Clostridium butyricum strain derived from korean microbiome having Anti-obesity efficacy and use thereof

Abstract

The present invention relates to a Korean microbiome-derived Clostridium butyricum strain having anti-obesity efficacy and a use thereof. The strain has an excellent effect of reducing the body weight and adipose tissue weight of experimental animals compared to a control group (strain-untreated) under high-fat diet conditions, has an excellent effect of reducing the content of total cholesterol, triglycerides, low-density lipoprotein (LDL), leptin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in serum, and has an excellent effect of reducing blood glucose and increasing the content of glucagon-like peptide-1 (GLP-1) in an oral glucose load test, and is thus expected to be effectively used as an ingredient in a composition for preventing, alleviating, or treating obesity and metabolic diseases caused by obesity.

Description

Clostridium butyricum strain derived from the Korean microbiome with anti-obesity efficacy and its use

[0001] The present invention relates to a Clostridium butyricum strain derived from a Korean microbiome having anti-obesity efficacy and its use.

[0002]

[0003] This research was supported by the Ministry of Education's National Research Foundation of Korea's Science and Technology Research Infrastructure Development Project (Project No. 2021R1A6A1A03045495) and the Ministry of Science and ICT's Korea Research Institute of Bioscience and Biotechnology Research Operation Support Project (Project No. KGM5232322).

[0004] Obesity is a condition characterized by excessive accumulation of fat tissue in the body. It is primarily caused by an energy imbalance, resulting from excessive nutrient intake relative to energy expenditure. It is also known to be induced by hormonal changes, genetics, mental health issues, and socioeconomic factors. The number of obese people is increasing worldwide, and obesity has recently been identified as a significant risk factor for adult diseases, making the severity of obesity a significant issue.

[0005] Current treatments for obesity include fenfluramine, which inhibits the serotonergic nervous system; ephedrine and caffeine, which act through the noradrenergic nervous system; sibutramine, which acts simultaneously on both serotonergic and noradrenergic nervous systems; and orlistat, which inhibits pancreatic lipase to reduce fat absorption. However, existing medications, such as fenfluramine, have been banned due to side effects such as primary pulmonary hypertension and heart valve disease. Other medications also cause hypotension and lactic acidosis, making them unsuitable for patients with heart failure or renal disease. Therefore, the development of substances for the prevention, improvement, or treatment of obesity with fewer side effects is urgently needed.

[0006] Recently, a study reported that obese people have about 20% more harmful gut bacteria called Firmicutes than lean people, and that consuming probiotics reduces subcutaneous fat and visceral fat.

[0007] Meanwhile, Korean Patent No. 2183841 discloses 'a novel Lactobacillus plantarum Ln4 strain and a pharmaceutical composition for preventing and treating obesity containing the same', and Korean Patent No. 1508586 discloses 'a novel Enterococcus ficalis MD366 strain with excellent anti-obesity activity', but the Korean microbiome-derived Clostridium butyricum strain with anti-obesity activity of the present invention and its use are not described.

[0008] The present invention was derived from the above-mentioned needs, and the inventors of the present invention confirmed that the Clostridium butyricum S-45-5 strain, deposited number KCTC12754BP, has an excellent effect on reducing body weight and adipose tissue weight of experimental animals under high-fat diet conditions compared to the control group (untreated strain), and has an excellent effect on reducing the contents of total cholesterol, triglycerides, low-density lipoprotein (LDL), leptin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the serum, and has an excellent effect on reducing blood sugar and increasing GLP-1 (glucagon-like peptide-1) contents in an oral glucose tolerance test. In addition, the present invention was completed by confirming that the anti-obesity activity of the Clostridium butyricum S-45-5 strain of the present invention is superior to that of other strains of the same genus and species.

[0009] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating obesity or metabolic disease, containing Clostridium butyricum S-45-5 strain having a deposit number of KCTC12754BP or a culture solution thereof as an active ingredient.

[0010] In addition, the present invention provides a health functional food composition for preventing or improving obesity or metabolic disease, which contains Clostridium butyricum S-45-5 strain having a deposit number of KCTC12754BP or a culture solution thereof as an active ingredient.

