New Faecalibacterium prausnitzii strain EB-FPDK9 and its uses

Abstract

The present invention relates to a new strain of Faecalibacterium prausnitzii EB-FPDK9 and its use, which has the effects of preventing, alleviating and treating inflammatory diseases, liver diseases or metabolic diseases when a composition containing one or more selected from the following is ingested: Faecalibacterium prausnitzii EB-FPDK9, a culture solution, lysate or extract of the strain.

Description

Technical Field

[0001] The present disclosure relates to a novel Faecalibacterium prausnitzii strain EB-FPDK9 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 together 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 pharmaceutical probiotics (pharmabiotics) have also attracted attention. "Pharmaceutical probiotics" is a compound word of a drug in the sense of "pharmaceutical" and a live bacterium 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 found in the intestinal mucus layer and have a high retention rate and number in humans. In addition, these bacteria are the main components of the intestinal flora.

[0004] Against this background, the present inventors have endeavored 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-FPDK9 strain (accession number: KCCM12620P).

[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 group consisting of: Faecalibacterium prausnitzii EB-FPDK9 strain, a culture of Faecalibacterium prausnitzii EB-FPDK9 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 comprising at least one selected from the group consisting of: a Faecalibacterium prausnitzii EB-FPDK9 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK9 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-FPDK9 strain (Accession No.: KCCM12620P).

[0011] In one embodiment of the present disclosure, the Faecalibacterium prausnitzii EB-FPDK9 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-FPDK9 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK9 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: a Faecalibacterium prausnitzii EB-FPDK9 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK9 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-FPDK9 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK9 strain, a lysate of the strain, and an extract of the strain.

[0015] Yet another aspect of the present disclosure provides a food composition comprising at least one selected from the group consisting of a Faecalibacterium prausnitzii EB-FPDK9 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK9 strain, a lysate of the strain, and an extract of the strain.

[0016] Another aspect of the present disclosure provides a food composition comprising at least one selected from the group consisting of a Faecalibacterium prausnitzii EB-FPDK9 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK9 strain, a lysate of the strain, and an extract of the strain.

[0017] Yet another aspect of the present disclosure provides a food composition comprising at least one selected from the group consisting of a Faecalibacterium prausnitzii EB-FPDK9 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK9 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-FPDK9 strain, a culture of Faecalibacterium prausnitzii EB-FPDK9 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 observation of a standard strain of Faecalibacterium prausnitzii and EB-FPDK9 is shown.

[0023] Figure 2 Shown are the results of electrophoresis after PCR using FP-specific primers for the Faecalibacterium prausnitzii standard strain and EB-FPDK9.

[0024] Figure 3 Shown are the results of electrophoresis after PCR using ERIC-1, ERIC-2, and (GTG)5 primers for the Faecalibacterium prausnitzii standard strain and EB-FPDK9.

[0025] Figure 4 This is a phylogenetic tree prepared using the 16S rRNA nucleotide sequence of Faecalibacterium prausnitzii EB-FPDK9.

[0026] Figure 5 Shown are the results of examining whether the Faecalibacterium prausnitzii standard strain and EB-FPDK9 cause hemolysis.

[0027] Figure 6 Graphs showing the results of analyzing short-chain fatty acids in Faecalibacterium prausnitzii standard strains and EB-FPDK9.

[0028] Figure 7 Graphs showing the results of analyzing mRNA expression of the inflammatory cytokine IL-8 in each of the Faecalibacterium prausnitzii standard strain and EB-FPDK9.

[0029] Figure 8Graphs showing the results of analyzing the concentration of the inflammatory cytokine IL-10 in each of the Faecalibacterium prausnitzii standard strain and EB-FPDK9.

[0030] Figure 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-FPDK9.

[0031] Figure 10 Shown are photographs and graphs showing the results of examining the degree of inhibition of lipid accumulation by cultures of each of the Faecalibacterium prausnitzii standard strain and EB-FPDK9.

[0032] Figure 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-FPDK9.

[0033] Figure 12 A graph shows a comparison of body weight and dietary intake between Faecalibacterium prausnitzii standard strain-administered mice and EB-FPDK9 strain-administered mice in non-alcoholic steatohepatitis-induced mice.

[0034] Figure 13 A graph showing a comparison of glucose tolerance between mice administered with the Faecalibacterium prausnitzii standard strain and mice administered with the EB-FPDK9 strain in non-alcoholic steatohepatitis-induced mice.

[0035] Figure 14 This graph compares liver weight and shape between mice administered with the standard strain of Faecalibacterium prausnitzii and mice administered with the EB-FPDK9 strain in mice induced with non-alcoholic steatohepatitis.

