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

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to a microbiome composition of the fermentation culture supernatant of the Lactiplantibacillus plantarum KM2 strain, which has anti-inflammatory effects. [Background technology]

[0002] Inflammation is a normal, protective defense mechanism that occurs locally in response to tissue damage caused by physical trauma, harmful chemicals, microbial infection, or irritants in the body's metabolism. Inflammation is triggered by a variety of chemical mediators produced by damaged tissue and migrating cells, and these chemical mediators vary widely depending on the type of inflammatory process. Under normal circumstances, the body neutralizes or eliminates pathogenic factors through the inflammatory response, regenerates damaged tissue, and restores normal structure and function.

[0003] However, if this inflammatory response occurs abnormally, it can progress to diseases such as chronic inflammation, or if inflammation is inappropriately triggered by harmless substances such as pollen or autoimmune reactions such as asthma and rheumatoid arthritis, the defense response itself can actually damage tissues and cause various diseases.

[0004] Currently, steroidal and non-steroidal therapeutic agents are widely used as the most common preventive or therapeutic agents for inflammatory diseases. However, most of them have the problem of causing side effects, and active development of therapeutic agents for inflammatory diseases that overcome these problems is currently underway. Summary of the Invention [Problem to be solved by the invention]

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

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

[0007] It is yet another object of the present invention to provide a method for preventing or treating inflammatory diseases. [Means for solving the problem]

[0008] To achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, 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] The present invention also provides a functional health food composition for preventing or improving inflammatory diseases, which contains as an active ingredient the fermentation culture supernatant of the strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof.

[0010] The present invention also provides a method for preventing or treating an inflammatory disease, the method comprising administering the pharmaceutical composition of claim 1 to a group of patients with an inflammatory disease having a decreased 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 compared to a normal group; or to a group of patients with an inflammatory disease having an increased number of Bifidobacterium pseudolongum strains compared to a normal group. [Effects of the Invention]

[0011] According to the present invention, it has been confirmed that the fermentation culture supernatant of Lactiplantibacillus plantarum KM2 strain exhibits anti-inflammatory activity by suppressing the activity of nitric oxide and regulating the expression of inflammatory cytokines, and therefore it can be usefully used as a composition for preventing, treating, or ameliorating inflammatory diseases. [Brief explanation of the drawings]

[0012] [Figure 1] The results show the cytotoxicity of the fermented culture supernatant (hereinafter referred to as the sample) of Lactiplastobacillus plantarum KM2 strain in macrophages. [Figure 2] The results are from an analysis of the effect of the sample on the production of nitric oxide (hereinafter referred to as NO) in macrophages. [Figure 3] The results are obtained by analyzing the effect of the sample on the production of prostaglandin E2 (hereinafter referred to as PGE2) in macrophages. [Figure 4] The results show the analysis of the effects of samples on the expression of iNOS (Inducible nitric oxide synthase) and COX-2 (Cyclooxygenase-2) in macrophages. [Figure 5] The results are from analyzing the effects of samples on the production of pro-inflammatory and anti-inflammatory cytokines in macrophages. [Figure 6] These are the results of analyzing the effect of samples on TEER (Trans-epithelial electrical resistance) in intestinal epithelial cells. [Figure 7] These are the results of analyzing the effect of samples on paracellular permeability in intestinal epithelial cells. [Figure 8] The results are from an analysis of the effect of samples on the expression of intestinal junction proteins in macrophages. [Figure 9] The results show the results of analyzing the effects of samples on body weight and disease activity in animal models. [Figure 10]The results are from an analysis of the effect of the samples on colon length in an animal model. [Figure 11] The results are from an analysis of the effects of the samples on the production of inflammatory cytokines in an animal model. [Figure 12] The results are from an analysis of the effects of the samples on intestinal tissue in an animal model. [Figure 13] The results are from an analysis of the effects of samples on intestinal microorganisms in an animal model.

