New Faecalibacterium prausnitzii strain EB-FPDK11 and its use

Abstract

The present disclosure relates to a new Faecalibacterium prausnitzii EB-FPDK11 strain and its use. Administration of a composition comprising at least one selected from the following has the effect of preventing, alleviating and treating inflammatory diseases, liver diseases or metabolic diseases: Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of the strain, a lysate of the strain and an extract of the strain.

Description

Technical Field

[0001] The present disclosure relates to a novel Faecalibacterium prausnitzii strain EB-FPDK11 and uses thereof. Background Art

[0002] Probiotics refer to all bacteria that show beneficial effects in the body, including lactic acid bacteria, and are involved in a variety of body functions for intestinal diseases and immune diseases. For a time, research that dietary fiber (i.e., prebiotics), which is a food for probiotics, works better when taken with probiotics has attracted attention. Recently, the claim that postbiotics, which are metabolites released by probiotics, are effective as therapeutic agents or for diagnosing diseases has attracted attention, and pharmabiotics have also attracted attention. "Pharmaceutical probiotics" is a compound word of drugs in the sense of "medicinal" and live bacteria in the sense of "probiotics", refers to a human microbiome that can be used for medical purposes of disease care, and includes both probiotics and postbiotics.

[0003] Meanwhile, Faecalibacterium bacteria are obligate anaerobic rods that are often present in the intestinal mucus layer, and their retention rate and number in humans are both high. In addition, these bacteria are the main components of the intestinal flora.

[0004] Against this background, the present inventors have made efforts to develop a technology capable of curing diseases using strains that are harmless to the human body, and as a result, have identified a Faecalibacterium prausnitzii strain that exhibits excellent anti-inflammatory effects and lipid accumulation inhibitory effects, and found that the identified strain is suitable for treating liver diseases and colitis, thereby completing the present disclosure. Summary of the invention

[0005] Technical issues

[0006] One object of the present disclosure is to provide Faecalibacterium prausnitzii EB-FPDK11 strain (Accession No.: KCCM12621P).

[0007] Another object of the present disclosure is to provide a pharmaceutical composition for preventing or treating inflammatory diseases, liver diseases or metabolic diseases, the pharmaceutical composition comprising at least one selected from the following: Faecalibacterium prausnitzii (F. prausnitzii) EB-FPDK11 strain, a culture of Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain and an extract of the strain.

[0008] Yet another object of the present disclosure is to provide a food composition for preventing or alleviating inflammatory diseases, liver diseases or metabolic diseases, the food composition comprising at least one selected from the group consisting of: Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain and an extract of the strain.

[0009] Technical Solution

[0010] One aspect of the present disclosure provides Faecalibacterium prausnitzii EB-FPDK11 strain (Accession No.: KCCM12621P).

[0011] In one embodiment of the present disclosure, the Faecalibacterium prausnitzii EB-FPDK11 strain has a 16S rRNA sequence of SEQ ID NO:1.

[0012] Another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating an inflammatory disease, the pharmaceutical composition comprising at least one selected from the group consisting of: a Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain, and an extract of the strain.

[0013] Yet another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating liver disease, the pharmaceutical composition comprising at least one selected from the group consisting of: Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain, and an extract of the strain.

[0014] Yet another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating a metabolic disease, the pharmaceutical composition comprising at least one selected from the group consisting of: a Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain, and an extract of the strain.

[0015] Yet another aspect of the present disclosure provides a food composition for preventing or alleviating an inflammatory disease, the food composition comprising at least one selected from the group consisting of: a Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain, and an extract of the strain.

[0016] Another aspect of the present disclosure provides a food composition for preventing or alleviating liver disease, the food composition comprising at least one selected from the group consisting of: a Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain, and an extract of the strain.

[0017] Yet another aspect of the present disclosure provides a food composition for preventing or alleviating metabolic diseases, the food composition comprising at least one selected from the group consisting of: a Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain, and an extract of the strain.

[0018] According to one embodiment of the present disclosure, the food composition may be prepared in the form of a health functional food.

[0019] According to one embodiment of the present disclosure, the food composition may be prepared in the form of a probiotic formulation.

[0020] Beneficial Effects

[0021] Administration of a composition comprising at least one selected from the group consisting of Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain, and an extract of the strain has the effect of preventing, alleviating, or treating inflammatory diseases, liver diseases, or metabolic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Microscopic observations of the Faecalibacterium prausnitzii standard strain and EB-FPDK11 are shown.

[0023] Figure 2 The results of electrophoresis after PCR with FP-specific primers for the Faecalibacterium prausnitzii standard strain and EB-FPDK11 are shown.

[0024] Figure 3 The results of electrophoresis after PCR using ERIC-1, ERIC-2, and (GTG)5 primers for the Faecalibacterium prausnitzii standard strain and EB-FPDK11 are shown.

[0025] Figure 4 This is a phylogenetic tree prepared using the 16 rRNA nucleotide sequence of Faecalibacterium prausnitzii EB-FPDK11.

[0026] Figure 5 Shown are the results of examining whether the Faecalibacterium prausnitzii standard strain and EB-FPDK11 cause hemolysis.

[0027] Figure 6 It is a graph showing the results of analyzing short-chain fatty acids in the Faecalibacterium prausnitzii standard strain and EB-FPDK11.

[0028] Figure 7 The graph shows the results of analyzing the mRNA expression of the inflammatory cytokine IL-8 in each of the Faecalibacterium prausnitzii standard strain and EB-FPDK11.

[0029] Figure 8 The graph shows the results of analyzing the concentration of the inflammatory cytokine IL-10 in each of the Faecalibacterium prausnitzii standard strain and EB-FPDK11.

[0030] Fig. 9 Shown are photographs and graphs showing the results of examining the degree of inhibition of lipid accumulation by each of the Faecalibacterium prausnitzii standard strain and EB-FPDK11.

[0031] Fig.10 Shown are photographs and graphs showing the results of examining the degree of inhibition of lipid accumulation by respective cultures of the Faecalibacterium prausnitzii standard strain and EB-FPDK11.

[0032] Fig.11 Shown are graphs showing the results of comparing and analyzing the expression levels of genes involved in adipocyte differentiation after induction of adipogenesis after each treatment with the Faecalibacterium prausnitzii standard strain and EB-FPDK11.

[0033] Fig.12 A graph showing comparison of body weight and dietary intake between Faecalibacterium prausnitzii standard strain-administered mice and EB-FPDK11-administered mice in nonalcoholic steatohepatitis-induced mice.

[0034] Fig.13 A graph showing a comparison of glucose tolerance between Faecalibacterium prausnitzii standard strain-administered mice and EB-FPDK11-administered mice in nonalcoholic steatohepatitis-induced mice.

[0035] Fig.14 It is a graph comparing the liver weight and shape between Faecalibacterium prausnitzii standard strain-administered mice and EB-FPDK11-administered mice in non-alcoholic steatohepatitis-induced mice.

[0036] Fig.15 It is a graph comparing the spleen weight and shape between Faecalibacterium prausnitzii standard strain-administered mice and EB-FPDK11-administered mice in non-alcoholic steatohepatitis-induced mice.

[0037] Fig.16 The results of analyzing and comparing blood lipid biochemical indices between mice administered with the standard strain of Faecalibacterium prausnitzii and mice administered with EB-FPDK11 in nonalcoholic steatohepatitis-induced mice are shown.

