Akkermansia muciniphila EB-AMDK19 strain and its use

Abstract

The present invention relates to a novel muciniphilic Akkermansia strain having the efficacy of preventing or treating inflammatory diseases or metabolic diseases, and a pharmaceutical composition comprising the above strain or a culture thereof for effectively preventing or treating inflammatory diseases or metabolic diseases. Traditional probiotics generally have little therapeutic effect on inflammatory or metabolic diseases. In comparison, the next-generation probiotic strain of the present invention can be used as a novel preventive and therapeutic tool for inflammatory diseases and / or metabolic diseases, and has excellent preventive or therapeutic effects.

Description

Technical Field

[0001] The present invention relates to an Akkermansia muciniphila strain and uses thereof, and more particularly, to a novel Akkermansia muciniphila strain having anti-inflammatory or metabolic disease prevention or treatment efficacy and a pharmaceutical composition comprising the same for preventing or treating inflammatory diseases or metabolic diseases. Background Art

[0002] Inflammatory diseases are diseases caused by the over-activation of the human immune system due to harmful stimuli such as pro-inflammatory factors or radiation, which leads to the secretion of inflammatory cytokines such as TNF-α (tumor necrosis factor-α), IL-1 (interleukin-1), IL-6, prostaglandin, leukotriene or nitric oxide (NO) in immune cells such as macrophages.

[0003] Inflammatory diseases such as inflammatory bowel disease (IBD) refer to a series of chronic, recurrent and tissue-destructive diseases characterized by dysfunction of mucosal T-cells, abnormal production of cytokines and cellular inflammation that cause mucosal damage. Inflammatory diseases are a disease that seriously reduces the quality of life, but their causes are still unclear.

[0004] Taking inflammatory bowel disease as an example, one of the current treatments is to remove colon ulcers through colectomy, but this method may reduce the quality of life and increase the risk of complications. On the other hand, another medical method is to control inflammation by suppressing the immune system with immunomodulators, but immunomodulators induce a low immune state, which makes the patient vulnerable to various diseases.

[0005] In order to overcome the side effects caused by conventional drugs or immunomodulators, research is being conducted on the use of probiotics alone or in combination with antibiotics. In terms of health promotion, research on the role of intestinal bacteria has attracted more attention than research on pathogens.

[0006] In order to overcome the side effects caused by conventional drugs or immunomodulators, research is being conducted on the use of probiotics alone or in combination with antibiotics. In terms of health promotion, as research on the role of intestinal bacteria is actively carried out, interest in lactic acid bacteria preparations is increasing.

[0007] As a technology for treating inflammation and obesity using probiotics, Korean Patent No. 2011-0095929 discloses a Lactobacillus reuteri strain that inhibits the absorption of lipids in the body, Korean Patent No. 0996577 discloses a Lactobacillus curvus that lowers blood cholesterol and inhibits obesity, Korean Patent No. 2011-0010015 discloses a Lactobacillus johnsonii that suppresses obesity while lowering blood cholesterol, and Korean Patent No. 2019-0034796 discloses a Lactobacillus plantarum strain with antioxidant and anti-inflammatory effects.

[0008] However, the probiotics described above have little effect on improving inflammatory diseases or metabolic diseases, and therefore have a limitation in that they are difficult to use as a new preventive and therapeutic tool. Summary of the invention

[0009] Technical issues

[0010] The present invention is used to overcome the limitations of the prior art as described above. One purpose of the present invention is to provide a new next-generation probiotic strain that inhibits inflammatory response by reducing or inhibiting the release of one or more inflammatory factors such as tumor necrosis factor (TNF-α), IL-8, IL-6 and IL-1β, and effectively prevents and treats inflammatory diseases, obesity, obesity complications or metabolic diseases by inhibiting the differentiation of adipocytes.

[0011] Another object of the present invention is to provide a pharmaceutical composition comprising Akkermansia muciniphila as a next-generation probiotic strain for effectively preventing or treating inflammatory diseases or metabolic diseases.

[0012] Another object of the present invention is to provide a composition for suppressing appetite comprising the Akkermansia muciniphila strain.

[0013] Still another object of the present invention is to provide a food comprising the Akkermansia muciniphila strain that helps improve inflammatory diseases or metabolic diseases.

[0014] Solutions to the problem

[0015] One aspect of the present invention for achieving the above-mentioned objects relates to the Akkermansia muciniphila EB-AMDK19 strain with a deposit number of KCTC13761BP.

[0016] Another aspect of the present invention for achieving the above-mentioned purpose relates to a pharmaceutical composition for preventing or treating inflammatory diseases or metabolic diseases, comprising the Akkermansia muciniphila EB-AMDK19 strain with a deposit number of KCTC13761BP, a culture or a dried product of the strain.

[0017] Another aspect of the present invention for achieving the object as described above relates to a composition for suppressing appetite comprising the Akkermansia muciniphila EB-AMDK19 strain (KCTC13761BP) of the present invention, or a culture or a dried product of the strain.

[0018] Another aspect of the present invention for achieving the above-mentioned purpose relates to a food for preventing or improving inflammatory diseases or metabolic diseases, comprising the Akkermansia muciniphila EB-AMDK19 strain (KCTC13761BP) of the present invention, a culture or a dried product of the strain.

[0019] Effects of the Invention

[0020] The novel Akkermansia muciniphila EB-AMDK19 strain of the present invention suppresses inflammatory response by reducing or inhibiting the release of inflammatory factors, and effectively treats inflammatory diseases, especially enteritis, by maintaining the stability of intestinal microbiota.

[0021] The pharmaceutical composition comprising the Akkermansia muciniphila EB-AMDK19 strain of the present invention as an active ingredient can be used as a pharmaceutical composition, a functional food composition, etc. for treating and / or preventing inflammatory diseases or metabolic diseases.