[0036] Figure 15 This graph compares the spleen weight and shape between mice administered with the standard strain of Faecalibacterium prausnitzii and mice administered with the EB-FPDK9 strain in non-alcoholic steatohepatitis-induced mice.

[0037] Figure 16 The results of analyzing and comparing blood lipid biochemical indicators in mice induced with nonalcoholic steatohepatitis are shown in Figure 1, which shows the results of analyzing and comparing blood lipid biochemical indicators in mice administered with the standard strain of Faecalibacterium prausnitzii and mice administered with the EB-FPDK9 strain.

[0038] Figure 17 Shown are the results of determining fat droplet formation in non-alcoholic steatohepatitis-induced mice by H&E staining of the livers of mice administered with the Faecalibacterium prausnitzii standard strain and mice administered with the EB-FPDK9 strain.

[0039] Figure 18Shown are the results of a comparison of collagen deposition between mice administered with the Faecalibacterium prausnitzii standard strain and mice administered with the EB-FPDK9 strain in non-alcoholic steatohepatitis-induced mice.

[0040] Figure 19 The results are shown, comparing the degree of liver damage between mice administered with the standard strain of Faecalibacterium prausnitzii and mice administered with the EB-FPDK9 strain by observing α-SMA expression in the liver in non-alcoholic steatohepatitis-induced mice.

[0041] Figure 20 Shown are the results of comparing liver triglyceride and total cholesterol levels between Faecalibacterium prausnitzii standard strain-administered mice and EB-FPDK9 strain-administered mice in non-alcoholic steatohepatitis-induced mice. DETAILED DESCRIPTION

[0042] In order to achieve the above objectives, one aspect of the present disclosure provides Faecalibacterium prausnitzii EB-FPDK9 strain (accession number: KCCM12620P).

[0043] In one embodiment of the present disclosure, the Faecalibacterium prausnitzii EB-FPDK9 strain has a 16S rRNA sequence of SEQ ID NO: 1.

[0044] Faecalibacterium prausnitzii is one of the most abundant bacteria in 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-FPDK9 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK9 strain, a lysate of the strain, and an extract of the strain.

[0046] The term "culture" as 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 the culture supernatant. In addition to the conventional components necessary for culturing Faecalibacterium prausnitzii, the culture composition may also include components that synergistically act on the growth of Faecalibacterium prausnitzii, and a person skilled in the art can easily select a composition comprising these components.

[0047] In addition, the strain may be in a liquid state or a dry state, and some examples of drying methods for the strain include, but are 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 with inflammation as a main pathology. For example, the inflammatory disease may be any one selected from the following: inflammatory skin disease, inflammatory bowel disease 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 disease, 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: a Faecalibacterium prausnitzii EB-FPDK9 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK9 strain, a lysate of the strain, and an extract of the strain.

[0050] The liver disease may be 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, the prevention or treatment of liver disease may refer to suppressing an abnormal increase in liver weight, and may refer to suppressing an abnormal increase in spleen length and weight. In addition, it may refer to controlling the concentration of triglycerides, cholesterol, GOT or GPT or suppressing an abnormal increase in the concentration, as well as suppressing 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-FPDK9 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK9 strain, a lysate of the strain, and an extract of the strain.

[0053] Metabolic disease 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 disease can be obesity, but is 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" as used herein refers to an amount that is sufficient to treat a disease at a reasonable benefit / risk ratio that is 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, as well as other factors known in the medical field. As will be appreciated by those skilled in the art, effective dose can vary based on the potential of treatment approach, excipient use, and use with other drugs.

[0055] The pharmaceutical compositions of the present disclosure can be prepared into pharmaceutical dosage forms using methods well known in the art so as to provide 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 composition.

[0057] Some examples of the carrier, excipient and diluent that can 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 diluent or excipient can be used, for example filler, extender, adhesive, wetting agent, disintegrant and surfactant are prepared preparation.

[0058] Yet another aspect of the present disclosure provides a food composition comprising at least one selected from the group consisting of a Faecalibacterium prausnitzii EB-FPDK9 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK9 strain, a lysate of the strain, and an extract of the strain.

[0059] Another aspect of the present disclosure provides a food composition comprising at least one selected from the group consisting of a Faecalibacterium prausnitzii EB-FPDK9 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK9 strain, a lysate of the strain, and an extract of the strain.