[0038] Fig.17 Shown are the results of determining fat droplet formation in nonalcoholic steatohepatitis-induced mice by H&E staining of the livers of Faecalibacterium prausnitzii standard strain-administered mice and EB-FPDK11-administered mice.

[0039] Fig.18Shown are the results of comparing collagen deposition between Faecalibacterium prausnitzii standard strain-administered mice and EB-FPDK11-administered mice in nonalcoholic steatohepatitis-induced mice.

[0040] Fig.19 Shown are the results of comparing the extent of liver damage indicated by hepatic α-SMA between Faecalibacterium prausnitzii standard strain-administered mice and EB-FPDK11-administered mice in nonalcoholic steatohepatitis-induced mice.

[0041] Fig. 20 Shown are the results of comparing liver triglyceride and total cholesterol levels between Faecalibacterium prausnitzii standard strain-administered mice and EB-FPDK11-administered mice in nonalcoholic steatohepatitis-induced mice. DETAILED DESCRIPTION

[0042] In order to achieve the above-mentioned object, one aspect of the present disclosure provides Faecalibacterium prausnitzii EB-FPDK11 strain (accession number: KCCM12621P).

[0043] In one embodiment of the present disclosure, the Faecalibacterium prausnitzii EB-FPDK11 strain has a 16S rRNA sequence of SEQ ID NO:1.

[0044] Faecalibacterium prausnitzii is one of the most abundant bacteria among the bacteria constituting the human intestinal flora and is a non-motile Firmicute. Faecalibacterium prausnitzii is characterized in that it is extremely sensitive to oxygen and therefore does not grow even in the presence of very small amounts of oxygen.

[0045] Another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating an inflammatory disease, the pharmaceutical composition comprising at least one selected from the group consisting of: a Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain, and an extract of the strain.

[0046] The term "culture" used herein may refer to a composition obtained after the culture is complete. More specifically, the culture medium may or may not contain cells. Therefore, the culture may include a culture supernatant, a composition from which the culture supernatant is removed, or a composition obtained by concentrating it. In addition to the conventional components necessary for culturing Faecalibacterium prausnitzii, the culture composition may also include components that act synergistically on the growth of Faecalibacterium prausnitzii, and a person skilled in the art may easily select a composition comprising these components.

[0047] In addition, the strain may be in a liquid state or a dry state, and the drying method for the strain includes, but is not limited to, air drying, natural drying, spray drying, and freeze drying.

[0048] The term "inflammatory disease" used herein is a general term for diseases having inflammation as a main pathology. For example, the inflammatory disease may be any one selected from inflammatory skin diseases, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, hepatitis, peritonitis, osteomyelitis, cellulitis, meningitis, encephalitis, pancreatitis, cystic fibrosis, stroke, acute bronchitis, bronchitis, arthritis, arthritis, hemochromatosis, sickle cell anemia and other hemoglobinopathies, and sepsis, and may preferably be inflammatory skin diseases, colitis, chronic bronchitis, hepatitis or osteoarthritis, but is not limited thereto.

[0049] Yet another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating liver disease, the pharmaceutical composition comprising at least one selected from the group consisting of: Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain, and an extract of the strain.

[0050] The liver disease includes liver fibrosis or cirrhosis, acute or chronic hepatitis, fatty liver or liver cancer, and may preferably be fatty liver or hepatitis, more preferably nonalcoholic steatohepatitis, but is not limited thereto.

[0051] In the present disclosure, preventing or treating liver disease may refer to inhibiting abnormal increase in liver weight, and may refer to inhibiting abnormal increase in spleen length and weight. In addition, it may refer to controlling the concentration of triglycerides, cholesterol, GOT or GPT or inhibiting abnormal increase in the concentration, and inhibiting fat droplet formation, liver fibrosis and α-SMA expression in hepatocytes. However, the preventive and therapeutic effects of the pharmaceutical composition are not limited thereto.

[0052] Yet another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating a metabolic disease, the pharmaceutical composition comprising at least one selected from the group consisting of: a Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain, and an extract of the strain.

[0053] Metabolic diseases can be hyperlipidemia, diabetes, gout, dementia, obesity, hypertension, hypoglycemia, hypercholesterolemia, hemochromatosis, amyloidosis or porphyria. Diabetes can include type 1 diabetes and type 2 diabetes. Preferably, metabolic diseases can be obesity, but are not limited thereto.

[0054] The pharmaceutical composition used in the present disclosure should be used in a pharmaceutically effective amount. The term "pharmaceutically effective amount" used herein refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level of the pharmaceutical composition can be determined based on factors including: type of subject, severity of disease, age and sex, type of infected virus, drug activity, sensitivity to the drug, time of administration, route of administration, excretion rate, duration of treatment, and the drug used in combination with the composition, and other factors known in the medical field. As will be appreciated by those skilled in the art, effective amount can vary based on the potential of treatment route, excipient use, and use with other drugs.

[0055] The pharmaceutical composition of the present disclosure can be prepared into a pharmaceutical dosage form using methods well known in the art so as to provide a rapid, sustained or delayed release of the active ingredient after administration to a mammal. In the preparation of the dosage form, the active ingredient is preferably mixed with a carrier or diluted with a carrier or encapsulated in a carrier in the form of a container.

[0056] Therefore, the pharmaceutical composition of the present disclosure can be formulated into oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups or aerosols, or in the form of external preparations and patches according to conventional methods, and may also contain suitable carriers, excipients or diluents commonly used for preparing the compositions.

[0057] Some examples of carriers, excipients and diluents that may be included in the pharmaceutical composition of the present disclosure include but are not limited to lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum, magnesium stearate and mineral oil. Commonly used diluents or excipients may be used, such as fillers, extenders, adhesives, wetting agents, disintegrants and surfactants to prepare the preparation.

[0058] Yet another aspect of the present disclosure provides a food composition for preventing or alleviating an inflammatory disease, the food composition comprising at least one selected from the group consisting of: a Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain, and an extract of the strain.

[0059] Another aspect of the present disclosure provides a food composition for preventing or alleviating liver disease, the food composition comprising at least one selected from the group consisting of: a Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain, and an extract of the strain.

[0060] Yet another aspect of the present disclosure provides a food composition for preventing or alleviating metabolic diseases, the food composition comprising at least one selected from the group consisting of: a Faecalibacterium prausnitzii EB-FPDK11 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK11 strain, a lysate of the strain, and an extract of the strain.

[0061] In the present disclosure, the food composition can be used in various forms, including pills, powders, granules, needles, tablets, capsules, or liquids and solutions, and the food to which the composition of the present disclosure can be added includes, for example, various foods such as beverages, chewing gum, tea, vitamin complexes, and health supplement foods.

[0062] Except that the food composition of the present disclosure includes the Faecalibacterium prausnitzii EB-FPDK11 strain, the culture of the Faecalibacterium prausnitzii EB-FPDK11 strain, the lysate of the strain, or the extract of the strain, or its active ingredient or its physiologically acceptable salt as an essential ingredient, there is no particular limitation on other ingredients. Similar to ordinary food, the food composition may also include additional ingredients, such as various herbal extracts, food supplement additives or natural carbohydrates.