[0022] The novel Akkermansia muciniphila EB-AMDK19 strain of the present invention and the pharmaceutical composition containing the same inhibit weight gain and body fat increase, reduce insulin resistance, reduce the concentration of total cholesterol in the blood, reduce the content of GPT as an indicator of hepatotoxicity in the blood, and reduce the concentrations of IL-8, TNF-α, IL-6 and IL-1β as inflammatory cytokines. Therefore, it can be effectively used for the prevention or treatment of diabetes, obesity, insulin resistance, fatty liver, hyperlipidemia or metabolic diseases related thereto.

[0023] In particular, the pharmaceutical composition of the present invention contains the Akkermansia muciniphila EB-AMDK19 strain as an active ingredient, which not only reduces the fat accumulated in cells and reduces the expression of PPARγ, a factor related to adipocyte differentiation, but also reduces the expression of mRNA of CEBPα, aP2, CD36, ACC1, lipoprotein lipase (LPL), LDLR, and FAS, thereby substantially exerting the effect of inhibiting fat accumulation.

[0024] The composition for suppressing appetite comprising the Akkermansia muciniphila EB-AMDK19 strain (KCTC13761BP) of the present invention can suppress appetite by promoting the secretion of appetite suppressing hormone. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The results of microscopic observation of the Akkermansia muciniphila EB-AMDK19 strain (KCTC13761BP) of the present invention and the Akkermansia muciniphila ATCC BAA-835 strain as a standard strain (type strain) are shown.

[0026] Figure 2 These are the PCR analysis results of the Akkermansia muciniphila EB-AMDK19 strain and the Akkermansia muciniphila ATCC BAA-835 strain of the present invention.

[0027] Figure 3 These are the hemolytic activity test results of the Akkermansia muciniphila EB-AMDK19 strain and the Akkermansia muciniphila ATCC BAA-835 strain of the present invention.

[0028] Figure 4 The results of random amplified polymorphic DNA (RAPD) analysis of genomic DNA of Akkermansia muciniphila EB-AMDK19 strain and Akkermansia muciniphila ATCC BAA-835 strain of the present invention are as follows:

[0029] Figure 5 Comparison of the phylogenetic relationship between the Akkermansia muciniphila EB-AMDK19 strain of the present invention and other Akkermansia muciniphila strains is shown.

[0030] Figure 6 The results of confirming the anti-inflammatory effect of the Akkermansia muciniphila EB-AMDK19 strain of the present invention are bar graphs showing the relative mRNA expression levels of IL-6, IL-8, IL-1β, and TNF-α, which are cytokines.

[0031] Figure 7 The photographs and bar graphs show the extent of fat accumulation formed in 3T3-L1 cells when the Akkermansia muciniphila EB-AMDK19 strain of the present invention is treated.

[0032] Figure 8 It is a bar graph showing the expression levels of PPARγ, CEBPα, aP2, CD36, ACC1, lipoprotein lipase (LPL), LDLR, and FAS mRNA in preadipocytes of the Akkermansia muciniphila EB-AMDK19 strain according to the present invention.

[0033] Fig. 9The results of analysis of body weight changes in the Akkermansia muciniphila EB-AMDK19 strain administration group (EB-AMDK19), the control group (DM), and the Akkermansia muciniphila ATCC BAA-835 strain administration group of the present invention.

[0034] Fig.10 The results are analysis of changes in subcutaneous fat, epididymal fat, and superior mesenteric fat in the Akkermansia muciniphila EB-AMDK19 strain administration group, the control group (DM), and the Akkermansia muciniphila ATCC BAA-835 strain administration group of the present invention.

[0035] Fig.11 The results are the analysis results of the changes in glucose tolerance of the Akkermansia muciniphila EB-AMDK19 strain administration group, the control group (DM) and the Akkermansia muciniphila ATCC BAA-835 strain administration group of the present invention.

[0036] Fig.12 The results are the results of measuring changes in the blood insulin, cholesterol and glutamic pyruvic transaminase (GPT) concentrations in the Akkermansia muciniphila EB-AMDK19 strain administration group of the present invention, the control group (DM) and the Akkermansia muciniphila ATCC BAA-835 strain administration group by ELISA.

[0037] Fig.13 The diagram is a bar graph showing photographs of a normal group, a high-fat diet group (HFD), a Garcinia cambogia administration group (GC), an Akkermansia muciniphila ATCC BAA-835 strain administration group (BAA-835), and an Akkermansia muciniphila EB-AMDK19 strain administration group (EB-AMDK19) of the present invention stained with Alcian blue, and the results of quantifying the positive area in each group based on these results.

[0038] Fig.14 The graph shows changes in the expression of TLR4, TLR2, GLP-1, PYY, IL-6, TNF-α, MCP-1, IL-10, ZO-1, and tight junction-related proteins in the intestinal cells of mice in each experimental group.

[0039] Figure 15-16 The figure shows photographs of hepatocytes and lipid droplets observed under an optical microscope by H&E staining, and a bar graph showing the liver steatosis scores in each group.

[0040] Fig.17The photographs and bar graphs show the degree of fat accumulation formed in the mesenteric adipose tissue of the animals in each experimental group when treated with the Akkermansia muciniphila EB-AMDK19 strain of the present invention.

[0041] Fig.18 Comparison of linear discriminant analysis results between experimental groups is shown.

[0042] Fig.19 The figure is a bar graph showing the relative ratio of microbiota in the intestine by administering Akkermansia muciniphila ATCC BAA-835 strain (BAA-835) and Akkermansia muciniphila EB-AMDK19 strain of the present invention according to an embodiment of the present invention. DETAILED DESCRIPTION

[0043] Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

[0044] In the scope of the invention claims, the term "includes / comprising" does not exclude the existence of other species or steps.

[0045] In the present invention, "inflammatory disease" refers to a disease caused by an inflammatory response in a mammal, and representative examples include respiratory diseases such as asthma, chronic obstructive pulmonary disease, and rhinitis; skin diseases such as atopic dermatitis; digestive system diseases such as gastritis and inflammatory enteritis; arteriosclerosis, sepsis, inflammatory arthritis, inflammatory encephalopathy, etc.