[0060] Yet another aspect of the present disclosure provides a food composition comprising at least one selected from the group consisting of a Faecalibacterium prausnitzii EB-FPDK9 strain, a culture of the Faecalibacterium prausnitzii EB-FPDK9 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 composition of the present disclosure can be added to, 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 contains the Faecalibacterium prausnitzii EB-FPDK9 strain, the culture of the Faecalibacterium prausnitzii EB-FPDK9 strain, the lysate of the strain, or the extract of the strain, or its active ingredient or a physiologically acceptable salt thereof as an essential ingredient, there is no particular limitation on other ingredients. Similar to ordinary foods, the food composition may contain various herbal extracts, food supplement additives or natural carbohydrates as additional ingredients.

[0063] In addition, the food composition may further include the food supplement additives as mentioned above, and the food supplement additives may be conventional food supplement additives known in the art, and examples thereof include flavoring agents, 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 flavorings (e.g., rebaudioside A, glycyrrhizin etc.) or synthetic flavorings (saccharin, aspartame etc.) also can be suitably used as flavorings.

[0065] Except above-mentioned composition, the food composition of present disclosure can also comprise the carbonizing agent etc. that uses in multiple nutrients, vitamin, mineral substance (electrolyte), flavoring agent such as synthetic flavoring agent and natural flavoring agent, coloring agent and filler (such as cheese or chocolate), pectic acid and salt thereof, alginic acid and salt thereof, organic acid, protective colloid thickener, pH adjusting agent, stabilizing agent, preservative, glycerine, alcohol, carbonated beverage.In addition, food composition can comprise natural fruit juice and the pulp that is used to produce fruit juice beverage and vegetable beverage.These compositions can be used individually or in combination.

[0066] According to one embodiment of the present disclosure, a food composition can be prepared in the form of a health functional food. The term "health functional food" as used herein has the same meaning as "food for special health use (FoSHU)", and refers to a food with high pharmaceutical and medical effects, which is processed to effectively exert a bioregulatory function in addition to nutritional 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 medicines that contain 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.

[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-FPDK9 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 flavorings. 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, condition 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 for illustrating 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-FPDK9 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 an extremely oxygen sensitive (EOS) strain was 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. As a result, Figure 1 As shown in FIG, Faecalibacterium prausnitzii DSM17677 was identified as a standard strain observed at 1,000x magnification. T strains ( Figure 1 A) and Faecalibacterium prausnitzii EB-FPDK9 strain ( Figure 1 B) Both have straight or curved rod-like cell shapes and therefore appear similar in shape.

[0076] PCR analysis

[0077] In order to determine whether the isolated strain was a Faecalibacterium prausnitzii strain, PCR analysis was performed on the isolated strain using the FP-specific primers (SEQ ID NO: 2 and SEQ ID NO: 3) shown in Table 1 below. Figure 2 As shown in , 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] 1.4. Random Amplified Polymorphic DNA (RAPD) Analysis

[0081] In order to check 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 the 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.

[0082] [Table 2]

[0083] SEQ ID NO name direction Sequence (5'→3') SEQ ID NO:4 ERIC-1 Forward 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

[0084] like Figure 3 As shown in , as a result of comparing DNA fragment patterns, it was determined that the Faecalibacterium prausnitzii EB-FPDK9 strain showed a pattern that was partially similar to but different from the standard strain Faecalibacterium prausnitzii DSM 17677 T Therefore, it was determined that the isolated strain was different from the reported standard strain Faecalibacterium prausnitzii DSM 17677 T Belongs to the same species, but is a different strain.

[0085] 1.5.16S rRNA BLAST

[0086] In order to determine whether the isolated strain is a Faecalibacterium prausnitzii strain, the isolated strain was subjected to 16SrRNA sequencing and subsequently analyzed by BLAST. As a result, the isolated strain had 99% or higher identity with the Faecalibacterium prausnitzii species. Based on these results, the isolated strain was named Faecalibacterium prausnitzii EB-FPDK9 strain and deposited in the Korean Culture Center of Microorganisms (KCCM) with accession number KCCM12620P.

[0087] Phylogenetic tree analysis using 16S rRNA nucleotide sequences

[0088] As a result of strain identification, there were strains similar to currently known strains, but no completely consistent results were obtained. Therefore, a phylogenetic tree analysis was performed. For full-length 16S rRNA gene sequencing of the isolated Faecalibacterium prausnitzii EB-FPDK9 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-FPDK9 strain and the standard strain, as well as the previously disclosed 16S rRNA gene sequences of other strains of the same species, a maximum likelihood method was used to prepare a phylogenetic tree. Figure 4 The phylogenetic tree shown in .