[0063] In addition, the food composition may further include food supplement additives as mentioned above, and the food supplement additives include conventional food supplement additives known in the art, such as flavoring agents, aromas, coloring agents, fillers and stabilizers.

[0064] Some examples of natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. In addition to the above ingredients, natural flavoring agents (e.g., rebaudioside A, glycyrrhizin, etc.) or synthetic flavoring agents (saccharin, aspartame, etc.) may also be suitably used as flavoring agents.

[0065] In addition to the above-mentioned ingredients, the food composition of the present disclosure may also include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers (such as cheese or chocolate), pectin acid and its salt, alginic acid and its salt, organic acid, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerine, alcohol, the carbonizing agent used in carbonated beverages, etc. In addition, the food composition may include natural fruit juice and pulp for producing fruit juice beverages and vegetable beverages. These ingredients may be used individually or in combination.

[0066] According to one embodiment of the present disclosure, the food composition can be prepared in the form of a healthy functional food. The term "healthy functional food" used herein has the same meaning as "food for special health use (food for special health use, FoSHU)", and means a food with high pharmaceutical and medical effects, which is processed to effectively play a biological regulation function in addition to nutrient supply. Here, "functional food" means obtaining an effect that can be used for health applications, such as nutritional control or physiological effects on the structure and function of the human body. The food of the present disclosure can be prepared by methods commonly used in the art, and can be prepared by adding raw materials and ingredients commonly used in the art. In addition, any preparation of the food can also be prepared without restriction, as long as it is acceptable as food. The food composition of the present disclosure can be prepared into various types of preparations, and because it is different from general drugs containing food as raw materials, it has the advantage of being free from the side effects that may occur when the drug is administered for a long time. In addition, due to its excellent portability, the food composition of the present disclosure can be carried as a supplement for enhancing the effect of preventing or alleviating inflammatory diseases, liver diseases or metabolic diseases.

[0067] According to one embodiment of the present disclosure, the food composition may be prepared in the form of a probiotic formulation.

[0068] Probiotic preparations can be prepared and administered in a variety of dosage forms according to various methods known in the art. For example, the Faecalibacterium prausnitzii EB-FPDK11 strain of the present disclosure, its culture or concentrate or dry product of the culture can be prepared and administered in the form of powders, liquids and solutions, tablets, capsules, syrups, suspensions or granules by mixing with carriers commonly used in the pharmaceutical field. Some examples of carriers include, but are not limited to, adhesives, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants and flavoring agents. In addition, the dosage of the probiotic preparation can be appropriately selected based on the in vivo absorption rate, inactivation rate and excretion rate of the active ingredient, the age, sex, type, symptoms and severity of the disease of the subject, etc.

[0069] Example

[0070] Hereinafter, one or more embodiments will be described in more detail with reference to examples. However, these examples are used to illustrate one or more embodiments, and the scope of the present disclosure is not limited to these examples.

[0071] Example 1: Isolation and identification of Faecalibacterium prausnitzii EB-FPDK11 strain

[0072] 1.1. Acquisition and isolation of Faecalibacterium prausnitzii samples

[0073] To isolate Faecalibacterium prausnitzii from the stool of a healthy Korean (female, 9 years old, BMI 15.5), the stool was cultured using YBHI medium [brain heart infusion medium supplemented with 0.5% w / v yeast extract (Difco), 0.1% w / v D-cellobiose, and 0.1% w / v D-maltose] according to the Martin method, and then extremely oxygen sensitive (EOS) strains were selected and isolated.

[0074] 1.2. Microscopic observation

[0075] In order to determine whether the isolated strain is a Faecalibacterium prausnitzii strain, the isolated strain was observed under a microscope. Figure 1 As shown in FIG, Faecalibacterium prausnitzii DSM17677 was determined as a standard strain observed at 1,000x magnification. T strain ( Figure 1 Both F. prausnitzii EB-FPDK11 strains ( FIG. 1B ) have straight or curved rod-shaped cell shapes and thus show similar shapes.

[0076] PCR analysis

[0077] In order to determine whether the isolated strain is a Faecalibacterium prausnitzii strain, the isolated strain was subjected to PCR analysis using the FP-specific primers (SEQ ID NO: 2 and SEQ ID NO: 3) shown in Table 1 below. Figure 2 As shown in FIG, it was determined that the isolated strain showed similarity to Faecalibacterium prausnitzii DSM17677 as a positive control strain. T strips.

[0078] [Table 1]

[0079]

[0080]

[0081] 1.4. Random Amplified Polymorphic DNA (RAPD) analysis

[0082] In order to examine whether the strains isolated as described above are different from previously reported standard strains of the same species, random amplified polymorphic DNA (RAPD) analysis (a type of molecular typing) was performed. For this purpose, genomic DNA (gDNA) extracted from cells was amplified using universal primers (SEQ ID NO: 4 to SEQ ID NO: 6) shown in Table 2 below and then electrophoresed on a 1% agarose gel for 90 minutes. Then, as Figure 3 As shown in , DNA fragmentation patterns were compared using a UV transilluminator.

[0083] [Table 2]

[0084] SEQ ID NO name direction Sequence (5'→3') SEQ ID NO: 4 ERIC-1 Positive ATG TAA GCT CCT GGG GAT TCA C SEQ ID NO: 5 ERIC-2 Reverse AAG TAA GTG ACT GGG GTG AGC G SEQ ID NO: 6 <![CDATA[(GTG)5]]> Forward / Reverse GTG GTG GTG GTG GTG

[0085] As Figure 3 The results of the pattern comparison showed that the Faecalibacterium prausnitzii EB-FPDK11 strain showed some similarities but differences from the standard strain DSM 17677. T The banding pattern shows that the strain Faecalibacterium prausnitzii EB-FPDK11 is different from the standard strain Faecalibacterium prausnitzii DSM 17677 T It belongs to the same species, but is a different strain.

[0086] 1.5.16S rRNA BLAST

[0087] In order to determine whether the isolated strain is a Faecalibacterium prausnitzii strain, the isolated strain is subjected to 16S rRNA sequencing and subsequently analyzed by BLAST. As a result, the isolated strain has 99% or higher identity with the Faecalibacterium prausnitzii species. Based on these results, the isolated strain is named Faecalibacterium prausnitzii EB-FPDK11 strain and is deposited in the Korean Culture Center of Microorganisms (KCCM) with accession number KCCM12621P.

[0088] 1.6. Phylogenetic tree analysis using 16S rRNA nucleotide sequences

[0089] As a result of strain identification, there were strains similar to currently known strains, but completely consistent results were not obtained. Therefore, a phylogenetic tree analysis was performed. For full-length 16S rRNA gene sequencing of the isolated Faecalibacterium prausnitzii EB-FPDK11 strain, the 16S rRNA gene was amplified using primers 27F (SEQ ID NO: 7) and 1492R (SEQ ID NO: 8) shown in Table 3 below, and then its nucleotide sequence was determined using a 3730xl DNA analyzer (Thermo Fisher Scientific, USA). Using the obtained 16S rRNA gene sequences of the EB-FPDK11 strain and the standard strain, as well as the previously published 16S rRNA gene sequences of other strains of the same species, a maximum likelihood method was prepared. Figure 4 The phylogenetic tree shown in .