[0046] In the present invention, unless otherwise specified, "treatment" refers to reversing, alleviating or inhibiting the progression of one or more symptoms of an inflammatory disease or metabolic disease or the above-mentioned disease. In this specification, "prevention" refers to including reducing the possibility of an inflammatory disease or metabolic disease.

[0047] In this specification, "cytokine" refers to a secreted protein that affects the function of other cells. In particular, it is related to the regulation of the interaction between cells involved in the immune system or cells involved in inflammatory responses. As cytokines, interleukin 1 (IL-1) can be exemplified, preferably IL-1β, interleukin-6 (IL-6), interleukin 8 (IL-8) and TNF-α (tumor necrosis factor-α) can be exemplified, but are not limited thereto. The term "appetite suppression" used in the present invention refers to all behaviors of suppressing or delaying appetite by promoting the secretion of diet-related hormones (e.g., appetite suppressant hormones) by administering a strain or composition.

[0048] One aspect of the present invention relates to a novel next-generation probiotic strain, which is an Akkermansia muciniphila EB-AMDK19 strain with a deposit number of KCTC13761BP.

[0049] The above strain has the 16S ribosomal RNA gene shown in Sequence 1.

[0050] The Akkermansia muciniphila EB-AMDK19 strain of the present invention is an oval cell with a size of 0.5-1 μm isolated from the feces of healthy Koreans, and is a monococcal or diplococcal, anaerobic, non-motile, Gram-negative, non-endospore-forming mucin-degrading bacterium. The Akkermansia muciniphila EB-AMDK19 strain produces several mucin-degrading enzymes, and can use mucus as a carbon source and a nitrogen source, including galactose, N-acetylglucosamine and lactose, thereby metabolizing a variety of carbon sources and generating short-chain fatty acids such as propionic acid and acetic acid as major metabolites.

[0051] The Akkermansia muciniphila EB-AMDK19 strain of one embodiment of the present invention inhibits the expression of inflammatory cytokines, specifically, it can produce anti-inflammatory effects by inhibiting the expression of IL-8, IL-6, IL-1β and TNF-α. The tight junctions formed between epithelial cells are most important for the barrier function of epithelial cells. Tight junctions are one of the ways of binding between cells, and their strength is so great that the cell membranes of the two cells appear to be fused together under an electron microscope. Barrier function refers to the function of preventing external substances from passing through epithelial cells, which is important in both blood vessels and the digestive tract. The membrane protein called occludin is a representative constituent molecule for the formation of such tight junctions, and it also participates in ZO proteins and proteins such as cingulin in the cytoplasm. The tight junction structure and function are completed through the interaction of these proteins. The Akkermansia muciniphila EB-AMDK19 strain of one embodiment of the present invention prevents inflammatory cells from moving into the tissue by increasing the expression of intestinal mucosal proteins ZO-1 (zonular occludens-1, p < 0.01) and tight junction-associated proteins (p < 0.01), thereby promoting mucosal regeneration and alleviating inflammation of the colon mucosa.

[0052] The induction of obesity leads to abnormalities in visceral adipose tissue, excessive secretion of tumor necrosis factor, infiltration of immune cells such as macrophages into adipose tissue, and increased expression of inflammatory cytokines. As a result, chronic inflammation occurs in adipose tissue, and chronic inflammatory reactions reduce insulin sensitivity and induce glucose tolerance, leading to the onset of diabetes. Therefore, anti-obesity effects can be expected by inhibiting the hypertrophy and inflammatory reactions of adipose tissue, and as a result, it can also help fight metabolic diseases.

[0053] The novel Akkermansia muciniphila EB-AMDK19 strain of the present invention inhibits weight gain and body fat gain, reduces insulin resistance, reduces the concentration of total cholesterol in the blood, reduces the content of GPT, which is an indicator of hepatotoxicity in the blood, and reduces the concentration of inflammatory cytokines. Therefore, it can be effectively used for the prevention or treatment of diabetes, obesity, insulin resistance, fatty liver, hyperlipidemia or metabolic diseases related thereto.

[0054] In particular, metabolic diseases can be diseases in which multiple metabolic diseases such as diabetes and obesity occur simultaneously in the same person. The Akkermansia muciniphila EB-AMDK19 strain of the present invention has therapeutic efficacy on both inflammatory diseases and metabolic diseases, and thus can provide a significant effect of being able to simultaneously and comprehensively treat diseases that are highly related to inflammation or obesity.

[0055] In particular, the novel Akkermansia muciniphila EB-AMDK19 strain of the present invention not only reduces the expression of PPARγ that causes intracellular fat accumulation, but also reduces the expression of mRNA of CEBPα, aP2, CD36, ACC1, lipoprotein lipase (LPL), LDLR, and FAS, thereby exerting the effect of substantially inhibiting fat accumulation.

[0056] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases or metabolic diseases, comprising the Akkermansia muciniphila EB-AMDK19 strain with a deposit number of KCTC13761BP, a culture or a dried product of the strain.

[0057] The pharmaceutical composition of the present invention may contain the probiotics of the above-mentioned Akkermansia muciniphila EB-AMDK19 strain or contain a strain that has been sterilized at low temperature. Low temperature sterilization of the Akkermansia muciniphila strain refers to heating at a temperature of more than 50°C and less than 100°C for more than 10 minutes. For example, low temperature sterilization can be performed at a temperature of 70°C for 30 minutes. Compared with probiotics, the low temperature sterilized form of the Akkermansia muciniphila EB-AMDK19 strain is more capable of reducing body fat accumulation. Although the reason why the low temperature sterilized form of the Akkermansia muciniphila EB-AMDK19 strain is more effective has not been accurately elucidated, it can be speculated that under low temperature sterilization, cell wall components or membrane proteins such as Amuc_1100 of the Akkermansia muciniphila EB-AMDK19 strain enhance the metabolic advantage in the host.