[0013] In all figures, treatment groups labeled with different letters (e.g., a, b, etc.) indicate that there is a statistical difference at the 95% confidence level between them. For example, if there is group 1 labeled a, group 2 labeled b, and group 3 labeled ab, this means that there is a statistical difference at the 95% confidence level between group 1 and group 3, but there is no statistical difference between group 1 and group 2; and between group 2 and group 3. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will now be described in more detail.

[0015] The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, which contains as an active ingredient a fermentation culture supernatant of a Lactiplantibacillus plantarum strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof.

[0016] The strain is also Lactiplantibacillus plantarum KM2 strain deposited under accession number KCTC 14637BP.

[0017] The inflammatory diseases include ulcerative colitis, ulcerative duodenitis, Crohn's disease, irritable bowel syndrome, intestinal Behcet's disease, hemorrhagic rectal ulcer, pouchitis, enteritis, ischemic colitis, acne, and extra-intestinal manifestations. manifestations) dermatitis, atopic dermatitis, allergic dermatitis, seborrheic dermatitis, papular urticaria, eczema, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, pneumonia, pancreatitis, gastritis, hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome, and multiple sclerosis.

[0018] The pharmaceutical composition may further comprise killed bacteria or spores of Lactipranthibacillus plantarum KM2 strain.

[0019] The pharmaceutical composition can also regulate one or more intestinal microorganisms selected from the group consisting of Weizmania coagulans, Clostridium perfringens, Clostridium botulinum, Clostridium sporogenes, Clostridium baratii, Clostridium butyricum, and Bifidobacterium pseudolongum.

[0020] In addition, the pharmaceutical composition may suppress the production or expression of one or more selected from the group consisting of prostaglandin E2, iNOS, COX-2, IL-1β (Interleukin-1β), TNF-α (tumor necrosis factor-α), and IL-6 (Interleukin-6), or may promote the production or expression of one or more selected from the group consisting of ZO-1, Occludin, Claudin-1, and IL-10 (Interleukin-10), but is not limited to these.

[0021] The pharmaceutical composition of the present invention can be prepared in a unit dose form or in a multi-dose container by formulating it with a pharmaceutically acceptable carrier by a method that can be easily carried out by a person skilled in the art.

[0022] The pharmaceutically acceptable carrier is one commonly used in formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginic acid, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, 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.

[0023] In the present invention, the content of the additives contained in the pharmaceutical composition is not particularly limited, and may be appropriately adjusted within the range of the content used in conventional formulations.

[0024] The pharmaceutical composition may be formulated into one or more external skin preparations selected from the group consisting of an injectable dosage form such as an aqueous solution, suspension, or emulsion, a pill, capsule, granule, tablet, cream, gel, patch, spray, ointment, plaster, lotion, liniment, paste, and cataplasm, but is not limited thereto.

[0025] The pharmaceutical compositions of the present invention may contain additional pharmaceutically acceptable carriers and diluents for formulation. Examples of pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugars, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbates, cetyl alcohol, and glycerol. Pharmaceutically acceptable carriers and diluents are also biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffer solutions, solvents, and / or dispersion media.

[0026] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., 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, pharmaceutical powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. For parenteral administration, it can be formulated as injections, suppositories, powders for respiratory inhalation, aerosols for spraying, ointments, powders for application, oils, creams, etc.

[0027] The dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition, body weight, age, sex, health condition, dietary habits, properties of the formulation, severity of the disease, administration time, administration method, administration period or interval, excretion rate, and drug form, and can be appropriately selected by those skilled in the art. For example, the dosage range is about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day.

[0028] The pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention vary depending on the formulation method, administration mode, administration time, administration route, etc., of the pharmaceutical composition, and those skilled in the art can easily determine or prescribe an effective dosage for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or in divided doses.