[0011] In addition, the present invention provides a feed additive for improving obesity or metabolic disease, which contains Clostridium butyricum S-45-5 strain having a deposit number of KCTC12754BP or a culture solution thereof as an active ingredient.

[0012] In addition, the present invention provides a veterinary composition for preventing or treating obesity or metabolic disease, which comprises Clostridium butyricum S-45-5 strain having a deposit number of KCTC12754BP or a culture solution thereof as an active ingredient.

[0013] Since the Clostridium butyricum S-45-5 strain of the present invention has excellent anti-obesity activity, it is expected that it can be usefully utilized as a material for a composition for preventing, improving, or treating obesity and metabolic diseases caused by obesity.

[0014] Figure 1 shows the results of body weight measurement for 12 weeks after oral administration of Clostridium butyricum S-45-5 strain to a high-fat diet animal model. ND: normal diet, HFD: high-fat diet.

[0015] Figure 2 shows the results of measuring the contents of total cholesterol (T-chol), triglyceride (TG), low-density cholesterol (LDL-C), and high-density cholesterol (HDL-C) by isolating serum after oral administration of Clostridium butyricum S-45-5 strain to a high-fat diet animal model.

[0016] Figure 3 shows the results of separating epididymal fat and perirenal fat after oral administration of Clostridium butyricum S-45-5 strain to a high-fat diet animal model, performing H&E (hematoxylin and eosin) staining, measuring the weight of each fat, and separating serum to measure the content of leptin.

[0017] Figure 4 shows the results of orally administering Clostridium butyricum S-45-5 strain to a high-fat diet animal model, isolating the liver, performing H&E staining, measuring the weight of the liver, and isolating the serum to measure the contents of ALT (alanine aminotransferase) and AST (aspartate aminotransferase).

[0018] Figure 5 shows the results of orally administering Clostridium butyricum S-45-5 strain to a high-fat diet animal model, orally administering glucose at week 8, fasting for 2 hours, collecting blood through the tail vein to measure blood sugar, islet of Langerhans isolation and performing H&E staining, measuring the content of GLP-1 (glucagon-like peptide-1), and measuring feed intake.

[0019] Figure 6 shows the results of measuring body weight for 12 weeks after orally administering Clostridium butyricum S-45-5 strain, Clostridium butyricum Miyairi strain, or Clostridium butyricum strain (KCTC 1871) to a high-fat diet animal model.

[0020] Figure 7 shows the results of measuring the contents of total cholesterol (T-chol), triglyceride (TG), low-density cholesterol (LDL-C), and high-density cholesterol (HDL-C) in serum after oral administration of Clostridium butyricum S-45-5 strain, Clostridium butyricum Miyairi strain, or Clostridium butyricum strain (KCTC 1871) to a high-fat diet animal model.

[0021] Figure 8 shows the results of separating the fat around the epididymis and the fat around the kidney, performing H&E staining, measuring the weight of each fat, and separating the serum to measure the leptin content after orally administering Clostridium butyricum S-45-5 strain, Clostridium butyricum Miyairi strain, or Clostridium butyricum strain (KCTC 1871) to a high-fat diet animal model.

[0022] Figure 9 shows the results of orally administering Clostridium butyricum S-45-5 strain, Clostridium butyricum Miyairi strain, or Clostridium butyricum strain (KCTC 1871) to a high-fat diet animal model, isolating the liver, performing H&E staining, measuring the weight of the liver, and isolating the serum to measure the contents of ALT and AST.

[0023] Figure 10 shows a taxonomic phylogenetic tree of the Clostridium butyricum S-45-5 strain of the present invention.

[0024] In order to achieve the purpose of the present invention, the present invention provides a pharmaceutical composition for preventing or treating obesity or metabolic disease, which contains Clostridium butyricum S-45-5 strain having a deposit number of KCTC12754BP or a culture solution thereof as an active ingredient.