[0058] The beneficial effect of novel strain of the present invention is relevant with the gene cluster (Amuc_1098-Amuc_1102) comprising Amuc_1100, is present in the surface of the immune cell near the barrier of intestinal mucosa, and it can be inferred that it is derived from the polypeptide interacting with the signaling pathway of Toll-like receptor 2 (toll-like receptor, TLR2) for regulating intestinal homeostasis and host metabolism. For example, it can be predicted that the Amuc-1100 polypeptide maintains the integrity of the barrier of intestinal mucosa, and interacts with the TLR2 present in the surface of immune cells to regulate or promote the TLR2-signaling pathway, thereby promoting the secretion of cytokines (for example, IL-6, IL-8 and IL-10) from immune cells. Prolipoprotein oxalyltransferase gene (Amuc_1104) is located at a position very close to the gene cluster (Amuc_1098-Amuc_1102). And, Amuc_1100 remains stable under the temperature conditions used during low temperature sterilization, so it can contribute to the influence of the bacterial strain sterilized through low temperature.

[0059] The pharmaceutical composition of the present invention is effective in treating or preventing inflammatory diseases, in particular, inflammatory bowel disease. Examples of such inflammatory bowel disease include Crohn's disease, ulcerative colitis, Behcet's disease, simple ulcer, simple ulcer and ischemic enteritis, and is particularly effective for Crohn's disease or ulcerative colitis.

[0060] In the present invention, preferably, the metabolic disease is obesity, insulin resistance, fatty liver, hyperlipidemia or their complications, but is not limited thereto. For example, the pharmaceutical composition of the present invention is effective in preventing or treating inflammatory bowel disease (IBD), insulin resistance, dyslipidemia or reducing cholesterol or body weight.

[0061] The pharmaceutical composition of the present invention contains 10 8 CFU to 10 12 The content of CFU contains the Akkermansia muciniphila EB-AMDK19 strain as an active ingredient, or contains a culture having an equivalent amount of live bacteria.

[0062] The Akkermansia muciniphila EB-AMDK19 strain of the present invention can be cultured, recovered by a separation process such as centrifugation, and prepared into a probiotic form by drying (eg, freeze-drying) for use.

[0063] The Akkermansia muciniphila EB-AMDK19 strain of the present invention is sensitive to oxygen and is preferably cultured under anaerobic conditions (90% nitrogen, 5% hydrogen, 5% carbon dioxide).

[0064] The components of the liquid culture medium during cultivation may affect the growth of the strain and the production of effective ingredients. Therefore, it is necessary to establish the components and content conditions of the liquid culture medium that optimize the cultivation of the novel Akkermansia muciniphila EB-AMDK19 strain of the present invention.

[0065] The carbon source in the liquid culture medium may include one or more of the group consisting of glucose, lactose, galactose, mannose, 1-fucose, lactate, formate, acetate, propionate, 1,2-propylene glycol, butyrate and N-acetylglucosamine, but is not limited thereto. Preferably, glucose and N-acetylglucosamine may be included.

[0066] The nitrogen source in the liquid culture medium may include one or more selected from the group consisting of tryptone, peptone, soy peptone, L-glutamic acid, and ammonium, but is not limited thereto.

[0067] The trace elements in the above-mentioned liquid culture medium may include one or more selected from the group consisting of KH2PH4, Na2HPO4, NaCl, MgCl2, CaCl2, FeCl2, ZnCl2, CuCl2, MnCl2, CoCl2, NiCl2, Na2SeO3, Na2WO4 and Na2MoO4, but is not limited to this.

[0068] The pH of the liquid culture medium may be 6.8 to 7.2. The pH is preferably 7.0. The pH may affect the activity of protein forms by changing the charge of the amino group or carboxyl group of amino acids, which are enzyme protein units that are important for cell metabolism. Moreover, in the external environment, changes in pH may affect the ionization of microbial nutrients, thereby affecting the microbial intake of nutrients.

[0069] The liquid culture medium is preferably a culture medium containing glucose, N-acetylglucosamine, threonine, soy peptone or any combination thereof.

[0070] The pharmaceutical composition of the present invention may contain pharmaceutically acceptable carriers and / or excipients in addition to the above-mentioned active ingredients, and may also be formulated together with various additives commonly used in pharmacy such as adhesives, disintegrators, coating agents, lubricants, etc.

[0071] The pharmaceutical composition of the present invention can be formulated into powder, granule, tablet, capsule or liquid form by mixing the Akkermansia muciniphila strain of the present invention with appropriate carriers, excipients, auxiliary effective ingredients, etc. The pharmaceutical composition of the present invention can be formulated into a product for enteral or oral administration. In addition, the pharmaceutical composition of the present invention is enterically coated and formulated by a known method so that it passes through the stomach to the small intestine and the microorganism as an active ingredient is quickly released into the intestine.

[0072] Excipients that can be used in the present invention include sugars such as sucrose, lactose, mannitol, glucose, and starches such as corn starch, potato starch, rice starch, and partially pregelatinized starch. Binders include polysaccharides such as dextrin, sodium alginate, carrageenan, guar gum, gum arabic, and agar; natural polymers such as tragacanth, gelatin, and gluten; cellulose derivatives such as hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, hydroxypropyl ethyl cellulose, and sodium carboxymethyl cellulose; and polymers such as polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate, polyethylene glycol, polyacrylic acid, polymethacrylic acid, and vinyl acetate resin.

[0073] As the disintegrator, there can be used cellulose derivatives such as carboxymethyl cellulose, carboxymethyl cellulose calcium, low-substituted hydroxypropyl cellulose, and starches such as sodium carboxymethyl starch, hydroxypropyl starch, corn starch, potato starch, rice starch, and partially pregelatinized starch.

[0074] Examples of lubricants that can be used in the present invention include talc, stearic acid, calcium stearate, magnesium stearate, colloidal silicon dioxide, hydrated silicon dioxide, various types of waxes, hydrogenated oils, and the like.