[0089] [Table 3]

[0090]

[0091] Example 2: Characterization of Faecalibacterium prausnitzii EB-FPDK9 strain

[0092] 2.1. Antimicrobial susceptibility testing

[0093] To examine the antimicrobial susceptibility of Faecalibacterium prausnitzii EB-FPDK9 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 Faecalibacterium prausnitzii EB-FPDK9 strain was examined according to the liquid culture microdilution method of the Clinical & Laboratory Standard Institute (CLSI) guidelines.

[0094] [Table 4]

[0095]

[0096]

[0097] As a result, it can be seen from Table 4 above that the Faecalibacterium prausnitzii EB-FPDK9 strain of the present disclosure showed resistance to ceftizoxime (CTZ), chloramphenicol (CHL), meropenem (MEM) and fluoroquinolone-based antibiotics moxifloxacin (MXF) and ciprofloxacin (CIP), and showed sensitivity to piperacillin-tazobactam (PTZ), clindamycin (CLI) and metronidazole (MTZ). For the antibiotic piperacillin-tazobactam (PTZ), the Faecalibacterium prausnitzii EB-FPDK9 strain showed similarity to the standard strain (DSM 17677 T ) significant differences.

[0098] 2.2. Evaluation of hemolytic activity

[0099] To verify the safety of the Faecalibacterium prausnitzii EB-FPDK9 strain, the strain was evaluated for hemolytic activity. To this end, the strain was cultured using blood agar medium prepared by adding 1.5% w / v bacto-agar and 5% w / v defibrinated sheep blood to 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 occurred 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-FPDK9 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-FPDK9 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. The analysis was performed using a gas chromatograph (Agilent 7890N) equipped with an FFAP column (30m×0.320mm, 0.25 μm phase) under the conditions shown in Table 5 below.

[0103] [Table 5]

[0104]

[0105] As a result of analyzing functional short-chain fatty acids, such as Figure 6 As can be seen in the figure, it was determined that Faecalibacterium prausnitzii strain EB-FPDK9 consumed acetate and produced butyrate.

[0106] Example 3: Evaluation of the anti-inflammatory effect of Faecalibacterium prausnitzii EB-FPDK9 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-FPDK9 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. To determine whether the Faecalibacterium prausnitzii EB-FPDK9 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 using the M-MLV cDNA Synthesis Kit (Enzynomics, Korea). Real-time PCR was performed using the Quant Studio 3 Real-Time PCR System (Applied Biosystems, USA).

[0112] Inflammatory cytokine gene expression was analyzed using SYBR Green TOPreal™ qPCR 2X PreMIX (Enzynomics, Korea), using GAPDH as an internal standard. PCR was performed under the following conditions: preincubation at 50°C for 4 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Data were analyzed using the ΔCT method using the built-in program in QuantStudio Design & Analysis Software v1.4.3.

[0113] The statistical program GraphPad Prism 7 (GraphPad Software Inc., USA) was used to calculate the mean and standard deviation of each experimental group. Differences between groups were analyzed using one-way ANOVA and Tukey's test. A p value ≤ 0.05 was considered significant. For some results, the AUC (area under the 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 significantly increased compared to the IL-8 expression in the normal group. However, it was shown that the IL-8 expression in the group treated with LPS and a culture of the standard strain of Faecalibacterium prausnitzii A2-165 (10%, v / v) was reduced compared to the IL-8 expression in the LPS-treated group, and the IL-8 expression in the group treated with LPS and a culture of the Faecalibacterium prausnitzii EB-FPDK9 strain was further significantly reduced compared to 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-FPDK9 strain significantly reduced the inflammatory cytokine IL-8.

[0115] 3.2. Evaluation of the Anti-inflammatory Effect of Mouse Bone Marrow-Derived Dendritic Cells

[0116] In order to observe anti-inflammatory response, the cytokine secretion from dendritic cells (Dendritic Cells, DC) was analyzed. In order to use mouse bone marrow-derived dendritic cells (bone marrow-derived dendritic cell, BMDC) to evaluate the anti-inflammatory effect of bacterial strain, BMDC was separated. After using 18G needle to pierce 0.5ml microtubule, 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 with 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, 20 μg GM-CSF) culture medium and cultivated in 150mm culture dish. On the second day, BMDC was transferred to 100ml culture dish and cultivated therein, and on the 5th day, 10ml culture was transferred to 15ml conical tubes, and then centrifuged for 15 minutes with 1,000xg. The supernatant was removed, and 10 ml of BMDC culture medium was added to the BMDCs and placed in a culture dish. On day 6 or 7, the BMDCs were used for the experiment. To evaluate the anti-inflammatory effect of the F. prausnitzii EB-FPDK9 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-FPDK9 strain (10 7 Each of the 100 cfu / ml, 10% v / v) cells was treated in 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 culture 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 , IL-10 secretion from cells treated with each of LPS and E. coli was similar to 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. IL-10 expression in the group treated with the EB-FPDK9 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 the Faecalibacterium prausnitzii strain increased the anti-inflammatory cytokine IL-10 and that the Faecalibacterium prausnitzii EB-FPDK9 strain further increased 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] Oil Red-O Staining of Differentiated Adipocytes