[0090] [Table 3]

[0091]

[0092] Example 2: Characterization of Faecalibacterium prausnitzii EB-FPDK11 strain

[0093] 2.1. Antimicrobial susceptibility testing

[0094] To examine the antimicrobial susceptibility of F. prausnitzii EB-FPDK11 strain, the minimum inhibitory concentration (MIC) of each of the antimicrobial agents piperacillin-tazobactam, ceftizoxime, chloramphenicol, clindamycin, meropenem, moxifloxacin, metronidazole, and ciprofloxacin for anaerobic microorganisms against F. prausnitzii EB-FPDK11 strain was examined according to the liquid culture microdilution method of the Clinical & Laboratory Standard Institute (CLSI) guidelines.

[0095] [Table 4]

[0096]

[0097] As a result, it can be seen from Table 4 above that the Faecalibacterium prausnitzii EB-FPDK11 strain of the present disclosure showed resistance to piperacillin-tazobactam (PTZ), cefuroxime (CTZ), meropenem (MEM) and fluoroquinolone-based antibiotics moxifloxacin (MXF) and ciprofloxacin (CIP), and showed sensitivity to chloramphenicol (CHL), clindamycin (CLI) and metronidazole (MTZ). For the antibiotic chloramphenicol (CHL), the Faecalibacterium prausnitzii EB-FPDK11 strain showed similarity to the standard strain (DSM17677 T ) significant difference.

[0098] 2.2. Evaluation of hemolytic activity

[0099] In order to verify the safety of the Faecalibacterium prausnitzii EB-FPDK11 strain, whether the strain has hemolytic activity was evaluated. To this end, the strain was cultured using a blood agar medium prepared by adding 1.5% w / v bacto-agar and 5% w / v defibrinated sheep blood to a YBHI medium [brain heart infusion medium supplemented with 0.5% w / v yeast extract (Difco), 0.1% w / v D-cellobiose, and 0.1% w / v D-maltose], and then observing whether hemolysis would occur around the colonies. As a positive control, Streptococcus pyogenes ATCC 19615, which causes β-hemolysis, was used for comparison.

[0100] As a result, Figure 5 As shown in FIG, the Faecalibacterium prausnitzii EB-FPDK11 strain of the present disclosure and the standard strain DSM17677 T Both showed no clearing zones around the colonies, indicating that these strains do not cause β-hemolysis associated with pathogenicity.

[0101] 2.3. Analysis of functional metabolites (short-chain fatty acids)

[0102] In order to analyze the functional metabolites in the isolated Faecalibacterium prausnitzii EB-FPDK11 strain, the content of short chain fatty acids (SCFA) in the culture of the strain was analyzed by gas chromatography. To this end, the strain was cultured in YBHI medium [brain heart infusion medium supplemented with 0.5% w / v yeast extract (Difco), 0.1% w / v D-cellobiose and 0.1% w / v D-maltose] for 24 hours and then centrifuged at 12,000 x g for 5 minutes. The supernatant was collected, filtered through a 0.2 μm syringe filter, and then used for analysis. A gas chromatograph (Agilent 7890N) equipped with a FFAP column (30m×0.320mm, 0.25 μm phase) was used for analysis under the conditions shown in Table 5 below.

[0103] [Table 5]

[0104]

[0105] As a result of analyzing the functional short-chain fatty acids, such as Figure 6 As can be seen in the figure, it was determined that Faecalibacterium prausnitzii strain EB-FPDK11 consumed acetate and produced butyrate.

[0106] Example 3: Evaluation of the anti-inflammatory effect of Faecalibacterium prausnitzii EB-FPDK11 strain

[0107] Evaluation of anti-inflammatory effects in HT-29 intestinal epithelial cells

[0108] Since cytokines are involved in the regulation of inflammatory responses in inflammatory bowel disease, the Faecalibacterium prausnitzii EB-FPDK11 strain was administered and changes in cytokine gene expression were examined. In order to evaluate the anti-inflammatory effect by in vitro experiments, HT-29 cells ( HTB-38 TM , USA). McCoy's 5A modified medium (Gibco, USA) supplemented with 10% FBS (fetal bovine serum, Hyclone, USA) and 10 μg / ml gentamicin was used as the basal medium, and the cells were cultured in an incubator (NUAIRE, USA) at 37°C under 5% CO2. In order to determine whether the Faecalibacterium prausnitzii EB-FPDK11 strain inhibits LPS-induced inflammatory cytokine IL-8 gene expression in HT-29 cells, real-time PCR was performed using the primers shown in Table 6 below (SEQ ID NOs: 9 to 12).

[0109] [Table 6]

[0110] SEQ ID NO Target Primer sequences SEQ ID NO: 9 GAPDH F: 5′-GAC ATC AAG AAG GTG GTG AAG CAG-3′ SEQ ID NO: 10 GAPDH R: 5′-ATA CCA GGA AAT GAG CTT GAC AAA-3′ SEQ ID NO: 11 IL-8 F: 5′-TTT TGC CAA GGA GTG CTA AAG A-3′ SEQ ID NO: 12 IL-8 R: 5′-AAC CCT CTG CAC CCA GTT TTC-3′

[0111] Total RNA was extracted using TRI reagent (Sigma, USA), and for cDNA synthesis, 1 μg of RNA was synthesized into cDNA by M-MLV cDNA Synthesis Kit (Enzynomics, Korea). Real-time PCR was performed using Quant Studio 3 Real-time PCR System (Applied Biosystems, USA).

[0112] The expression of inflammatory cytokine genes was analyzed using SYBR Green TOPrealTM qPCR 2X PreMIX (EnZynomics, Korea), and GAPDH was used as an internal standard. PCR was performed under the following conditions: preincubation at 50°C (for UDG) for 4 minutes and preincubation at 95°C for 10 minutes, and 40 cycles, each consisting of 95°C for 15 seconds and 60°C for 1 minute. Data were analyzed by the ΔCT method using a program built into QuantStudio Design & Analysis Software v1.4.3.

[0113] The results obtained in all experiments were calculated as the mean and standard deviation of each experimental group using the statistical program GraphPad Prism 7 (GraphPad software Inc, USA), and the differences between groups were analyzed using one-way ANOVA and Tukey's test. P values ​​≤ 0.05 were considered significant. In some results, AUC (area under curve) was calculated.

[0114] As a result, Figure 7 As shown in, when HT-29 cells were treated with LPS alone (100 μg / ml) for 6 hours, the expression of the representative inflammatory cytokine IL-8 in the cells was increased compared with the IL-8 expression in the normal group. However, it was shown that the IL-8 expression in the group treated with LPS and the culture of the standard strain of Faecalibacterium prausnitzii A2-165 (10%, v / v) was reduced compared with the IL-8 expression in the LPS-treated group, and the IL-8 expression in the group treated with LPS and the culture of the Faecalibacterium prausnitzii EB-FPDK11 strain was further reduced compared with the IL-8 expression in the group treated with LPS and the A2-165 standard strain. Therefore, it was determined that the culture of the Faecalibacterium prausnitzii EB-FPDK11 strain reduced the inflammatory cytokine IL-8.