[0025] In the pharmaceutical composition of the present invention, the metabolic disease refers to diabetes, hyperlipidemia or non-alcoholic fatty liver disease caused by obesity, and the non-alcoholic fatty liver disease may be any one selected from the group consisting of obesity-induced fatty liver disease, diabetic fatty liver disease, steatohepatitis, simple fatty liver disease, nutritional fatty liver disease, starvation-induced fatty liver disease, liver fibrosis and liver cirrhosis, but is not limited thereto.

[0026] In the pharmaceutical composition of the present invention, the Clostridium butyricum S-45-5 strain having the deposit number KCTC12754BP is characterized by an excellent effect in reducing body weight and adipose tissue weight of experimental animals compared to the control group (untreated strain) under high-fat diet conditions, an excellent effect in reducing the contents of total cholesterol, triglycerides, LDL (low-density lipoprotein), leptin, ALT (alanine aminotransferase), and AST (aspartate aminotransferase) in serum, and an excellent effect in reducing blood sugar and increasing GLP-1 (glucagon-like peptide-1) contents in an oral glucose tolerance test.

[0027] In addition, the Clostridium butyricum S-45-5 strain with the above-mentioned accession number KCTC12754BP is characterized by its lack of bile salt decomposition ability and hemolysis.

[0028] The method for culturing the Clostridium butyricum S-45-5 strain of the present invention can be performed according to a method commonly used in the art, and is not limited to a specific method.

[0029] In the present invention, the strain culture means the strain cultured in a culture medium, and may include both a concentrate of the culture or a dried product thereof.

[0030] The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above-mentioned effective ingredient, and may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include capsules, powders, granules, tablets, pills, etc., and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, emulsions, syrups, and aerosols. In addition to the commonly used simple diluents, such as water and liquid lapine, various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can be witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin. For parenteral administration, it is preferable to select a method such as topical application to the skin or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine, epidural, or intracerebrovascular injection.

[0031] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The level of the effective amount may be determined based on factors including the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0032] The present invention also provides a health functional food composition for preventing or improving obesity or metabolic disease, containing Clostridium butyricum S-45-5 strain having a deposit number of KCTC12754BP or a culture solution thereof as an active ingredient.

[0033] In the health functional food composition of the present invention, the metabolic disease is as described above.

[0034] The health functional food composition of the present invention is preferably manufactured in any one dosage form selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage, but is not limited thereto. The health functional food composition of the present invention may be manufactured by adding the active ingredient as it is or mixing it with other foods or food ingredients, and may be manufactured appropriately according to a conventional method. Examples of foods to which the above-mentioned active ingredient may be added include dairy products including caramel, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense. That is, there is no particular limitation on the type of the above-mentioned food. The above health functional food composition may contain various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavorings, colorings and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain fruit pulp for producing natural fruit juice and vegetable beverages. The above ingredients may be used independently or in combination. In addition, the health functional food composition of the present invention may contain various flavorings or natural carbohydrates as additional ingredients, and the natural carbohydrates are 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. The ratio of the above natural carbohydrates is not particularly important, but is preferably 0.01 to 0.04 g, more preferably 0.02 to 0.03 g, per 100 g of the composition of the present invention, but is not limited thereto.Sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame.

[0035] The present invention also provides a feed additive for improving obesity or metabolic disease, which comprises Clostridium butyricum S-45-5 strain having a deposit number of KCTC12754BP or a culture solution thereof as an active ingredient.

[0036] The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. The term 'feed' in the present invention may mean any natural or artificial diet, meal, etc., or ingredients of the meal, which are suitable for animals to eat, ingest, and digest. The type of the feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of the feed include plant feeds such as grains, roots, fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal feeds such as proteins, inorganic substances, oils and fats, mineral substances, oils and fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

[0037] The present invention also provides a veterinary composition for preventing or treating obesity or metabolic disease, comprising Clostridium butyricum S-45-5 strain having a deposit number of KCTC12754BP or a culture thereof as an active ingredient.

[0038] The veterinary composition of the present invention may further comprise suitable excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil. The veterinary composition according to the present invention may further include fillers, anticoagulants, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc., and the veterinary composition according to the present invention may be formulated using a method well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile topical preparations, etc. The effective amount of the veterinary composition according to the present invention may be appropriately selected depending on the individual animal. It may be determined according to factors including the severity of the disease or condition, the sensitivity to the active ingredient of the present invention depending on the age, weight, health condition or sex of the individual, the route of administration, the period of administration, other compositions combined with or used simultaneously with the composition, and other factors well known in the field of physiology or veterinary medicine.