[0029] The present invention also provides a functional health food composition for preventing or improving inflammatory diseases, which contains as an active ingredient a fermentation culture supernatant of a Lactiplantibacillus plantarum strain, a concentrate thereof, a dried product thereof, a fermentation metabolite thereof, or a mixture thereof.

[0030] The strain is also Lactiplantibacillus plantarum KM2 strain deposited under accession number KCTC 14637BP.

[0031] The present invention is generally used as a conventional food product.

[0032] The food composition of the present invention can be used as a functional health food. The term "functional health food" refers to a food manufactured and processed using raw materials or ingredients that have functional properties beneficial to the human body as defined by the Act on Functional Health Foods. The term "functional" refers to the food being ingested for the purpose of regulating nutrients for the structure and function of the human body or obtaining beneficial health effects such as physiological effects.

[0033] The functional health food composition may contain common food additives, and unless otherwise specified, the suitability of a food additive shall be determined in accordance with the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety.

[0034] Examples of items listed in the "Food Additives Code" include chemically synthesized substances such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as indigo dye, licorice extract, crystalline cellulose, sorghum pigment, and guar gum; and mixed preparations such as monosodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.

[0035] The food composition of the present invention can be manufactured and processed into the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among capsule-type health functional foods, hard capsules can be manufactured by mixing the composition of the present invention with additives such as excipients and filling them into a conventional hard capsule, while soft capsules can be manufactured by mixing the composition of the present invention with additives such as excipients and filling them into a capsule base such as gelatin. The soft capsules can contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc. as needed.

[0036] The definitions of the terms excipient, binder, disintegrant, lubricant, flavoring agent, flavoring agent, etc. are those described in literature known to those skilled in the art, and include those having the same or similar functions, etc. The type of the food is not particularly limited, and includes any health functional food in the usual sense.

[0037] In the present invention, the term "prevention" refers to any action of administering a composition according to the present invention to suppress or delay the onset of an inflammatory disease. In the present invention, the term "treatment" refers to any action of administering a composition according to the present invention to improve or favorably alter the symptoms of an inflammatory disease. In the present invention, the term "amelioration" refers to any action of administering a composition according to the present invention to improve the adverse state of an inflammatory disease.

[0038] The present invention also provides a method for preventing or treating an inflammatory disease, comprising administering the pharmaceutical composition to a group of patients with an inflammatory disease in which the number of one or more strains selected from the group consisting of Weizmania coagulans, Clostridium perfringens, Clostridium botulinum, Clostridium sporogenes, Clostridium baratii, and Clostridium butyricum is reduced compared to a normal group; or to a group of patients with an inflammatory disease in which the number of Bifidobacterium pseudolongum strains is increased compared to a normal group.

[0039] The present invention will be described in detail below with reference to examples to aid in understanding the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

[0040] [Experimental Example 1] Sample production

[0041] To produce the fermentation culture supernatant of the KM2 strain (Lactiplantibacillus plantarum KM2), the sample for this experiment, KM2 stock stored at -70°C was activated and subjected to primary seed culture in test tubes and flasks. Secondary seed culture was then performed in a 50L fermenter with a 20L working volume at 2% (v / v) for 6 hours. For main culture, a 500L fermenter with a 350L working volume was inoculated at 2% (v / v) for 12 hours, with glucose feeding once after 6 hours of culture. After the cultivation was completed, the cell slurry was removed by primary centrifugation using a disk centrifuge at 7,200 rpm and 2 L / min. The supernatant was then subjected to secondary centrifugation using a tubular centrifuge at 15,000 rpm and 1.5 L / min to remove the cell cake and recover the supernatant. The recovered supernatant was then filtered through a 0.2 μm sterilization filter to obtain the fermentation culture supernatant of the KM2 strain, from which the bacterial cells had been removed. Trehalose was added to the sterilized fermentation culture supernatant at 3% (w / v) as an excipient and freeze-dried for 96 to 120 hours to obtain a powdered fermentation culture supernatant.