[0122] In order to examine the effect of the Faecalibacterium prausnitzii EB-FPDK9 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 The cells were distributed in a 24-well plate at a density of 100 μg / well. 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% FBS, 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 (Faerobacterium prausnitzii strains or their cultures) of 10 cells / well and subsequently 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 Faerobacterium prausnitzii strains and their cultures at the same time. The strains and their cultures (10 mg / ml insulin) were cultured at a concentration of 10% v / v. 7 cfu / ml) treated cells.

[0123] Oil Red-O staining is a method for measuring fat production in differentiated 3T3-L1 cells by staining them with Oil Red-O reagent. 3T3-L1 cells, which are mouse preadipocytes, were cultured (Korea Cell Line Bank, Korea). Dulbecco's Modified Eagle's Medium (DMEM, 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 adipocyte differentiation from preadipocytes 3T3-L1 was induced for 10 days by insulin (1 μg / ml), IBMX (0.5 mM) and dexamethasone (1 μM), 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 fat droplets.

[0124] After cell staining, the cells were washed three times with 40% isopropanol (Duksan, Korea) and dried, and the size of the fat droplets in the cells was observed using an optical microscope. The fat droplet 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 Figure 9 and 10 middle.

[0125] like Figure 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-FPDK9 strain more significantly inhibited lipid accumulation than the group treated with the Faecalibacterium prausnitzii A2-165 standard strain.

[0126] Similarly, if Figure 10 As shown in , as a result of treating 3T3-L1 cells with a culture of the Faecalibacterium prausnitzii A2-165 standard strain during cell differentiation, lipid accumulation in the treated cells was significantly suppressed compared to lipid accumulation in the control group. Treatment with a culture of the Faecalibacterium prausnitzii EB-FPDK9 strain more significantly suppressed lipid accumulation than the group treated with a culture of the Faecalibacterium prausnitzii A2-165 standard strain.

[0127] It was determined that compared with the standard strain A2-165 of Faecalibacterium prausnitzii and its culture, the Faecalibacterium prausnitzii EB-FPDK9 strain and its culture had a better effect on inhibiting the adipogenic differentiation of 3T3-L1 cells.

[0128] 4.2. Evaluation of the Effects on Biomarker Gene Expression

[0129] To evaluate the effect of the strain on inhibiting adipocyte differentiation, real-time PCR was performed using the gene-specific primers shown in Table 7 below (SEQ ID NOs: 13 to 26) to analyze the mRNA expression levels of transcription factors C / EBPα (CCAAT / enhancer binding protein α) and SREBP1c (sterol regulatory element binding protein 1c), which are involved in adipocyte differentiation and maturation, as well as lipogenic genes aP2 (adipocyte protein 2), FAS (fatty acid synthase), ACC1 (acetyl-CoA-carboxylase), and LPL (lipoprotein lipase).

[0130] [Table 7]

[0131]

[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 consisting of 95°C for 15 seconds and 60°C for 1 minute. Data were analyzed using the program built into QuantStudio Design & Analysis Software v1.4.3 by the ΔCT method.

[0133] like Figure 11As shown in , when the increased expression levels of C / EBPa, SREBP1c, aP2, FAS, ACC1, and LPL, genes involved in adipocyte differentiation after adipogenic differentiation induction, were expressed as 100%, the expression levels of C / EBPα, aP2, FAS, ACC1, and LPL were reduced in the group treated with the culture of the Faecalibacterium prausnitzii A2-165 standard strain, and the expression levels of C / EBPα, SREBP1c, aP2, FAS, ACC1, and LPL were significantly reduced in the group treated with the culture of the Faecalibacterium prausnitzii EB-FPDK9 strain. Compared with the control group, the expression level of SREBP1c was reduced only in the group treated with the culture of the Faecalibacterium prausnitzii EB-FPDK9 strain, and the Faecalibacterium prausnitzii EB-FPDK9 strain further reduced the expression of all the above genes compared with the Faecalibacterium prausnitzii A2-165 standard strain. It was determined that both the Faecalibacterium prausnitzii A2-165 standard strain and the Faecalibacterium prausnitzii EB-FPDK9 strain have the effect of inhibiting the expression of adipogenic differentiation-related genes in 3T3-L1 cells.