[0075] Coating agents include water-insoluble polymers such as dimethylaminoethyl methacrylate-methacrylic acid copolymer, polyvinyl acetaldehyde diethylamine acetate, ethyl acrylate-methacrylic acid copolymer, ethyl acrylate-methyl methacrylate-ethyl methacrylate trimethylammonium chloride copolymer, and ethyl cellulose; enteric polymers such as methacrylic acid-ethyl acrylate copolymer, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; and water-soluble polymers such as methylcellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, and polyethylene glycol, but are not limited to these.

[0076] The dosage of the above-mentioned strain as an active ingredient in the composition for preventing or treating inflammatory diseases or metabolic diseases of the present invention can be determined by taking into account factors including the type of disease, age, weight, sex, medical condition of the patient, severity of the condition, and route of administration. Therefore, the dosage regimen can vary in many ways, but can be determined daily using standard methods. Generally, for adult patients, 1×10 6The above live bacteria or low temperature sterilized bacteria, preferably, 1×10 8 Up to 1×10 12 Live bacteria or low temperature sterilized bacteria can be administered once or in divided doses as needed. Individual physicians can select the correct dosage form, administration route and dosage of the pharmaceutical composition disclosed herein considering the patient's condition.

[0077] Another aspect of the present invention relates to a composition for suppressing appetite comprising the Akkermansia muciniphila EB-AMDK19 strain (KCTC13761BP) of the present invention, or a culture or a dried product of the strain.

[0078] Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are appetite suppressant hormones that are associated with reduced food intake (Wren and Bloom, Gastroenterology 132: 2116-2130 (2007)). The Akkermansia muciniphila EB-AMDK19 strain of one embodiment of the present invention can suppress appetite by increasing the expression and secretion of glucagon-like peptide 1 (GLP-1) and peptide YY (PYY), which are appetite suppressant hormones.

[0079] Still another aspect of the present invention relates to a food product comprising the Akkermansia muciniphila EB-AMDK19 strain, a culture thereof, or a dried product thereof.

[0080] The strain-containing food of the present invention can be taken as a food or nutritional product such as milk or dairy products, or as a food supplement or health functional food. According to an embodiment of the present invention, as the above-mentioned product, dairy products, beverages, juices, soups or children's food can be exemplified, but it is not limited thereto.

[0081] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited to the following examples.

[0082] Example

[0083] Example 1: Isolation and identification of Akkermansia muciniphila strains

[0084] 1.1. Isolation and identification of strains

[0085] In order to isolate Akkermansia muciniphila from the feces of a healthy Korean (female, 35 years old, BMI 23.3), the strain was selected and cultured according to the method of Derrien using a mucin medium (0.4 g / L monopotassium phosphate, 0.53 g / L sodium phosphate dichloride, 0.3 g / L sodium chloride, 0.3 g / L aluminum chloride, 0.1 g / L magnesium chloride, 0.11 g / L calcium chloride, 4.0 g / L sodium bicarbonate, 1 mL acidic trace element solution, 1 mL alkaline trace element solution, 1 mL vitamin solution, 2.5 g / L porcine gastric mucus (type III) and 0.25 g / L sodium sulfide nonahydrate) for isolation (Derrien et al., 2004).

[0086] In order to confirm whether the isolated strain is a strain of Akkermansia muciniphila, the isolated strain was observed under a microscope and the results were Figure 1 PCR analysis was performed using the AM-specific primers in Table 1 below, and the results were Figure 2 Shown in.

[0087] exist Figure 1 In the figure, part A is the Akkermansia muciniphila ATCC BAA-835 strain, and part B is a microscope photo of the Akkermansia muciniphila EB-AMDK19 strain at a magnification of 1000 times. Figure 2 In the figure, Yongdao M is a DNA size marker, Yongdao 1 is a positive control group (ATCC BAA-835), Yongdao 2 is the Akkermansia muciniphila EB-AMDK19 strain, and Yongdao 3 is the result of the negative control group (distilled water).

[0088] Table 1

[0089]

[0090] 1.2. Confirmation of sugar utilization of isolated A. muciniphila strains

[0091] In order to investigate the sugar utilization of the above-isolated Akkermansia muciniphila EB-AMDK19 strain of the present invention, the API50CH kit (Biomerieux, France) was used for culture, and the growth using various sugars was compared with that of the standard strain (ATCC BAA-835). The results are shown in the following Table 2.

[0092] Table 2

[0093]

[0094]

[0095]

[0096] As can be seen from Table 2, compared with the standard strain (ATCC BAA-835), the Akkermansia muciniphila EB-AMDK19 strain of the present invention has differences in the ability to utilize ribose, D-galactose, D-fructose and D-mannose.

[0097] 1.3. Whole genome sequencing

[0098] In order to analyze the variation between the Akkermansia muciniphila EB-AMDK19 strain isolated as described above and the Akkermansia muciniphila ATCC BAA-835 strain at the genome level, the full base sequence of the genome was analyzed using PacBio technology and compared with the standard strain (Tables 3 and 4).

[0099] Table 3

[0100]

[0101]

[0102] Table 4

[0103]

[0104]

[0105] As can be seen from Table 3, when comparing the whole genome statistical data of the Akkermansia muciniphila EB-AMDK19 strain of the present invention with that of the Akkermansia muciniphila ATCC BAA-835 strain, there are differences between the two.

[0106] As is well known, Amuc_1100 is associated with therapeutic activity against inflammatory or metabolic diseases and is a thermostable protein that acts on Toll-like receptor 2 (Plovier et al., 2017). As confirmed by the above Table 4, the Akkermansia muciniphila EB-AMDK19 strain of the present invention and the Akkermansia muciniphila ATCC BAA-835 strain also have slight differences in Amuc_1100-related genes.

[0107] 1.4. Confirmation of antimicrobial susceptibility of isolated strains

[0108] In order to investigate the antimicrobial susceptibility of the Akkermansia muciniphila EB-AMDK19 strain isolated as described above, the minimum inhibitory concentration (MIC) of anaerobic antimicrobial agents (penicillin-tazobactam (PTZ), cefuroxime (CTZ), chloramphenicol (CHL), clindamycin (CLI), meropenem (MEM), moxifloxacin (MXF), metronidazole (MTZ), ciprofloxacin (CIP)) was determined by the broth microdilution method according to the guidelines of the Clinical & Laboratory Standard Institute (CLSI, 2017), and the results are shown in the following Table 5.