[0039]

[0040] Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0041]

[0042] Materials and Methods

[0043] 1. Strain preparation

[0044] In the present invention, Clostridium butyricum S-45-5 strain (KCTC 12754BP), Clostridium butyricum Miyairi strain (CBM588), or Clostridium butyricum strain (KCTC 1871) was used in the experiment. Each strain was cultured in PYG (Peptone Yeast Glucose) liquid medium at 37°C for 48 hours, and the culture solution was centrifuged at 4,000 rpm at 4°C for 20 minutes to obtain a pellet. The pellet was washed three times with PBS (phosphate buffered saline), and then diluted to the desired concentration with PBS for use in the experiment.

[0045]

[0046] 2. High-fat diet animal testing

[0047] Four-week-old C57 / BL6J mice were acclimated to constant conditions (temperature: 22±2℃, humidity: 50±5%, light / dark cycle: 12 hours) in an animal breeding room for one week, and then randomly divided into groups of 10 mice each according to Table 1 below for the experiment. Each group was fed a normal diet (ND) or a high-fat diet (HFD), and PBS or each strain was administered orally daily for 12 weeks (Fig. 1A and Fig. 6A). Body weight was measured daily, and after the experiment, serum, liver tissue, and adipose tissue were extracted and used in subsequent experiments. After the first experiment to analyze the anti-obesity activity of Clostridium butyricum S-45-5 strain, the second experiment was performed to compare the anti-obesity activity of each Clostridium butyricum strain.

[0048] High-fat diet animal experiment 1st 2nd Normal group ND + PBS Normal group ND + PBS Control group HFD + PBS Control group HFD + PBS Experimental group HFD + S-45-5 (2x10 8CFU) Experimental group HFD+S-45-5 (5x10 8 CFU) Experimental group HFD+S-45-5 (5x10 8 CFU)Comparative groupHFD+Miyairi(5x10 8 CFU)--Comparative groupHFD+KCTC 1871(5x10 8 CFU)

[0049] ND: normal diet, HFD: high-fat diet.

[0050]

[0051] 3. Serum analysis

[0052] Blood obtained from experimental animals was centrifuged at 12,000 rpm for 20 minutes to separate serum. Afterwards, total cholesterol and triglyceride contents were measured using a dry automatic biochemical analyzer (Fuji dri-chem 7000i), and low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, leptin, and glucagon-like peptide-1 (GLP-1) contents were measured using ELISA analysis.

[0053]

[0054] 4. Genomic DNA analysis

[0055] A taxonomic phylogenetic tree based on 100 core genes was generated using AutoMLST based on whole-genome sequences of genetically homologous related strains, and maximum-likelihood phylogenetic analysis was performed using IQ-tree. Average nucleotide identity (ANI) values ​​were calculated using an ANI calculator, and digital DNA-DNA hybridization (dDDH) was estimated using a genome-to-genome homology calculator. In addition, whole-genome sequences of strains, along with their respective GenBank numbers, were used to perform ANI, dDDH, and species phylogenetic analyses.

[0056]

[0057] 5. Bile salt deconjugation analysis

[0058] Bile salt degradation was evaluated using a plate assay. The medium used for the analysis was prepared by adding 1 mM sodium salts of taurocholic acid (TCA), glycocholic acid (GCA), taurodeoxycholic acid (TDCA), and taurodeoxycholic acid (TCDCA) to Reinforced Clostridial Medium (RCM). Clostridium butyricum S-45-5 strain and Clostridium butyricum Miyairi strain were inoculated into the prepared culture medium and cultured at 37°C for 72 h, after which the formation of degradation zones around the colonies was observed.

[0059]

[0060] 6. Hemolytic assay

[0061] Hemolytic assay was performed by culturing Clostridium butyricum S-45-5 strain and Clostridium butyricum Miyairi strain in RCM medium containing blood at 37℃ for 48 hours, and analyzing blood hemolytic reaction according to the type of ring formed around the colony.