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

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

[0136] Animal experiments were conducted in accordance with the animal use and care protocols of the Institutional Animal Care and Use Committee (IACUC). Eight-week-old male C57BL / 6 mice (9 mice / group) were purchased and acclimated for 1 week as experimental animals. The mice were then housed for 12 weeks. The culture environment was maintained at a constant temperature (22°C) and relative humidity (40% to 60%) with a 12-hour light / 12-hour dark cycle for 1 week.

[0137] To induce nonalcoholic steatohepatitis, mice consumed a high-fat diet (60 kcal% fat; Research Diets Inc., NJ, USA) and 30% fructose in drinking water as an experimental diet (NASH) 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 viable cells of Faecalibacterium prausnitzii.

[0142] Mice in the normal diet group (Normal) were fed a 10% fat diet. As positive controls, silymarin, a functional ingredient known to help alleviate non-alcoholic fatty liver disease, or the standard strain of Faecalibacterium prausnitzii A2-165 were administered. Both the normal diet group and the experimental diet group were orally administered the same amount of phosphate-buffered saline (25% glycerol and 0.05% cysteine / PBS) daily to eliminate the effects of stress caused by administration.

[0143] 5.2. Changes in Body Weight and Food Intake

[0144] Sixteen weeks after the nonalcoholic steatohepatitis induction experiment, the body weight changes of the experimental group were measured, and the results are shown in Figure 12 middle.

[0145] refer to Figure 12 , the body weight of all groups of animals with non-alcoholic steatohepatitis induced by the experimental diet increased compared to that of the normal diet group. When the weight gain from week 8 (when silymarin or Faecalibacterium prausnitzii strain was administered) to week 16 was calculated as mass (g) and percentage (%), it was observed that the weight gain was slightly reduced in the silymarin-administered group and the Faecalibacterium prausnitzii EB-FPDK9 strain-administered group compared to the non-alcoholic steatohepatitis induced group, but no significant decrease in weight gain was found. Compared with the normal diet group, the percentage weight gain observed in the Faecalibacterium prausnitzii EB-FPDK9 strain-administered group was the smallest. 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-FPDK9 strain on glucose tolerance, 16 weeks after the start of the experiment, glucose (2 g / kg) was orally administered to mice 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 Figure 13 middle.

[0148] refer to Figure 13 , among the administration groups, the group that was administered with Faecalibacterium prausnitzii EB-FPDK9 strain immediately before glucose administration showed the greatest reduction in blood glucose levels. 30 minutes after glucose administration, blood glucose levels were 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, the Faecalibacterium prausnitzii A2-165 standard strain administration group, and the EB-FPDK9 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 Faecalibacterium prausnitzii EB-FPDK9 strain can improve the blood glucose control ability that is reduced by non-alcoholic fatty liver inflammation-induced 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 Figure 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-FPDK9 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 with the normal diet group. However, the liver tissue weight of the Faecalibacterium prausnitzii EB-FPDK9 strain-administered group was most similar to that of the normal diet group, and did have a significant difference from the liver tissue weight of the non-alcoholic steatohepatitis-induced group. By Figure 14 The results in the EB-FPDK9 group showed that the liver shape and weight of the group administered with the Faecalibacterium prausnitzii strain were similar to those of the normal diet group. Therefore, it can be concluded that the EB-FPDK9 strain of Faecalibacterium prausnitzii can alleviate non-alcoholic steatohepatitis.

[0152] like Figure 15As shown in , both spleen length and weight increased in the non-alcoholic steatohepatitis-induced group compared to the normal diet group. As with liver tissue, spleen length increased in the groups treated with silymarin and the Faecalibacterium prausnitzii A2-165 standard strain compared to the normal diet group. However, the increase in spleen length in the Faecalibacterium prausnitzii EB-FPDK9 strain-administered group was so low as to be insignificant. It was confirmed that 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 Figure 16 The concentrations of TG, TC, GOT, and GPT, which are indicators of lipid composition, were quantified using separate measurement kits purchased from Asan Pharmaceutical Co., Ltd.