[0042] [Experimental Example 2] Cell culture

[0043] Raw 264.7 cells, a mouse macrophage cell line, were provided by the Korean Cell Line Bank (KCLB). Cells were subcultured every two days in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) at 37°C and 5% CO2 in an incubator.

[0044] IPEC-J2 cells, porcine intestinal epithelial cells, were provided by the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). Cells were subcultured every two days in Dulbecco's Modified Eagle's Medium-Nutrient Mixture F-12 (DMEM / F-12) medium supplemented with 10% FBS and 1% P / S in an incubator at 37°C and 5% CO2.

[0045] [Experimental Example 3] Preparation of animal model

[0046] C57BL / 6 mice were purchased from Orient Bio. Healthy animals were used in the experiments after confirming the number of mice, observing general symptoms, weighing, and checking the test results provided by the animal supplier. All animals were checked for any abnormalities and underwent a 7-day acclimation period to adapt to the animal room environment. During the acclimation period, all animals were observed daily for general symptoms, and on the final day of acclimation, their health status was assessed by monitoring their weight changes. Animals were separated into four groups, each with nine mice, so that the average weight of each experimental group was equal. Individual animals were labeled with a five-color permanent marker on their tails, and individual identification cards were attached to their cages. Animal models were housed at a temperature of 20–26°C, a relative humidity of 30–70%, and a 12-hour light / dark cycle (9:00 AM–9:00 PM) and provided with food and water ad libitum. Laboratory chow (Lab DFiet #5053, PMI Nutrition International, USA) was provided ad libitum. Animal experiments were performed with the approval of the Animal Experiment Ethics Committee of the Korea Food Industry Cluster Promotion Institute (IACUC-22-014).

[0047] [Experimental Example 4] Statistical analysis

[0048] All experimental results are expressed as mean ± standard error (SEM), and all statistical analyses were performed using the SAS program (release 9.4, SAS Institute Inc., Cary, NC, USA). Significant differences (p < 0.05) between experimental groups were analyzed using one-way ANOVA and Duncan's multiple range test.

[0049] [Example 1] Analysis of the physiological activity of samples (in vitro)

[0050] 1-1. Cytotoxicity analysis

[0051] To confirm the cytotoxicity of the samples in macrophages and intestinal epithelial cells, the EZ-Cytox kit (DAEIL lab, Korea) was used to determine the concentration of the sample that affected cell viability based on the decrease in absorbance at each sample treatment concentration relative to the control (untreated sample group). Raw 264.7 and IPEC-J2 cells were each plated at 1 × 10 4 After seeding at a density of 100 cells / well and culturing for 24 hours in an incubator at 5% CO2 and 37°C, samples were treated at different concentrations and cultured for 24 hours. The culture medium was then removed, and 90 μL of DMEM and 10 μL of EZ-Cytox reagent were mixed and dispensed. The mixture was incubated in an incubator at 5% CO2 and 37°C in the dark for 1 hour. The absorbance was then measured at 450 nm using a microplate reader, and cell viability was calculated using the following equation.

[0052] [Number 1] Cell viability = (absorbance of control group) / (absorbance of sample-treated group) x 100

[0053] As a result, in the case of Raw 264.7 cells, no significant cytotoxicity was observed in the 0.1-50 μl / ml sample treatment group, and in the case of IPEC-J2 cells, no significant cytotoxicity was observed in the 0.1-10 μl / ml sample treatment group, as shown in Figure 1. Based on these results, experiments were conducted to evaluate the physiological activity of the samples in the concentration range where no cytotoxicity was observed.