[0062]

[0063] Example 1. Anti-obesity activity of Clostridium butyricum S-45-5 strain

[0064] The anti-obesity activity of Clostridium butyricum S-45-5 strain was analyzed in a high-fat diet animal model. The normal group was fed a normal diet instead of a high-fat diet, and the control group was fed a high-fat diet and orally administered PBS instead of the strain.

[0065]

[0066] 1-1. Weight analysis

[0067] 2x10 in a high-fat diet animal model 8 CFU or 5x10 8 Clostridium butyricum S-45-5 strain was administered orally for 12 weeks, and body weight was measured daily.

[0068] As a result, it was confirmed that the body weight of the control group increased compared to the normal group, while the body weight of the Clostridium butyricum S-45-5 strain administration group decreased compared to the control group (Fig. 1).

[0069]

[0070] 1-2. Serum analysis

[0071] Dyslipidemia is a condition in which the levels of total cholesterol, low-density cholesterol, and triglycerides in the blood increase or the levels of high-density cholesterol decrease, and is known to be caused by obesity, diabetes, etc.

[0072] 2x10 in a high-fat diet animal model 8 CFU or 5x10 8After oral administration of CFU of Clostridium butyricum S-45-5 strain for 12 weeks, serum was separated and the contents of total cholesterol, triglycerides, low-density cholesterol, and high-density cholesterol were measured.

[0073] As a result, it was confirmed that the contents of total cholesterol, neutral fat, and low-density cholesterol in the control group increased compared to the normal group, whereas the contents of total cholesterol, neutral fat, and low-density cholesterol in the Clostridium butyricum S-45-5 strain administration group decreased compared to the control group, and it was confirmed that there was no significant difference in the contents of high-density cholesterol in all experimental groups (Fig. 2).

[0074] Based on the above results, it was found that the Clostridium butyricum S-45-5 strain was excellent in reducing blood lipid content.

[0075]

[0076] 1-3. Adipose tissue analysis

[0077] It is known that obesity leads to excessive fat accumulation, and leptin, a hormone secreted from fat cells, is known to suppress appetite and increase energy consumption.

[0078] 2x10 in a high-fat diet animal model 8 CFU or 5x10 8 After oral administration of CFU of Clostridium butyricum S-45-5 strain for 12 weeks, epididymal fat and perirenal fat were separated, stained with H&E (hematoxylin and eosin), and the weight of each fat was measured. Serum was separated and the leptin content was measured.

[0079] As a result, it was confirmed that the weight of the fat around the epididymis and the fat around the kidney and the leptin content of the control group increased compared to the normal group, whereas the weight of the fat around the epididymis and the fat around the kidney and the leptin content of the Clostridium butyricum S-45-5 strain administration group significantly decreased compared to the control group (Fig. 3).

[0080]

[0081] 1-4. Liver function analysis

[0082] 2x10 in a high-fat diet animal model 8 CFU or 5x10 8 After oral administration of CFU of Clostridium butyricum S-45-5 strain for 12 weeks, the liver was isolated, H&E stained, and weighed, and the serum was isolated and the contents of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) were measured.

[0083] As a result, it was confirmed that the liver weight and ALT and AST contents of the control group increased compared to the normal group, whereas the liver weight and ALT and AST contents of the Clostridium butyricum S-45-5 strain administration group decreased compared to the control group. In addition, it was confirmed that the control group formed a large number of lipid droplets in the liver tissue, whereas the normal group and the Clostridium butyricum S-45-5 strain administration group did not form lipid droplets in the liver tissue (Fig. 4).

[0084]

[0085] 1-5. Oral glucose tolerance test

[0086] The islets of Langerhans are clusters of cells that are responsible for the exocrine function of the pancreas, and they secrete hormones such as insulin and glucagon to regulate blood sugar levels. GLP-1 is a substance secreted in the small intestine that increases insulin secretion depending on glucose concentration, and is attracting attention as a new candidate drug for the treatment of diabetes.