[0115] 3.2. Evaluation of anti-inflammatory effects in mouse bone marrow-derived dendritic cells

[0116] In order to observe the anti-inflammatory response, the cytokine secretion from dendritic cells (Dendritic Cells, DC) was analyzed. In order to evaluate the anti-inflammatory effect of strains using mouse bone marrow derived dendritic cells (bone marrow-derived dendritic cell, BMDC), BMDC was separated. After piercing 0.5ml microtubes with 18G needles, the femur and tibia of 6-week-old C57BL / 6 mice were separated, and placed in 1.5ml precipitation tubes, and then centrifuged for 15 seconds at 10,000xg. The precipitation in the 1.5ml precipitation tube was washed 3 times with PBS, and then the precipitation was added to RPMI-1640 (10% FBS, 1% P / S, culture medium, 1X mercaptoethanol, 20μg GM-CSF) culture medium and cultivated in 150mm culture dishes. On the second day, BMDC was transferred to a 100ml culture dish and cultured therein, and on the 5th day, 10ml culture was transferred to a 15ml conical tube, and then centrifuged for 15 minutes at 1,000xg. The supernatant was removed, and 10 ml of BMDC medium was added to the BMDC and placed in a culture dish. On day 6 or 7, the BMDC was used for the experiment. To evaluate the anti-inflammatory effect of the F. prausnitzii EB-FPDK11 strain, the secretion of the representative anti-inflammatory cytokine IL-10 was analyzed by mIL-10 ELISA (Invitrogen, USA).

[0117] BMDCs were treated with LPS (100 μg / ml), Escherichia coli, Faecalibacterium prausnitzii A2-165 standard strain and EB-FPDK11 strain (10 7 cfu / ml, 10% v / v) in an antibiotic-free medium for 1 hour, and then the medium was replaced with a medium containing penicillin / streptomycin antibiotics. Next, the cells were cultured for 24 hours, and the medium was centrifuged at 1,000 x g. The supernatant was used to measure IL-10 secretion by ELISA.

[0118] like Figure 8 As shown in , the IL-10 secretion from cells treated with each of LPS and E. coli was similar to the IL-10 secretion from the normal group, with no difference. However, the group treated with the standard strain of Faecalibacterium prausnitzii A2-165 showed a significant increase in IL-10 secretion compared to the normal group. The IL-10 expression in the group treated with the EB-FPDK11 strain of Faecalibacterium prausnitzii was further increased to a significant level compared to the IL-10 expression in the group treated with the A2-165 standard strain. Therefore, it was determined that treatment with the Faecalibacterium prausnitzii strain resulted in a significant increase in the anti-inflammatory cytokine IL-10.

[0119] Example 4: Evaluation of lipid accumulation inhibitory effect

[0120] It was examined whether the expression of lipid accumulation- and obesity-related biomarkers was affected by administration of the strains of the present disclosure.

[0121] 4.1. Oil Red-O Staining of Differentiated Adipocytes

[0122] In order to examine the effect of the Faecalibacterium prausnitzii EB-FPDK11 strain of the present disclosure on adipocyte differentiation and adipogenesis from 3T3-L1 cells, an Oil Red-O (ORO) staining experiment was performed. First, in order to differentiate 3T3-L1 preadipocytes into adipocytes, the cells were cultured at 2×10 4 / well in a 24-well plate. The cells were cultured in DMEM medium containing 10% FBS for 4 days. When the cells reached saturation in the plate, the medium was replaced with differentiation medium [DMEM, 10% fetal bovine serum, 0.5 mM IBMX (3-isobutyl-1-methylxanthine, Sigma I5879), 1 μM dexamethasone (Sigma D4902, FW392.5), 10 mg / ml insulin], and the cells were incubated with 50 μl (1×10 7 The cells were treated with samples (Faecalis strains or their cultures) of 10 cells / well and then cultured at 37°C under 5% CO2 for 2 days. Thereafter, the culture medium was replaced with insulin medium (10% FBS, 10 mg / ml insulin) every two days, and the cells were cultured under the same conditions for 8 days. Whenever the culture medium was replaced, the cells were treated with Faecalis strains and their cultures at the same time. The strains and their cultures (10 7 cfu / ml) treated cells.

[0123] Oil Red-O staining is a method for staining differentiated 3T3-L1 cells with Oil Red-O reagent to measure the fat produced in the cells. 3T3-L1 cells (Korea Cell Line Bank, Korea) as mouse preadipocytes were cultured. DMEM (Dulbecco's modified Eagle medium, Welgene, Korea) supplemented with 10% FBS (fetal bovine serum, Hyclone, USA) and 1% penicillin / streptomycin was used as the basal medium, and the cells were cultured in a 5% CO2 incubator (NUAIRE, USA) at 37°C. After inducing adipocyte differentiation from preadipocytes 3T3-L1 by insulin (1 μg / ml), IBMX (0.5 mM) and dexamethasone (1 μM) for 10 days, the culture medium was removed by washing three times with PBS, and 10% formalin (Sigma, USA) was added to the cells, and then the cells were reacted with Oil red O (Sigma, USA) solution for 1 hour and washed with distilled water to stain lipid droplets.

[0124] After the cell staining was completed, the cells were washed 3 times with 40% isopropanol (Duksan, Korea) and dried, and the size of lipid droplets in the cells was observed using an optical microscope. The adipocyte sample stained with Oil Red-O solution was melted by adding isopropanol thereto, and the absorbance at 500 nm was measured using a spectrophotometer (Epoch, BioTek, USA). The results are shown in Fig. 9 and 10 middle.

[0125] like Fig. 9 As shown in, as a result of treating 3T3-L1 cells with the Faecalibacterium prausnitzii A2-165 standard strain of the present disclosure during cell differentiation, lipid accumulation in the treated cells was inhibited compared to lipid accumulation in the control group. It was determined that treatment with the Faecalibacterium prausnitzii EB-FPDK11 strain more significantly inhibited lipid accumulation than in the group treated with the Faecalibacterium prausnitzii A2-165 standard strain.

[0126] Similarly, if Fig.10 As shown in, as a result of treating 3T3-L1 cells with a culture of the standard strain of Faecalibacterium prausnitzii A2-165 during cell differentiation, lipid accumulation in the treated cells was significantly inhibited compared to lipid accumulation in the control group. Treatment with the culture of the Faecalibacterium prausnitzii EB-FPDK11 strain more significantly inhibited lipid accumulation than the group treated with the culture of the standard strain of Faecalibacterium prausnitzii A2-165.

[0127] As a result of this experiment, it was determined that the Faecalibacterium prausnitzii EB-FPDKl1 strain and its culture had a better effect on inhibiting adipogenesis of 3T3-L1 cells than the Faecalibacterium prausnitzii A2-165 standard strain and its culture.

[0128] 4.2. Evaluation of the effect on biomarker gene expression

[0129] In order to evaluate the effect of the strain on inhibiting adipocyte differentiation, real-time PCR was performed using the gene-specific primers (SEQ ID NOs: 13 to 24) shown in Table 7 below to analyze the mRNA expression levels of the transcription factor C / EBPα (CCAAT / enhancer binding protein α) involved in adipocyte differentiation and maturation and the lipogenic genes aP2 (adipocyte protein 2), FAS (fatty acid synthase), ACC1 (acetyl-CoA-carboxylase) and LPL (lipoprotein lipase) during the adipocyte differentiation stage.