[0054] 1-2. NO production analysis

[0055] To confirm the effect of the samples on nitric oxide (NO) production, NO activity was measured using an NO assay kit (Promega Corp, USA, Cat. #G2930). Raw 264.7 cells were cultured in a 24-well plate at 2.5 × 10 5 Cells were seeded at a density of 1000 cells / well and incubated in an incubator at 5% CO2 and 37°C for 24 hours. Afterwards, DMEM medium supplemented with 1% FBS and 1% P / S was used to dilute 1 μg / ml of LPS (Lipopolysaccharides, Escherichia coli 055:B5, Sigma-Aldrich, USA) and the samples were incubated for 24 hours. The amount of NO produced during cell culture was then measured. According to the kit protocol, 50 μL of cell culture medium was mixed with 100 μL of Griess reagent (Sigma-Aldrich Co.) and incubated for 10 minutes at room temperature. The absorbance of the colored reaction mixture was measured at 520 nm using a microplate reader. The nitrite concentration was calculated using a standard curve.

[0056] As a result, as shown in Figure 2, NO production was significantly increased in the LPS-only treated group compared to the control group (group untreated with LPS and sample), and NO production was significantly decreased in a concentration-dependent manner in the sample-treated group compared to the LPS-only treated group.

[0057] 1-3. Analysis of PGE2 production inhibitory activity

[0058] To confirm the effect of the sample on PGE2 (Prostaglandin E2) production, 2.5 × 10 Raw 264.7 cells were cultured in a 24-well plate. 5 Cells were seeded at a density of 1000 cells / well and cultured in an incubator at 5% CO2 and 37°C for 24 hours. Then, LPS (derived from Escherichia coli 055:B5) (Sigma-Aldrich, USA) was added to DMEM medium supplemented with 1% FBS and 1% P / S at 1 μg / ml, and the samples were diluted and cultured for 24 hours. Each cell culture was then centrifuged at 13,000 rpm for 10 minutes, and the PGE2 content of the supernatant was measured. All samples were stored frozen at -20°C until quantification. 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 for the standard was 0.99 or higher.

[0059] As a result, as shown in Figure 3, PGE2 production was significantly increased in the LPS-only treated group compared to the control group (group untreated with LPS and sample), and PGE2 production was significantly decreased in the sample-treated group compared to the LPS-only treated group.

[0060] 1-4. Analysis of iNOS and COX-2 expression inhibitory activity

[0061] qPCR was performed to confirm the effect of the samples on the expression of iNOS and COX-2. Total RNA was isolated from Raw 264.7 cells using a Trizol reagent kit (Invitrogen, USA). 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 nanospectrophotometer. cDNA was synthesized using Maxime RT pre-Mix (Oligo dt 15 Primer) (iNtRON Biotechnology, Korea) according to the manufacturer's instructions. The KAPA SYBR fast qPCR Kit (KAPA biosystems, USA) was used to analyze gene expression of iNOS (NCBI Gene ID: 396859) and COX-2 (NCBI Gene ID: 808504). The qPCR conditions were 95°C for 5 minutes, 35 cycles of 96°C for 20 seconds, 60°C for 20 seconds, 72°C for 20 seconds, and 72°C for 5 minutes. Data analysis was performed using the Light Cycler 96 software provided by the manufacturer (Roche Applied Science). Quantitative results were reported in 2 -ΔΔCT The results were expressed relative to a reference mRNA (GAPDH) using the method (Livak & Schmittgen, 2001).

[0062] As a result, as shown in Figure 4, the expression of iNOS and COX-2 significantly increased in the LPS-only treated group compared to the control group (group untreated with LPS and sample), and the expression of iNOS and COX-2 significantly decreased in a concentration-dependent manner in the sample-treated group compared to the LPS-only treated group.

[0063] 1-5. Analysis of pro-inflammatory and anti-inflammatory cytokine production

[0064] To assess the effects of the samples on the production of pro-inflammatory 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) ELISA kits were used to measure the production of these cytokines. Experiments were performed using Raw 264.7 cell culture supernatants according to the kit protocol. The absorbance of the colored reaction mixture was measured at 450 nm using a microplate reader. The concentration of each cytokine was calculated using a standard curve.

