In addition, alcian blue staining was performed to analyze the degree of intestinal mucus secretion.

As a result, as shown in Figure 12, the structure and size of the epithelial tissue forming the colon surface changed irregularly in the negative control group (DSS) compared to the control group (CON), and the infiltration of inflammatory cells increased. On the other hand, it was confirmed that symptoms appearing in the negative control group were improved in the positive control group (DSS+ASA) and sample treatment group (DSS+KM2) compared to the negative control group. In addition, the degree of blue staining decreased in the negative control group compared to the control group in which the mucosal layer was stained blue, whereas the degree of blue staining increased in the positive control group and sample treatment group compared to the negative control group.

2-6. Gut microbiome analysis

To confirm the effect of the sample on the intestinal microorganisms, an animal model was sacrificed, the cecum was removed, and changes in the intestinal microorganisms were analyzed using shotgun metagenome sequencing.

As a result, as shown in Figure 13, in the α-diversity analysis results, which can confirm the diversity of microorganisms present in a sample, it was confirmed that there was no significant difference in the Shannon index between the experimental groups. Additionally, there was no significant difference between the experimental groups in the F/B (Firmicutes/Bacteroidetes) ratio, which is an inflammation level. The results of β-diversity analysis based on unweighted UniFrac principal coordinate analysis (PCA), which can confirm the microbial community pattern, showed significant differences between experimental groups. In order to identify significantly different microbiomes between each experimental group, linear discriminant analysis (LDA) and KEGG pathway analysis were performed. As a result, as shown in Figure 13, the sample treatment group (DSS+) compared to the negative control group (DSS) KM2), weizmannia coagulans , Welch's bacillus ( clostridium perfringens ), clostridium botulinum , clostridium sporogenes , clostridium baratii And the number of Clostridium butyricum strains increased significantly. In addition, the number of Bifidobacterium pseudolongum strains significantly increased in the negative control group compared to the sample treatment group.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. do. That is, the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

1. Lactiplantibacillus Plantarum A pharmaceutical composition for preventing or treating inflammatory diseases, comprising as an active ingredient the fermentation culture supernatant of the plantarum strain, its concentrate, its dried product, its fermentation metabolite, or a mixture thereof.
2. The pharmaceutical composition according to claim 1, wherein the strain is Lactiplantibacillus plantarum KM2 ( Lactiplantibacillus plantarum KM2) deposited under the accession number KCTC 14637BP.
3. The method of claim 1, wherein the inflammatory disease is ulcerative colitis, ulcerative duodenitis, Crohn's disease, irritable bowel syndrome, and intestinal Behcet's disease. ), hemorrhagic rectal ulcer, pouchitis, enteritis, ischemic colitis, acne, extra-intestinal manifestations dermatitis, 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, A pharmaceutical composition comprising at least one selected from the group consisting of cystitis, nephritis, and Sjogren's syndrome multiple sclerosis.
4. The pharmaceutical composition according to claim 1, further comprising dead cells or spores of Lactiplantibacillus plantarum KM2 ( Lactiplantibacillus plantarum KM2) strain.
5. The method of claim 1, wherein the pharmaceutical composition is Weizmannia coagulans , Welch's bacillus ( clostridium perfringens ), Clostridium botulinum , Clostridium sporogenes , Clostri. A pharmaceutical composition characterized in that it regulates one or more intestinal microorganisms selected from the group consisting of clostridium baratii, clostridium butyricum , and bifidobacterium pseudolongum .
6. A health functional food composition for preventing or improving inflammatory diseases, comprising the 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 ( Lactiplantibacillus plantarum KM2) strain deposited under the accession number KCTC 14637BP.
8. Compared to the normal group, weizmannia coagulans , Welch's bacillus ( clostridium perfringens) , Clostridium botulinum , Clostridium sporogenes , Clostridium baratii ( A group of patients with inflammatory diseases in which the number of one or more strains selected from the group consisting of clostridium baratii and clostridium butyricum is reduced; Or a method for preventing or treating an inflammatory disease, comprising treating the pharmaceutical composition of claim 1 to a group of inflammatory disease patients with an increased number of Bifidobacterium pseudolongum strains compared to a normal group.

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