[0087] 2x10 in a high-fat diet animal model 8 CFU or 5x10 8 CFU of Clostridium butyricum S-45-5 strain was administered orally daily, and glucose was administered orally at 2 g / kg at week 8. After fasting for 2 hours, blood was collected through the tail vein to measure blood glucose, islets of Langerhans were isolated and stained with H&E, the content of GLP-1 was measured, and daily feed intake was measured.

[0088] As a result, it was confirmed that the blood sugar level of the control group increased compared to the normal group, whereas the blood sugar level of the Clostridium butyricum S-45-5 strain administration group decreased compared to the control group (Fig. 5A to Fig. 5C). In addition, it was confirmed that the control group had irregular and increased islets of Langerhans, whereas the Clostridium butyricum S-45-5 strain administration group had decreased islets of Langerhans (Fig. 5D). It was also confirmed that the content of GLP-1 in the control group decreased compared to the normal group, whereas the content of GLP-1 in the Clostridium butyricum S-45-5 strain administration group increased compared to the control group (Fig. 5E). It was also confirmed that the feed intake of the Clostridium butyricum S-45-5 strain administration group decreased compared to the control group (Fig. 5F).

[0089]

[0090] Example 2. Comparison of anti-obesity activity among Clostridium butyricum strains.

[0091] The anti-obesity activity of the Clostridium butyricum S-45-5 strain, which was confirmed to have anti-obesity activity in Example 1, was analyzed by comparing it with the Clostridium butyricum Miyairi strain or the Clostridium butyricum strain (KCTC 1871).

[0092]

[0093] 2-1. Weight analysis

[0094] 5x10 in a high-fat diet animal model 8 CFU of Clostridium butyricum S-45-5 strain, 5x10 8 CFU of Clostridium butyricum Miyairi strain or 5x10 8 Clostridium butyricum strain (KCTC 1871) of 10 CFU was administered orally for 12 weeks, and body weight was measured daily.

[0095] As a result, it was confirmed that the body weight of the Clostridium butyricum S-45-5 strain administration group was lower than that of the Clostridium butyricum Miyairi strain or the Clostridium butyricum strain (KCTC 1871) administration group (Fig. 6).

[0096]

[0097] 2-2. Serum analysis

[0098] 5x10 in a high-fat diet animal model 8 CFU of Clostridium butyricum S-45-5 strain, 5x10 8 CFU of Clostridium butyricum Miyairi strain or 5x10 8 After oral administration of Clostridium butyricum strain (KCTC 1871) of CFU for 12 weeks, serum was separated and the contents of total cholesterol, triglycerides, low-density cholesterol, and high-density cholesterol were measured.

[0099] As a result, it was confirmed that the contents of total cholesterol, neutral fat, and low-density cholesterol in the Clostridium butyricum S-45-5 strain administration group were lower than those in the Clostridium butyricum Miyairi strain or Clostridium butyricum strain (KCTC 1871) administration group, and it was confirmed that there was no significant difference in the contents of high-density cholesterol in all experimental groups (Fig. 7).

[0100]

[0101] 2-3. Fat tissue analysis

[0102] 5x10 in a high-fat diet animal model 8 CFU of Clostridium butyricum S-45-5 strain, 5x10 8 CFU of Clostridium butyricum Miyairi strain or 5x10 8 After oral administration of CFU of Clostridium butyricum strain (KCTC 1871) for 12 weeks, the fat around the epididymis and the fat around the kidney were separated, stained with H&E (hematoxylin and eosin), and the weight of each fat was measured. The serum was separated and the leptin content was measured.

[0103] As a result, it was confirmed that the weight of the fat around the epididymis and the fat around the kidney and the leptin content of the Clostridium butyricum S-45-5 strain administration group were lower than those of the Clostridium butyricum Miyairi strain or the Clostridium butyricum strain (KCTC 1871) administration group (Fig. 8).

[0104]

[0105] 2-4. Liver function analysis

[0106] 5x10 in a high-fat diet animal model 8 CFU of Clostridium butyricum S-45-5 strain, 5x10 8 CFU of Clostridium butyricum Miyairi strain or 5x10 8After oral administration of CFU of Clostridium butyricum strain (KCTC 1871) for 12 weeks, the liver was isolated, stained with H&E, and weighed, and the serum was isolated and the contents of ALT and AST were measured.