[0109] Table 5

[0110]

[0111] As can be seen from Table 5, the Akkermansia muciniphila EB-AMDK19 strain of the present invention exhibits moderate resistance to clindamycin, resistance to moxifloxacin and ciprofloxacin, which are fluoroquinolone antibiotics, and sensitivity to all other antibacterial agents. Compared with the standard strain, the antibacterial resistance pattern is slightly different. It can be confirmed that the Akkermansia muciniphila strain of the present invention is a safe strain that is not resistant to most antibiotics.

[0112] 1.5. Confirmation of hemolytic activity of isolated strains

[0113] In order to verify the safety of the Akkermansia muciniphila EB-AMDK19 strain isolated as described above, it was evaluated whether the strain had hemolytic activity. To this end, the strain was cultured using a blood agar medium prepared by adding 5% w / v defibrinated sheep blood to tryptic soy agar (17.0 g / L of pancreatic digest of casein, 3.0 g / L of pancreatic digest of soybean, 2.5 g / L of glucose, 5.0 g / L of sodium chloride, 2.5 g / L of potassium phosphate, 15 g / L of agar). The results are shown in Table 1. Figure 3 shown.

[0114] pass Figure 3 It was found that the Akkermansia muciniphila EB-AMDK19 strain of the present invention did not show β-hemolysis (completely transparent portion around the colonies) associated with pathogenicity.

[0115] 1.6. Random Amplified Polymorphic DNA (RAPD) analysis

[0116] In order to verify whether the Akkermansia muciniphila EB-AMDK19 strain isolated as described above is the same as the reported standard strain of the same species, RAPD as a molecular typing was performed. For this purpose, genomic DNA extracted from the bacteria was used as the object, DNA was amplified using the universal primers in Table 6 below, and then electrophoresed in 1% agarose gel for 1 hour and 30 minutes, and the DNA fragmentation pattern was compared on a UV perforator. The results are as follows: Figure 4 shown.

[0117] Table 6

[0118]

[0119] pass Figure 4 It can be seen that the Akkermansia muciniphila EB-AMDK19 strain of the present invention shows a different banding pattern compared to the standard strain of Akkermansia muciniphila (ATCC BAA-835). It is known that the RAPD banding patterns of Akkermansia muciniphila are different from each other when the species is different, and therefore, it was confirmed that the Akkermansia muciniphila strain isolated in the present invention is different from the standard strain (ATCC BAA-835).

[0120] 1.7. Phylogenetic tree analysis using full-length 16S ribosomal RNA gene base sequence

[0121] In order to analyze the full-length 16S ribosomal RNA gene base sequence of the Akkermansia muciniphila EB-AMDK19 strain isolated as described above, the 16S ribosomal RNA gene was amplified using the 27F and 1541R primers in Table 7 below, and the base sequence was determined using a 3730xl DNA analyzer. A phylogenetic tree was prepared using the 16S ribosomal RNA gene base sequences of the EB-AMDK19 and other strains of the same species disclosed in the above manner, as shown in FIG. Figure 5 shown.

[0122] Table 7

[0123]

[0124] like Figure 5As shown, the evolutionary relationship between the 16S ribosomal RNA gene base sequences was analyzed by phylogenetic tree, and the analysis results confirmed that the Akkermansia muciniphila EB-AMDK19 strain is a strain that genetically belongs to the species Akkermansia muciniphila (A. muciniphila) ( Figure 6 ).

[0125] Akkermansia muciniphila T The Akkermansia muciniphila EB-AMDK19 strain isolated from human feces was identified by biochemical methods (API) and molecular biological methods (16S ribosomal RNA sequence analysis, RAPD and full-length screening) with (ATCC BAA-835) as the control group, and the strain was confirmed to be a safe strain that can function as a probiotic by antibiotic resistance testing. Based on this result, the isolated Akkermansia muciniphila strain was named Akkermansia muciniphila EB-AMDK19 strain and deposited in the Microbial Resource Center (KCTC) of the Korea Institute of Biotechnology with the deposit number KCTC12398BP.

[0126] Furthermore, the strain was finally confirmed to belong to the species Akkermansia muciniphila (A. muciniphila) through 16S ribosomal RNA gene base sequence analysis and was named "A. muciniphila EB-AMDK19".

[0127] Example 2: Confirmation of the anti-inflammatory efficacy of Akkermansia muciniphila strains

[0128] 2.1. Preparation of Pasteurized Cells for Evaluation of Anti-inflammatory Efficacy

[0129] The cells were collected by centrifugation at 12000 x g and 4°C for 5 minutes, suspended in PBS, adjusted to an OD value of 0.25±0.03 (8 log CFU / mL), sterilized by heat treatment at 70°C for 30 minutes, and stored in an ultra-low temperature refrigerator for later use.

[0130] 2.2. Evaluation of anti-inflammatory efficacy in HT-29 intestinal epithelial cells

[0131] Cytokines and other immunomodulators are involved in the regulation of inflammatory responses in inflammatory bowel disease, and therefore, whether these genes are affected by administration of the strain of the present invention was investigated. For in vitro anti-inflammatory efficacy evaluation experiments, HT-29 cells (ATCC HTB-38) as human colon epithelial cells were cultured. TM, the United States (USA)). McCoy's 5A modified media (Gibco, the United States (USA)) supplemented with 10% FBS (fetal bovine serum, Hyclone, the United States (USA)) and 10 μg / ml gentamicin was used as the basal medium and cultured in an incubator (NUAIRE, the United States (USA)) at 37°C and 5% CO2. In order to determine whether the Akkermansia muciniphila EB-AMDK19 strain inhibits the expression of IL-8, TNF-α, IL-6, and IL-1β genes, which are pro-inflammatory cytokines induced by LPS, real-time PCR was performed using the primers in Table 8 below.