[0155] It was determined that the triglyceride concentration was significantly increased in the non-alcoholic steatohepatitis induction group. However, compared with the non-alcoholic steatohepatitis induction group, the triglyceride concentration was significantly reduced in the silymarin-administered group and the Faecalibacterium prausnitzii EB-FPDK9 strain-administered group. Compared with the normal group, the total cholesterol level was higher in the non-alcoholic steatohepatitis induction group and the group treated with the Faecalibacterium prausnitzii A2-165 standard strain. However, compared with the non-alcoholic steatohepatitis induction group and the group treated with the Faecalibacterium prausnitzii A2-165 standard strain, the total cholesterol level was significantly reduced in the group treated with the Faecalibacterium prausnitzii EB-FPDK9 strain. 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 induction group, and the GPT concentration was significantly reduced only in the silymarin-administered group and the Faecalibacterium prausnitzii EB-FPDK9 strain-administered group. Through the analysis of blood lipid biochemical indicators, it was determined that the administration of Faecalibacterium prausnitzii EB-FPDK9 strain reduced the concentrations of triglycerides, total cholesterol, GOT and GPT, which are closely related to non-alcoholic steatohepatitis.

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

[0157] In order to observe the effect of administering Faecalibacterium prausnitzii EB-FPDK9 strain on alleviating non-alcoholic fatty liver disease, 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 each staining. The degree of liver damage observed by each staining was expressed as a percentage positive area (%) by the Image J program.

[0158] like Figure 17 As shown in , H&E staining analysis of mouse liver tissue revealed that the liver tissue of the normal group lacked fat droplets, as the hepatocyte structure is typically dense. However, in the liver tissue of mice induced with nonalcoholic steatohepatitis, the formation of numerous fat droplets was clearly observed compared to that in the normal group. It was observed that fat droplet formation decreased in all administration groups compared to the nonalcoholic steatohepatitis-induced group, and further reductions were confirmed in the silymarin-administered group and the Faecalibacterium prausnitzii EB-FPDK9 strain-administered group.

[0159] like Figure 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 liver disease induction groups, silymarin-administered groups, and Faecalibacterium prausnitzii A2-165 standard strain-administered groups compared to the normal group. However, it was determined that collagen production was significantly inhibited in the Faecalibacterium prausnitzii EB-FPDK9 strain-administered group compared to the non-alcoholic fatty liver disease induction group and the Faecalibacterium prausnitzii A2-165 standard strain-administered group, indicating that liver damage caused by liver fibrosis was significantly inhibited in the Faecalibacterium prausnitzii EB-FPDK9 strain-administered 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, as shown in FIG. Figure 19 As shown in Figure 2, α-SMA expression was observed to be reduced in all administration groups compared to the non-alcoholic steatohepatitis-induced group, indicating that liver damage in these groups was suppressed. Furthermore, α-SMA expression was observed to be more significantly reduced in the silymarin-administered group and the Faecalibacterium prausnitzii EB-FPDK9 strain-administered group compared to the Faecalibacterium prausnitzii A2-165 standard strain-administered group.

[0161] 5.7. Analysis of Triglyceride and Total Cholesterol Levels in Liver Tissue

[0162] Triglycerides and total cholesterol as lipid extracts in mouse liver tissue were analyzed. 120 μl of PBS was added to 30 mg of liver tissue and then chopped using a homogenizer, and then 320 μl of chloroform and 160 μl of 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 evaporated therefrom. Afterwards, the supernatant from which the solvent had been evaporated was dissolved in 1 ml of isopropanol and subsequently quantified using a TG / TC measurement kit (Asan Pharmaceutical Co., Ltd., Korea) relative to the total liver weight of each mouse.

[0163] As a result, Figure 20 As shown in the figure, it was determined that the triglyceride level in the liver tissue of the non-alcoholic steatohepatitis-induced group was significantly increased. However, the triglyceride level was significantly reduced in the silymarin-administered group, the Faecalibacterium prausnitzii A2-165 standard strain-administered group, and the Faecalibacterium prausnitzii EB-FPDK9 strain-administered group. In addition, the total cholesterol level in the liver tissue was higher in the non-alcoholic steatohepatitis-induced group than in the normal group. However, compared with the non-alcoholic steatohepatitis-induced group, the total cholesterol level in the liver tissue of the silymarin-administered group, the Faecalibacterium prausnitzii A2-165 standard strain-administered group, and the Faecalibacterium prausnitzii EB-FPDK9 strain-administered group was significantly reduced.

[0164] As a result of analyzing lipid accumulation in liver tissue, it was determined that administering the Faecalibacterium prausnitzii EB-FPDK9 strain together with silymarin most significantly inhibited the production of triglycerides and cholesterol and had the effect of alleviating non-alcoholic steatohepatitis.