[0065] 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 production of these three cytokines was significantly decreased in the sample-treated group compared to the LPS-only treatment group. In addition, the production of IL-10 was significantly increased in both the LPS-only treatment group and the sample-treated group, and the increase in IL-10 production was even more significant in the sample-treated group compared to the LPS-only treatment group.

[0066] TEER Analysis

[0067] To confirm the effect of the sample on TEER, the TEER of IPEC-J2 cell sections cultured on 0.4 μm transwell membranes (Corning, NY, USA) was analyzed using a Millicell-ERS-2 instrument (Millipore, MA, USA). 2 × 10 IPEC-J2 cells were cultured on 0.4 μm transwell membranes (Corning, NY, USA). 5The cells were distributed into 24 transwell plates at a density of 100 cells / well and incubated for 24 hours at 37°C and 5% CO2. The top wells were then replaced with 500 μL of the sample at each concentration, and the bottom wells were replaced with 1.5 mL of medium. The plates were then incubated for an additional 24 hours (37°C, 5% CO2, and 95% humidity). The cells were then treated with 1 μg / mL of LPS (derived from Escherichia coli 055:B5) (Sigma-Aldrich, USA) dissolved in HBSS (Hank's Balanced Salt Solution). TEER values ​​were measured at each time point, starting immediately after sample treatment, using a Miller-ERS instrument (Millipore, MA, USA). TEER values ​​were calculated using the following equation:

[0068] [Number 2] TEER=resistance(Ohm)×filter area(cm 2 )=Ω×cm 2

[0069] As a result, as shown in Figure 6, TEER values ​​decreased over time in all experimental groups, with the most significant decrease in TEER values ​​observed in the LPS-only treatment group, and it was confirmed that the decrease in TEER values ​​was suppressed in the sample-treated groups compared to the LPS-only treatment group.

[0070] 1-7. Analysis of paracellular permeability

[0071] To assess the effect of the sample on paracellular permeability, IPEC-J2 cells were treated with 1 μg / mL LPS for 48 hours and then measured for paracellular permeability using 4 kDa FITC-dextran (Sigma-Aldrich, USA). FITC-dextran was dissolved in HBSS at a concentration of 1 mg / mL and dispensed into the top well of the LPS-treated cells. After 4 hours of incubation, paracellular permeability was measured using a Tecan Reader (excitation, 492 nm; emission, 520 nm; Tecan Group Ltd., Switzerland).

[0072] As a result, as shown in Figure 7, the FITC-dextran flux was significantly increased in the LPS-only treated group compared to the control group (group untreated with LPS and sample), and the FITC-dextran flux was significantly decreased in a concentration-dependent manner in the sample-treated group compared to the LPS-only treated group.

[0073] 1-8. Expression analysis of intestinal junction proteins

[0074] To confirm the effect of the sample on the expression of intestinal junction proteins, IPEC-J2 cells were cultured with LPS for 48 hours and washed once with Dulbecco's phosphate-buffered saline (DPBS). 100-150 μl of PRO-PREP reagent was dispensed into each well, and the cells were collected into tubes using a scraper. The collected cells were then left on ice for 30 minutes and centrifuged at 13,000 rpm and 4°C for 5 minutes to obtain the supernatant. The supernatant was then subjected to a Bradford assay to quantify protein content. 50 μg of protein was separated using 8% SDS-PAGE and transferred to a PVDF membrane (Polyvinylidene Fluoride membrane). Next, the sections were blocked for 1 hour using blocking buffer (4% nonfat dry milk, 10 mM Tris, 100 mM NaCl, and 0.1% Tween 20, pH 7.5), washed three times for 15 minutes with 1% Tween 20-PBS, and then treated with antibodies against ZO-1 (NCBI Gene ID: 100736682), Occludin (NCBI Gene ID: 397236), Claudin-1 (NCBI Gene ID: 100625166), and β-actin (primary antibodies) at room temperature for 3 hours, followed by washing with 1% Tween 20-PBS. The cells were then treated with a secondary antibody for 1-2 hours, washed, and the expression levels of ZO-1 (Zonula occludens-1), Occludin, and Claudin-1 proteins were measured using an LAS 4000 instrument using an enhanced chemiluminescence (ECL) kit for detection.