[0132] Total RNA was extracted using TRI reagent (Sigma, USA), and 1 μg of RNA was synthesized into cDNA using M-MLV cDNA synthesis kit (Enzynomics, Korea). Real-time PCR was performed using Quantity Studio 3 real time PCR system (Applied Biosystems, USA).

[0133] Using SYBR Green TOPreal TM qPCR 2X PreMIX (Enzynomics, Korea) was used to analyze the expression of genes for inflammatory cytokines, using GAPDH as an internal standard. The PCR conditions were as follows: pre-incubation (for UDG) at 50°C for 4 minutes, 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute. Data were analyzed by the delta CT method using the program built into QuantStudio Design & Analysis Software v1.4.3, and the results are shown in Figure 2. Figure 6 shown.

[0134] The results obtained by all experiments of this embodiment were calculated as the mean and standard deviation of each experimental group using the statistical program GraphPad Prism 7 (GraphPad software Inc., USA), and the differences between the groups were analyzed using one-way ANOVA and Tukey's test. When the p value was below 0.05, it was determined to be significant. In some results, the area under the curve (AUC) was calculated.

[0135] Table 8

[0136]

[0137] Reference Figure 6 When HT-29 cells were treated with LPS (100 μg / ml) alone for 6 hours, the expression of IL-8, TNF-α, IL-6 and IL-1β increased compared with the normal group (p < 0.001). In contrast, in the group treated with LPS and dead cells of Akkermansia muciniphila BAA-835 strain or Akkermansia muciniphila EB-AMDK19 (10%, v / v) together, the expression of each of IL-8 (P < 0.001), TNF-α (P < 0.01), IL-6 (P < 0.01) and IL-1β (P < 0.001) was significantly reduced compared with the LPS-treated group.

[0138] The novel strain of the present invention reduces the concentration of pro-inflammatory cytokine indicators such as IL-8, TNF-α, IL-6 and IL-1β in a test tube, thus proving that the strain of the present invention is effective in treating inflammatory diseases.

[0139] Example 3: Confirmation of the fat accumulation inhibitory effect of Akkermansia muciniphila strains

[0140] It was investigated whether the expression of biomarkers associated with fat accumulation and obesity was affected by the administration of the strain of the present invention.

[0141] 3.1. Oil red-O staining of cells

[0142] In order to evaluate the effect of the Akkermansia muciniphila EB-AMDK19 strain of the present invention on adipocyte differentiation and adipogenesis in 3T3-L1 cells, an oil red-O staining experiment was performed (Jeon T. et al., Red yeast rice extracts suppress adipogenesis by downregulating adipogenic transcription factors and gene expression in 3T3-L1, Life Sci, 12; 75(26), pp.3195-3203, 2004).

[0143] Oil red O staining is a method for measuring fat produced in cells by staining differentiated 3T3-L1 cells with an oil red O reagent. 3T3-L1 cells (KCLB (Korean Cell Line Bank), Korea) as mouse adipocyte precursor cells were cultured in an incubator (NUAIRE, USA) at 37°C and 5% CO2 using DMEM medium (Dulbecco's Modified Eagle's Medium, Welgene, Korea) supplemented with 10% FBS (fetal bovine serum, Hyclone, USA) and 1% penicillin / streptomycin as a basal medium. After inducing adipogenic differentiation in 3T3-L1 cells in which Akkermansia muciniphila EB-AMDK19 strain is adipocytes by insulin (1 μg / ml), IBMX (0.5 mM), and dexamethasone (1 μM) for 10 days, the culture medium was washed three times with PBS to remove the culture medium, and 10% formalin (Sigma, USA) was added to react the cells with Oil Red O (Sigma, USA) solution for 1 hour, and the cells were washed with distilled water to stain lipid droplets.

[0144] After the cells were stained, they were washed three times with 40% isopropanol (Duksan, Korea), dried, and the size of the intracellular lipid droplets was observed under an optical microscope. Figure 7 As shown in Part A of FIG. Isopropanol was added to dissolve the lipid droplet pattern stained with Oil Red O solution, and the absorbance at 500 nm was measured using a spectrophotometer (Epoch, BioTek, USA). The results are shown in FIG. Figure 7 As shown in Part B.

[0145] The experimental results are as follows Figure 7 As shown in Part B, the Akkermansia muciniphila EB-AMDK19 strain of the present invention was treated during the differentiation process of 3T3-L1 cells, and the results showed that fat accumulation was significantly inhibited compared with the control group.

[0146] 3.2. Efficacy evaluation of biomarker gene expression

[0147] In order to confirm the inhibitory effect of the strain of the present invention on adipocyte differentiation, real-time PCR was performed using the gene-specific primers listed in Table 9 below to analyze the mRNA expression of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT / enhancer binding protein alpha (CEBPα), adipocyte protein 2 (aP2), CD36 (cluster differentiating 36), fatty acid synthase (FAS), acetyl-coenzyme A-carboxylase (ACC1), lipoprotein lipase (LPL), and low-density lipoprotein receptor (LDLR) genes.

[0148] Specifically, total RNA was extracted from the cell monolayer using TRI reagent (Sigma, USA) according to the manufacturer's instructions, and 1 μg of total RNA was synthesized into cDNA using the M-MLV cDNA synthesis kit (Enzynomics, Korea). Quantity Studio 3 real time PCR system (Quant Studio 3 real time PCR system, Applied Biosystems, USA) was used for PCR reaction. The conditions for PCR were as follows: pre-incubation (for UDG) was performed at 50°C for 4 minutes, at 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, and at 60°C for 1 minute. Data were analyzed using the program built into QuantStudio Design & Analysis Software v1.4.3 and by the delta CT method.

[0149] Table 9

[0150]

[0151] Reference Figure 8 When 3T3-L1 cells were induced to differentiate into adipocytes (differentiation medium, DM), the electron microscopic images of lipid droplets and the degree of fat accumulation in cells were reduced in the groups treated with Akkermansia muciniphila BAA-835 strain or Akkermansia muciniphila EB-AMDK19 dead bacteria (10% v / v) compared with the control group (p < 0.001), and were also significantly reduced in the EB-AMDK19 dead bacteria treated group compared with the positive control group (ATCC BAA-835) (P < 0.01).