[0130] [Table 7]

[0131] SEQ ID No Target Primer sequences SEQ ID NO: 13 GAPDH F: 5′-GAC ATC AAG AAG GTG GTG AAG CAG-3′ SEQ ID NO: 14 GAPDH R: 5′-ATA CCA GGA AAT GAG CTT GAC AAA-3′ SEQ ID NO: 15 C / EBPα F:5′-AGC AAC GAG TAC CGG GTA CG-3′ SEQ ID NO: 16 C / EBPα R: 5′-TGT TTG GCT TTA TCT CGG CTC-3′ SEQ ID NO: 17 aP2 F: 5′-AGT GAA AAC TTC GAT GAT TAC ATG AA-3′ SEQ ID NO: 18 aP R: 5′-GCC TGC CAC TTT CCT TGT G-3′ SEQ ID NO: 19 FAS F: 5′-AGG GGT CGA CCT GGT CCT CA-3′ SEQ ID NO: 20 FAS R: 5′-GCC ArG CCC AGA GGG TGG TT-3′ SEQ ID NO: 21 ACC1 F:5′-CCT CCG TCA GCT CAG ATA CA-3′ SEQ ID NO: 22 ACC1 R: 5′-TTT ACT AGG TGC AAG CCA GAC A-3′ SEQ ID NO: 23 LPL F: 5′-TTG CCC TAA GGA CCC CTG AA-3′ SEQ ID NO: 24 LPL R: 5′-ACA GAG TCT GCT AAT CCA GGA AT-3′

[0132] Specifically, total RNA was extracted from the cell monolayer using TRI reagent (Sigma, USA) according to the manufacturer's instructions, and cDNA was synthesized from 1 μg of total RNA using the M-MLV cDNA synthesis kit (Enzynomics, Korea). PCR reactions were performed using the Quant Studio 3 real-time PCR system (Applied Biosystems, USA). PCR was performed under the following conditions: pre-incubation at 50°C for 4 minutes and pre-incubation at 95°C for 10 minutes, and 40 cycles, each cycle consisting of 95°C for 15 seconds and 60°C for 1 minute. Data were analyzed using the built-in program in QuantStudio Design & Analysis Software v1.4.3 by the ΔCT method.

[0133] like Fig.11As shown in, when the increased expression levels of C / EBPa, aP2, FAS, ACC1 and LPL, which are genes involved in adipocyte differentiation after induced adipogenesis, are expressed as 100%, the expression levels of C / EBPα, aP2, FAS, ACC1 and LPL in the group treated with each of the cultures of the Faecalibacterium prausnitzii A2-165 standard strain and the Faecalibacterium prausnitzii EB-FPDK11 strain are significantly reduced. It was determined that both the Faecalibacterium prausnitzii A2-165 standard strain and the Faecalibacterium prausnitzii EB-FPDK11 strain have excellent effects of inhibiting the expression of adipogenesis-related genes in 3T3-L1 cells.

[0134] Example 5: Evaluation of the effect on non-alcoholic steatohepatitis

[0135] 5.1. Construction of animal model of nonalcoholic steatohepatitis

[0136] Animal experiments were performed in accordance with the animal use and care protocols of the Institutional Animal Care and Use Committee (IACUC). As experimental animals, 8-week-old male C57BL / 6 mice (9 mice / group) were purchased and adapted for 1 week. Then, the mice were cultured for 12 weeks. For the culture environment, the mice were adapted for 1 week at a constant temperature (22°C) and relative humidity (40% to 60%) and a 12-hour light / 12-hour dark cycle.

[0137] To induce nonalcoholic steatohepatitis, mice consumed drinking water containing a high-fat diet (60 kcal% fat; Research Diets Inc., NJ, USA) as an experimental diet (NASH) and 30% fructose for 16 weeks and were allowed ad libitum access to drinking water.

[0138] The experimental mice were randomly divided into 5 groups as shown in Table 8 below.

[0139] [Table 8]

[0140]

[0141] In the case of experimental groups III, IV, and V, silymarin (30 mg / kg) or 1×10 8 CFU / 150 μl PBS (25% glycerol and 0.05% cysteine / PBS) of live cells of Faecalibacterium prausnitzii.

[0142] Normal mice (Normal) were made to consume a 10% fat diet. As a positive control, silymarin, which is known as a functional raw material that helps alleviate non-alcoholic fatty liver, or a standard strain of Faecalibacterium prausnitzii A2-165 were administered. At this time, the normal group and the experimental diet group were orally administered with the same amount of phosphate buffered saline (25% glycerol and 0.05% cysteine / PBS) every day to exclude the effects of stress caused by administration.

[0143] 5.2. Changes in body weight and food intake

[0144] After 16 weeks of the nonalcoholic steatohepatitis induction experiment, the weight changes of the experimental groups were measured. The results are shown in Fig.12 middle.

[0145] refer to Fig.12 , the body weight of all groups of animals with non-alcoholic steatohepatitis induced by the experimental diet was increased compared with that of the normal diet group. When the weight gain during the 8th week (when silymarin or Faecalibacterium prausnitzii strain was administered) to the 16th week was calculated as mass (g) and percentage (%), it was observed that the weight gain was slightly reduced in the silymarin administration group and the Faecalibacterium prausnitzii EB-FPDK11 strain administration group compared with the non-alcoholic steatohepatitis induced group, but no significant decrease in weight gain was found. The percentage weight gain observed in the Faecalibacterium prausnitzii EB-FPDK11 administration group was the smallest compared with the normal diet group. There were no significant differences in food intake and calorie intake between the groups with non-alcoholic steatohepatitis induced by the experimental diet.

[0146] 5.3. Changes in glucose tolerance (Oral Glucose Tolerance Test (OGTT))

[0147] To evaluate the effect of administration of Faecalibacterium prausnitzii EB-FPDK11 strain on glucose tolerance, glucose (2 g / kg) was orally administered to mice 16 weeks after the start of the experiment in a state where the mice were fasted for 18 hours. Blood was collected from the tail vein immediately before glucose administration and 30, 60, 90 and 120 minutes after glucose administration, and blood glucose levels were measured with a glucometer. The measurement results are shown in Fig.13 middle.

[0148] refer to Fig.13, among the administration groups, the group to which the Faecalibacterium prausnitzii EB-FPDK11 strain was administered immediately before glucose administration showed the greatest reduction in blood glucose levels. 30 minutes after glucose administration, blood glucose levels increased in all administration groups compared with the normal diet group, but as a result of calculating the area under the curve (AUC) of blood glucose levels within 120 minutes, as time increased to 60 minutes, 90 minutes, and 120 minutes, blood glucose levels were significantly reduced in the silymarin administration group or the Faecalibacterium prausnitzii A2-165 standard strain administration group or the EB-FPDK11 strain administration group compared with the non-alcoholic fatty liver inflammation induced group. As a result of this study, it was determined that oral administration of the Faecalibacterium prausnitzii EB-FPDK11 strain can improve the blood glucose control ability reduced by non-alcoholic fatty liver inflammation induction, and can improve glucose tolerance.

[0149] 5.4. Observation of steatohepatitis and tissue weight changes

[0150] At the end of the experiment, the liver and spleen were removed under anesthesia with CO 2 , washed with physiological saline, dehydrated, and then weighed, and their size and color were visually observed.