[0107] As a result, it was confirmed that the liver weight and ALT and AST contents of the Clostridium butyricum S-45-5 strain administration group were lower than those of the Clostridium butyricum Miyairi strain or the Clostridium butyricum strain (KCTC 1871) administration group. In addition, it was confirmed that the formation of lipid droplets in the liver tissue of the Clostridium butyricum S-45-5 strain administration group was suppressed more than those of the Clostridium butyricum Miyairi strain or the Clostridium butyricum strain (KCTC 1871) administration group (Fig. 9).

[0108] Based on the above results, it was found that the anti-obesity activity of the Clostridium butyricum S-45-5 strain of the present invention was superior to that of the Clostridium butyricum strain of the same genus and species.

[0109]

[0110] Example 3. Genomic DNA analysis of Clostridium butyricum S-45-5 strain

[0111] It was confirmed that the Clostridium butyricum S-45-5 strain showed the highest sequence homology of 99.1% with the Clostridium butyricum DSM 10702T strain (Fig. 10). In addition, the ANI values ​​of the Clostridium butyricum S-45-5 strain and related strains ranged from 76.6 to 99.1%, and the dDDH values ​​ranged from 20.4 to 91.8% (Table 2). Through the above results, it was found that the Clostridium butyricum S-45-5 strain of the present invention belongs to the Butyricum species within the genus Clostridium.

[0112]

[0113]

[0114] Example 4. Analysis of bile salt decomposition ability and hemolysis of Clostridium butyricum S-45-5 strain.

[0115] Both Clostridium butyricum S-45-5 strain and Clostridium butyricum MIYAIRI strain were confirmed to have no ability to decompose taurocholic acid (TCA), glycocholic acid (GCA), taurodeoxycholic acid (TDCA), and taurodeoxycholic acid (TCDCA), and were also confirmed to have no hemolysis (Table 3).

[0116] Bile salt decomposition and hemolytic activity analysis of Clostridium butyricum S-45-5 Clostridium butyricum MIYAIRI Bile salt decomposition activity TCA--TCDCA--TDCA--GCA--Hemolytic activity--

[0117]

[0118] [Accession number]

[0119] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0120] Accession number: KCTC12754BP

[0121] Date of acceptance: 20150203

[0122]

Claims

1. A pharmaceutical composition for the prevention or treatment of obesity or metabolic disease, containing Clostridium butyricum S-45-5 strain or a culture thereof with the deposit number KCTC12754BP as an active ingredient.

2. A pharmaceutical composition for preventing or treating obesity or metabolic disease, characterized in that the metabolic disease in paragraph 1 is diabetes, hyperlipidemia, or non-alcoholic fatty liver disease.

3. A pharmaceutical composition for preventing or treating obesity or metabolic disease, characterized in that in the second paragraph, the non-alcoholic fatty liver disease is any one selected from the group consisting of obesity-induced fatty liver disease, diabetic fatty liver disease, steatohepatitis, simple fatty liver disease, nutritional fatty liver disease, starvation-induced fatty liver disease, liver fibrosis, and liver cirrhosis.

4. A pharmaceutical composition for preventing or treating obesity or metabolic disease, characterized in that, in addition to the effective ingredient in paragraph 1, it further comprises a pharmaceutically acceptable carrier, excipient or diluent.

5. A health functional food composition for preventing or improving obesity or metabolic disease, containing Clostridium butyricum S-45-5 strain or a culture thereof with the deposit number KCTC12754BP as an active ingredient.

6. A health functional food composition for preventing or improving obesity or metabolic disease, characterized in that the health functional food composition in paragraph 5 is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage.

7. A feed additive for improving obesity or metabolic disease, comprising Clostridium butyricum S-45-5 strain or its culture solution with the deposit number KCTC12754BP as an active ingredient.

8. A veterinary composition for the prevention or treatment of obesity or metabolic disease, comprising Clostridium butyricum S-45-5 strain with deposit number KCTC12754BP or a culture solution thereof as an active ingredient.

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