[0075] As a result, as shown in Figure 8, the expression of ZO-1, Occludin, and Claudin-1 proteins was reduced in the LPS-only treated group compared to the control group (group untreated with LPS and sample), and the expression of these three proteins was increased in a concentration-dependent manner in the sample-treated group compared to the LPS-only treated group.

[0076] [Example 2] Analysis of the physiological activity of samples (in vivo)

[0077] 2-1. Creation of an animal model of intestinal inflammation

[0078] To prepare an animal model for inducing enteritis, 3% DSS (dextran sulfate sodium salt) was provided in the drinking water of the animal model in Experimental Example 3 to induce enteritis. 50 mg / kg of 5-aminosalicyclic acid (5-ASA) was used as a positive control. The sample and 5-ASA were orally administered into the stomach of the animal model once daily for 5 weeks using a disposable syringe (1 ml) equipped with an oral administration probe, starting from the start of 3% DSS administration. The experimental groups were specifically set as follows:

[0079] 1) Normal group (CON)

[0080] 2) Negative control group (DSS): 3% DSS intake

[0081] 3) Positive control group (DSS + ASA): 3% DSS intake + 5-ASA 50 mg / kg oral administration

[0082] 4) Sample treatment group (DSS + KM2): 3% DSS intake + oral administration of 2 g / kg sample

[0083] 2-2. Analysis of body weight and disease activity

[0084] To confirm the effect of the sample on body weight and disease activity (DAI score; Disease Activity Index score), the body weight of the animal model was measured once a day for 5 weeks (DSS intake period), and the stool condition (fecal form and blood color) was checked using a blood stool kit, and disease activity (fecal form, blood color, and weight loss) was measured.

[0085] As a result, as shown in Figure 9, body weight decreased in the negative control group (DSS) compared to the control group (CON), and body weight increased in the positive control group (DSS + ASA) and sample-treated group (DSS + KM2) compared to the negative control group. In addition, from day 5 after enteritis induction (DSS ingestion), weight loss and bloody stool occurrence significantly increased in the negative control group, positive control group, and sample-treated group, indicating increased disease activity compared to the control group, and decreased disease activity in the positive control group and sample-treated group compared to the negative control group.

[0086] 2-3. Analysis of colon length

[0087] To confirm the effect of the sample on colon length, the animal model was fasted for 24 hours and then sacrificed. After dissection and cardiac blood collection, the appearance, abdominal cavity, and thoracic cavity were macroscopically observed. The colon was then excised and its length was measured.

[0088] As a result, as shown in Figure 10, the colon length was significantly reduced in the negative control group (DSS) compared to the control group (CON), and the colon length was increased in the positive control group (DSS + ASA) and the sample treatment group (DSS + KM2) compared to the negative control group.

[0089] 2-4. Blood biochemical analysis (analysis of inflammatory cytokine production)

[0090] To confirm the effect of the sample on the production of inflammatory cytokines, the animal model was sacrificed, and blood was collected from the heart and immediately centrifuged to separate the plasma. The separated plasma was then stored in a deep freezer at -80°C until analysis. The cytokine content was then measured using a spectrophotometer and an analysis kit.

[0091] As a result, as shown in Figure 11, the production of IL-6, IL-1β, and TNF-α was significantly increased and the production of IL-10 was significantly decreased in the negative control group (DSS) compared to the control group (CON). On the other hand, the production of IL-6, IL-1β, and TNF-α was significantly decreased and the production of IL-10 was significantly increased in the positive control group (DSS + ASA) and the sample-treated group (DSS + KM2) compared to the negative control group.