[0151] refer to Fig.14 , it was observed that the liver tissue of the normal diet group showed a bright red healthy liver shape, while the liver of the group with non-alcoholic steatohepatitis induced by the experimental diet became turbid in color due to lipid accumulation and lost its original bright red color. However, the silymarin-administered group, the Faecalibacterium prausnitzii A2-165 standard strain-administered group, and the EB-FPDK11 strain-administered group showed a bright red liver shape close to that of the normal diet group. As a result of measuring the liver weight, it was observed that the weight in each of the non-alcoholic steatohepatitis-induced group and the silymarin-administered group increased compared to the normal diet group. However, the liver tissue weight of the Faecalibacterium prausnitzii EB-FPDK11-administered group was most similar to that of the normal group, and did have a significant difference from the liver tissue weight of the non-alcoholic steatohepatitis-induced group. By Fig.14 The results in , it was determined that the Faecalibacterium prausnitzii EB-FPDK11-administered group exhibited liver shape and weight similar to those of the normal diet group. Therefore, it can be concluded that the Faecalibacterium prausnitzii EB-FPDK11 strain can alleviate non-alcoholic steatohepatitis.

[0152] like Fig.15As shown in , the length and weight of the spleen in the non-alcoholic steatohepatitis induced group were increased compared with the normal diet group. As in the case of liver tissue, it was observed that the spleen length of the group treated with each of silymarin and Faecalibacterium prausnitzii A2-165 standard strains was also increased compared with the spleen length of the normal diet group, but the increase in spleen length in the Faecalibacterium prausnitzii EB-FPDK11 administered group was so low that it was not significant. It was determined that the spleen weight was lower than that of the non-alcoholic steatohepatitis induced group.

[0153] 5.5. Analysis of blood lipid biochemical indicators

[0154] After fasting for 18 hours, blood was collected from each experimental animal, and then the concentrations of triglyceride (TG) and total cholesterol (TC) as indicators of lipid content, and the concentrations of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) as indicators of liver function were measured in the serum separated from the blood. The measurement results are shown in Fig.16 The concentrations of TG, TC, GOT, and GPT, which are indicators of lipid composition, were quantified using individual measurement kits purchased from Asan Pharmaceutical Co., Ltd.

[0155] It was determined that the triglyceride concentration was significantly increased in the non-alcoholic steatohepatitis induced group. However, compared with the non-alcoholic steatohepatitis induced group, the triglyceride concentration was significantly reduced in the silymarin administration group and the EB-FPDK11 strain administration group. Compared with the normal group, the total cholesterol level was higher in the non-alcoholic steatohepatitis induced group and the A2-165 standard strain treated group. However, compared with the non-alcoholic steatohepatitis induced group and the A2-165 standard strain treated group, the total cholesterol level was significantly reduced in the EB-FPDK11 strain treated group. It was observed that the GOT concentration, which indicates the degree of hepatocellular damage, was reduced in all administration groups compared with the non-alcoholic steatohepatitis induced group, and the GPT concentration was significantly reduced only in the silymarin administration group and the EB-FPDK11 strain administration group. Through the analysis of blood lipid biochemical indicators, it was determined that the concentrations of triglycerides, total cholesterol, GOT and GPT, which are closely related to non-alcoholic steatohepatitis, were reduced by administering the EB-FPDK11 strain.

[0156] 5.6. Analysis of pathological severity of steatohepatitis in liver tissue

[0157] In order to observe the effect of administering EB-FPDK11 strain on alleviating non-alcoholic fatty liver inflammation, hematoxylin and eosin (H&E) staining of liver tissue sections and Sirius red staining (Sirius red staining) that can measure liver fibrosis were performed, and the expression of alpha-smooth muscle actin (alpha-smooth muscle actin, α-SMA) that occurs during liver damage was observed by staining. The liver tissue isolated from each mouse was cut into a thickness of about 5 μm, and then embedded in paraffin, and the difference in morphological changes was observed by staining each time. The degree of liver damage observed by each staining was expressed as a percentage positive area (%) by the Image J program.

[0158] like Fig.17 As shown in , as a result of analyzing mouse liver tissue by H&E staining, it was observed that the liver tissue of the normal group had no fat droplets because its hepatocyte structure was generally dense. However, in the liver tissue of the non-alcoholic steatohepatitis-induced mouse, a large amount of fat droplet formation was clearly observed compared with that in the normal group. It was observed that fat droplet formation was reduced in all administration groups compared with the non-alcoholic steatohepatitis-induced group, and it was determined that the liver tissue of each of the silymarin administration group and the EB-FPDK11 strain administration group was more dense, indicating that fat droplets were reduced in these groups.

[0159] like Fig.18 As shown in , the amount of deposited collagen was analyzed by Sirius red staining of mouse liver tissue. The amount of collagen deposited in the liver is known to be a sensitive indicator reflecting the degree of fibrosis. In this experiment, liver fibrosis was increased in all non-alcoholic fatty hepatitis induction groups, silymarin administration groups, and Faecalibacterium prausnitzii A2-165 standard strain administration groups compared with the normal group. However, it was determined that collagen production was significantly inhibited in the Faecalibacterium prausnitzii EB-FPDK11 strain administration group compared with the non-alcoholic fatty hepatitis induction group and the Faecalibacterium prausnitzii A2-165 standard strain administration group, indicating that liver damage caused by liver fibrosis was significantly inhibited in the Faecalibacterium prausnitzii EB-FPDK11 strain administration group.

[0160] In addition, as a result of observing the degree of liver damage by staining and observing the expression of α-SMA in mouse liver tissue, such as Fig.19 As shown in , it was observed that α-SMA expression was reduced in all administration groups compared with the non-alcoholic steatohepatitis induced group, indicating that liver damage in these groups was suppressed. In addition, it was determined that α-SMA expression was more significantly reduced in the silymarin administration group and the Faecalibacterium prausnitzii EB-FPDK11 strain administration group compared with the Faecalibacterium prausnitzii A2-165 standard strain administration group.

[0161] 5.7. Analysis of triglyceride and total cholesterol levels in liver tissue

[0162] Triglyceride and total cholesterol as lipid extract in mouse liver tissue were analyzed. 120 μl PBS was added to 30 mg liver tissue, and then it was chopped using a homogenizer, and then 320 μl chloroform and 160 μl MeOH were added thereto to obtain a mixture. The mixture was incubated at room temperature for one day in a shaking incubator, and then centrifuged at 2,000 rpm, and only the supernatant was separated and the solvent was evaporated therefrom. After this, the supernatant from which the solvent had been evaporated was dissolved in 1 ml of isopropanol, and then quantified relative to the total liver weight of each mouse using a TG / TC measurement kit (Asan Pharmaceutical Col., Ltd., Korea).

[0163] As a result, Fig. 20 As shown in the figure, it was determined that the triglyceride level in the liver tissue was significantly increased in the non-alcoholic fatty hepatitis induced group. However, in the silymarin administration group, the Faecalibacterium prausnitzii A2-165 standard strain administration group, and the Faecalibacterium prausnitzii EB-FPDK11 strain administration group, the triglyceride level was significantly reduced. In addition, the total cholesterol level in the liver tissue was higher in the non-alcoholic fatty hepatitis induced group than in the normal group. However, compared with the non-alcoholic fatty hepatitis induced group, the total cholesterol level in the liver tissue was significantly reduced in the silymarin administration group, the Faecalibacterium prausnitzii A2-165 standard strain administration group, and the Faecalibacterium prausnitzii EB-FPDK11 strain administration group.