[0165] As a result of analyzing liver tissue, it was confirmed that, among the administration groups, the progression of steatohepatitis and liver damage induced by non-alcoholic steatohepatitis were most significantly suppressed in the Faecalibacterium prausnitzii EB-FPDK9 strain-administered group. <110> Enterobiome Co., Ltd <120> New Faecalibacterium prausnitzii EB-FPDK9 strain and its use <130> PN200150 <160> 26 <170> KoPatentIn 3.0 <210> 1 <211> 1433 <212> DNA <213> Artificial Sequence <220> <223> Faecalibacterium prausnitzii EB-RPDK9 16S rRNA <400> 1 gacgaacgct ggcggcgcgc ctaacacatg caagtcgaac gagcgagaga gagcttgctt 60 tctcgagcga gtggcgaacg ggtgagtaac gcgtgaggaa cctgcctcaa agagggggac 120 aacagttgga aacgactgct aataccgcat aagcccacga ctcggcatcg ggtagaggga 180 aaaggagcaa tccgctttga gatggcctcg cgtccgatta gctagttggt gaggtaacgg 240 cccaccaagg cgacgatcgg 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 catgggctca acccatgaac tgctttcaaa actgtttttc 600 ttgagtagtg cagaggtagg cggaattccc ggtgtagcgg tggaatgcgt agatatcggg 660 aggaacacca gtggcgaagg cggcctactg ggcaccaact gacgctgagg ctcgaaagtg 720 tgggtagcaa acaggattag ataccctggt agtccacact gtaaacgatg attactaggt 780 gttggaggat tgaccccttc agtgccgcag ttaacacaat aagtaatcca cctggggagt 840 acgaccgcaa ggttgaaact caaaggaatt gacggggcc cgcacaagca gtggagtatg 900 tggtttaatt cgacgcaacg cgaagaacct taccaagtct tgacatcctg cgacgcacat 960 agaaatatgt gtttccttcg ggacgcagag acaggtggtg catggttgtc gtcagctcgt 1020 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttatggtca gttactacgc 1080 aagaggactc tggccagact gccgttgaca aaacggagga aggtggggat gacgtcaaat 1140 catcatgccc tttatgactt gggctacaca cgtactacaa tggcgttaaa caaagagaag 1200 caagaccgcg aggtggagca aaactcagaa acaacgtccc agttcggact gcaggctgca 1260 actcgcctgc acgaagtcgg aattgctagt aatcgcagat cagcatgctg cggtgaatac 1320 gttcccgggc cttgtacaca ccgccccgtca caccatgaga gccggggga cccgaagtcg 1380 gtagtctaac cgcaaggagg acgccgccga aggtaaaact ggtgattggg gtg 1433 <210> 2 <211> 17 <212> DNA <213> Artificial sequence <220> <223> FP1 forward <400> 2 actcaacaag gaagtga 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> 17 <212> DNA <213> Artificial sequence <220> <223> SREBP1c positive <400> 17 gatgtgcgaa ctggaca 17 <210> 18 <211> 19 <212> DNA <213> Artificial sequence <220> <223> SREBP1c reverse <400> 18 catagggggc gtcaaacag 19 <210> 19 <211> 26 <212> DNA <213> Artificial sequence <220> <223> aP2 forward <400> 19 agtgaaaact tcgatgatta catgaa 26 <210> 20 <211> 19 <212> DNA <213> Artificial sequence <220> <223> aP2 reverse <400> 20 gcctgccact ttccttgtg 19 <210> twenty one <211> 20 <212> DNA <213> Artificial sequence <220> <223> FAS forward <400> twenty one aggggtcgac ctggtcctca 20 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <220> <223> FAS Reverse <400> twenty two gccatgccca gagggtggtt 20 <210> twenty three <211> 20 <212> DNA <213> Artificial sequence <220> <223> ACC1 forward <400> twenty three cctccgtcag ctcagataca 20 <210> twenty four <211> twenty two <212> DNA <213> Artificial sequence <220> <223> ACC1 reverse <400> twenty four tttactaggt gcaagccaga ca 22 <210> 25 <211> 20 <212> DNA <213> Artificial sequence <220> <223> LPL positive <400> 25 ttgccctaag gacccctgaa 20 <210> 26 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> LPL Reverse <400> 26 acagagtctg ctaatccagg aat 23

Claims

1. Faecalibacterium prausnitzii strain EB-FPDK9, with the accession number KCCM12620P.

2. A 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 the pharmaceutical composition in the preparation of a medicament for the treatment of non-alcoholic steatohepatitis, 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 the pharmaceutical composition in the preparation of a medicament for the treatment of obesity and diabetes, the pharmaceutical composition comprising at least one selected from the following: the strain according to claim 1 and a culture of the strain.

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