[0092] 2-5.Histological analysis

[0093] To confirm the effects of the sample on intestinal tissue, colonic tissue from an animal model was fixed in 10% formaldehyde, paraffin blocks were prepared, and H&E (Hematoxylin & Eosin) staining was performed. The stained tissue slides were photographed and quantitative image analysis was performed. Additionally, alcian blue staining was performed to analyze the level of intestinal mucus secretion.

[0094] As a result, as shown in Figure 12, the structure and size of the epithelial tissue forming the colonic surface were irregularly altered and inflammatory cell infiltration increased in the negative control group (DSS) compared to the control group (CON). Meanwhile, the symptoms observed in the negative control group were improved in the positive control group (DSS + ASA) and the sample-treated group (DSS + KM2) compared to the negative control group. Furthermore, the degree of blue staining in the mucosal layer decreased in the negative control group compared to the control group, where the mucosal layer was stained blue, while the degree of blue staining increased in the positive control group and the sample-treated group compared to the negative control group.

[0095] 2-6. Analysis of intestinal microorganisms

[0096] To confirm the effects of the samples on the gut microbiome, the animal models were sacrificed, the cecum was removed, and changes in the gut microbiome were analyzed using shotgun metagenome sequencing.

[0097] As a result, (Figure 13) α-diversity analysis, which can confirm the diversity of microorganisms present within a sample, confirmed that the Shannon index did not show significant differences between the experimental groups. Furthermore, the F / B (Firmicutes / Bacteroidetes) ratio, an inflammation index, also showed no significant differences between the experimental groups. β-diversity analysis based on unweighted UniFrac principal coordinate analysis (PCA), which can confirm the microbial community structure, showed significant differences between the experimental groups. Linear discriminant analysis (LDA) and KEGG pathway analysis were performed to identify significant differences in the microbiome between each experimental group. As shown in (Figure 13), the number of Weizmania coagulans, Clostridium perfringens, Clostridium botulinum, Clostridium sporogenes, Clostridium baratii, and Clostridium butyricum strains was significantly increased in the sample treatment group (DSS+KM2) compared to the negative control group (DSS). Additionally, the number of Bifidobacterium pseudolongum strains significantly increased in the negative control group compared to the sample-treated group.

[0098] Although the specific details of the present invention have been described above, it is obvious to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention, and the substantial scope of the present invention is defined by the claims and their equivalents.

[0099] JPEG2025536317000002.jpg182166

Claims

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

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

3. 2. The pharmaceutical composition of claim 1, wherein the inflammatory disease is at least one selected from the group consisting of ulcerative colitis, ulcerative duodenitis, Crohn's disease, irritable bowel syndrome, intestinal Behcet's disease, bleeding rectal ulcer, pouchitis, enteritis, ischemic colitis, acne, extraintestinal 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, Sjogren's syndrome, and multiple sclerosis.

4. The pharmaceutical composition according to claim 1, further comprising killed bacteria or spores of Lactiplantibacillus plantarum KM2 strain.

5. The pharmaceutical composition of claim 1, characterized in that the pharmaceutical composition regulates one or more intestinal microorganisms selected from the group consisting of Weizmania coagulans, Clostridium perfringens, Clostridium botulinum, Clostridium sporogenes, Clostridium baratii, Clostridium butyricum, and Bifidobacterium pseudolongum.

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

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

8. A method for preventing or treating an inflammatory disease, comprising the step of administering the pharmaceutical composition of claim 1 to a group of patients with an inflammatory disease in which the number of one or more strains selected from the group consisting of Weizmania coagulans, Clostridium perfringens, Clostridium botulinum, Clostridium sporogenes, Clostridium baratii, and Clostridium butyricum is reduced compared to a normal group; or to a group of patients with an inflammatory disease in which the number of Bifidobacterium pseudolongum strains is increased compared to a normal group.

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