[0164] As a result of analyzing lipid accumulation in liver tissue, it was determined that administration of Faecalibacterium prausnitzii EB-FPDK11 strain together with silymarin most significantly inhibited the production of triglycerides and cholesterol and had an effect of alleviating nonalcoholic steatohepatitis.

[0165] As a result of analyzing liver tissues, it was determined that, among the administration groups, in the Faecalibacterium prausnitzii EB-FPDK11 strain administration group, the progression of steatohepatitis and liver damage induced by nonalcoholic steatohepatitis were most significantly suppressed.

[0166]

[0167] <110> Enterobiome Co., Ltd. <120> New Faecalibacterium prausnitzii EB-FPDK11 and its use <130> PN200151 <160> twenty four <170> KoPatentIn 3.0 <210> 1 <211> 1433 <212> DNA <213> Artificial sequence <220> <223> Faecalibacterium prausnitzii EB - FPDK11 16S rRNA <400> 1 gacgaacgct ggcggcgcgc ctaacacatg caagtcgaac gagcgagaga gagcttgctt 60 tctcgagcga gtggcgaacg ggtgagtaac gcgtgaggaa cctgcctcaa agagggggac 120 aacagttgga aacgactgct aataccgcat aagcccacag gtcggcatcg accagaggga 180 aaaggagcaa tccgctttga gatggcctcg cgtccgatta gctagttggt gaggtaacgg 240 cccaccaagg caacgatcgg tagccggact gagaggttga acggccacat tgggactgag 300 acacggccca gactcctacg ggaggcagca gtggggaata ttgcacaatg ggggaaaccc 360 tgatgcagcg acgccgcgtg gaggaagaag gtcttcggat tgtaaactcc tgttgttgag 420 gaagataatg acggtactca acaaggaagt gacggctaac tacgtgccag cagccgcggt 480 aaaacgtagg tcacaagcgt tgtccggaat tactgggtgt aaagggagcg caggcgggaa 540 gacaagttgg aagtgaaatc tatgggctca acccataaac tgctttcaaa actgtttttc 600 ttgagtagtg cagaggtagg cggaattccc ggtgtagcgg tggaatgcgt agatatcggg 660 aggaacacca gtggcgaagg cggcctactg ggcaccaact gacgctgagg ctcgaaagtg 720 tgggtagcaa acaggattag ataccctggt agtccacacc gtaaacgatg attactaggt 780 gttggaggat tgaccccttc agtgccgcag ttaacacaat aagtaatcca cctggggagt 840 acgaccgcaa ggttgaaact caaaggaatt gacggggcc cgcacaagca gtggagtatg 900 tggtttaatt cgacgcaacg cgaagaacct taccaagtct tgacatccct tgacgaacat 960 agaaatgtgt tttctcttcg gagcaaggag acaggtggtg catggttgtc gtcagctcgt 1020 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttactgtca gttactacgc 1080 aagaggactc tggcaggact gccgttgaca aaacggagga aggtggggat gacgtcaaat 1140 catcatgccc tttatgactt gggctacaca cgtactacaa tggcgttaaa caaagagaag 1200 caagaccgcg aggtggagca aaactcagaa acaacgtccc agttcggact gcaggctgca 1260 actcgcctgc acgaagtcgg aattgctagt aatcgtggat cagcatgcca cggtgaatac 1320 gttcccgggc cttgtacaca ccgcccgtca caccatgaga gccgggggga cccgaagtcg 1380 gtagtctaac cgcaaggagg acgccgccga aggtaaaact ggtgattggg gtg 1433 <210> 2 <211> 17 <212> DNA <213> Artificial sequence <220> <223> FP1 forward <400> 2 ACTCAACAAGAAGTGGA 17 <210> 3 <211> 17 <212> DNA <213> Artificial sequence <220> <223> FP2 Reverse <400> 3 cagaggtagg cggaatt 17 <210> 4 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> ERIC-1 forward <400> 4 atgtaagctc ctggggattc ac 22 <210> 5 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> ERIC-2 reverse <400> 5 aagtaagtga ctggggtgag cg 22 <210> 6 <211> 15 <212> DNA <213> Artificial sequence <220> <223> (GTG)5 forward / reverse <400> 6 gtggtggtgg tggtg 15 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> 27F forward <400> 7 agagtttgat cmtggctcag 20 <210> 8 <211> 19 <212> DNA <213> Artificial sequence <220> <223> 1492R Reverse <400> 8 ggttaccttg ttacgactt 19 <210> 9 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> GAPDH positive <400> 9 gacatcaaga aggtggtgaa gcag 24 <210> 10 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> GAPDH reverse <400> 10 ataccaggaa atgagcttga caaa 24 <210> 11 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> IL-8 positive <400> 11 ttttgccaag gagtgctaaa ga 22 <210> 12 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> IL-8 reverse <400> 12 aaccctctgc acccagtttt c 21 <210> 13 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> GAPDH positive <400> 13 gacatcaaga aggtggtgaa gcag 24 <210> 14 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> GAPDH reverse <400> 14 ataccaggaa atgagcttga caaa 24 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <220> <223> C / EBPa positive <400> 15 agcaacgagt accgggtacg 20 <210> 16 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> C / EBPa reverse <400> 16 tgtttggctt tatctcggct c 21 <210> 17 <211> 26 <212> DNA <213> Artificial sequence <220> <223> aP2 forward <400> 17 agtgaaaact tcgatgatta catgaa 26 <210> 18 <211> 19 <212> DNA <213> Artificial sequence <220> <223> aP2 reverse <400> 18 gcctgccact ttccttgtg 19 <210> 19 <211> 20 <212> DNA <213> Artificial sequence <220> <223> FAS forward <400> 19 aggggtcgac ctggtcctca 20 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> FAS Reverse <400> 20 gccatgccca gagggtggtt 20 <210> twenty one <211> 20 <212> DNA <213> Artificial sequence <220> <223> ACC1 forward <400> twenty one cctccgtcag ctcagataca 20 <210> twenty two <211> twenty two <212> DNA <213> Artificial sequence <220> <223> ACC1 reverse <400> twenty two tttactaggt gcaagccaga ca 22 <210> twenty three <211> 20 <212> DNA <213> Artificial sequence <220> <223> LPL forward <400> twenty three ttgccctaag gacccctgaa 20 <210> twenty four <211> twenty three <212> DNA <213> Artificial sequence <220> <223> LPL Reverse <400> twenty four acagagtctg ctaatccagg aat 23

Claims

1. Faecalibacterium prausnitzii EB-FPDK11 strain, whose registration number is KCCM12621P.

2. Use of a pharmaceutical composition in the preparation of a medicament for treating an inflammatory disease, the pharmaceutical composition comprising at least one selected from the following: the strain according to claim 1 and a culture of the strain.

3. Use of a pharmaceutical composition in the preparation of a medicament for treating liver disease, the pharmaceutical composition comprising at least one selected from the following: the strain according to claim 1 and a culture of the strain.

4. Use of a pharmaceutical composition in the preparation of a medicament for treating a metabolic disease, the pharmaceutical composition comprising at least one selected from the following: the strain according to claim 1 and a culture of the strain, wherein the metabolic disease is hyperlipidemia, diabetes, obesity, hypertension or hypercholesterolemia.

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