[0152] Usually, during the stage of differentiation into adipocytes, the expressions of peroxisome proliferator-activated receptorγ (PPARγ), CCAAT / enhancer binding proteinα (CEBPα), activator protein 2alpha (aP2α), CD36, ACC1, LPL, LDLR and fatty acid synthase (FAS) increase.

[0153] And, if Figure 8 As shown in the results, after induction of adipocyte differentiation, when the increased expression of genes PPARγ, CEBPα, aP2, CD36, ACC1, LPL, LDLR, and FAS involved in adipocyte differentiation was numerically expressed as 100%, in the group treated with Akkermansia muciniphila BAA-835 strain, PPARγ (p < 0.001), CEBPα (p < 0.001), aP2 (p < 0.001), CD36 (p < 0.001), ACC1 (p < 0.001), and FAS (p < 0.001) were significantly higher than those in the group treated with BAA-835 strain. 1), LPL (p < 0.001), LDLR (p < 0.05), FAS (p < 0.001) expression was significantly reduced, and in the group treated with EB-AMDK19 dead bacteria, PPARγ (p < 0.001), CEBP (p < 0.001), aP2 (p < 0.001), CD36 (p < 0.001), ACC1 (p < 0.001), LPL (p < 0.001), LDLR (p < 0.01), FAS (p < 0.001) expression was significantly reduced. In addition, compared with the BAA-835 strain treatment group, the expression of PPARγ (p < 0.001), CEBPα (p < 0.001), CD36 (p < 0.01), LPL (p < 0.01) expression was also significantly reduced. That is, it was confirmed that the Akkermansia muciniphila EB-AMDK19 strain of the present invention has a superior adipogenic differentiation inhibitory effect as compared with 3T3-L1 cells.

[0154] Example 4: Confirmation of anti-obesity effect

[0155] 4.1. Animal model and sampling

[0156] Animal experiments were performed in accordance with the Animal Use and Care Protocol of the Institutional Animal Care and Use Committee (IACUC). Eight-week-old male C57BL mice (6 mice per group) were purchased as experimental animals. After acclimation for 1 week, the experimental animals were kept for 8 weeks. The temperature in the keeping environment was maintained at a specified temperature ( and relative humidity (40-60%)), and acclimatized for 1 week by adjusting light and dark in a 12-h cycle.

[0157] To induce obesity, animals were fed a high-fat diet (60 kcal% fat; Research Diets Inc, NJ, USA) and had free access to drinking water.

[0158] The experimental animals were divided into the following 5 groups:

[0159] - Experimental Group I (Normal): Normal diet control group

[0160] - Experimental group II (HFD): Obesity-induced group fed with a high-fat diet

[0161] - Experimental group II (GC): high-fat diet-fed obesity-induced group + Garcinia cambogia-administered group

[0162] - Experimental group IV (BAA-835) High-fat diet feeding induced obesity + Akkermansia muciniphila BAA-835 standard strain administration group.

[0163] -Experimental group V (EB-AMDK19) was a high-fat diet-fed obesity-inducing group + the Akkermansia muciniphila EB-AMDK19 strain (live bacteria) was administered.

[0164] In the case of experimental group V, after inducing obesity by high-fat diet, dead Akkermansia muciniphila EB-AMDK19 cells were added at a concentration of 1×10 8 CFU / 150 μl PBS (25% glycerol, 0.05% cysteine / PBS) concentration was orally administered every day.

[0165] Normal mice (Normal) were fed a 10% fat diet. Garcinia Cambogia (GC, 60% HCA 1000 mg / kg), a health functional food known to help reduce body fat, and Akkermansia muciniphila BAA-835 strain (live bacteria) were administered to the positive control group. At this time, in order to eliminate the effects of stress caused by administration, the normal group and the high-fat diet group were orally administered an equal amount of phosphate buffer (25% glycerol, 0.05% cysteine / PBS) every day.

[0166] 4.2. Changes in body weight and food intake

[0167] After 12 weeks of obesity induction, the changes in body weight, body fat, and tissue weight of each experimental group caused by the administration of Akkermansia muciniphila EB-AMDK19 strain were measured. The results are as follows Fig. 9 shown.

[0168] Reference Fig. 9 A slight decrease in body weight was also observed in experimental animals when Garcinia cambogia and Akkermansia muciniphila BAA-835 were administered, but a significant decrease in body weight could not be confirmed.

[0169] In contrast, the body weight was significantly reduced in the case of administration of Akkermansia muciniphila EB-AMDK19 compared with the high-fat diet group (HFD) (P < 0.05). Fig. 9Compared with the high-fat diet group, the food intake of the group administered with the Akkermansia muciniphila EB-AMDK19 strain of the present invention was significantly reduced. This result confirmed that the Akkermansia muciniphila EB-AMDK19 strain of the present invention induces sustained food intake and weight loss.

[0170] 4.3. Changes in tissue and body fat weight

[0171] When the experiment was over, the liver and spleen were removed under CO2 anesthesia, washed with saline, and weighed after removing water. Fig.10 shown.

[0172] Please refer to Fig.10 It was confirmed that all the administration groups showed a significant decrease compared with the high-fat diet group. This result indicates that the Akkermansia muciniphila strain of the present invention reduces fat accumulation and inflammation in spleen and liver cells, thereby contributing to the suppression of weight gain.

[0173] according to Fig.10 Compared with the normal group, the liver weight, spleen weight, subcutaneous fat, epididymal fat and mesenteric fat of the high-fat diet group (HFD) were significantly increased, while the liver weight, spleen weight, epididymal fat and mesenteric fat weight of the Akkermansia muciniphila EB-AMDK19 group were significantly suppressed. In particular, for subcutaneous fat, the body fat of the Akkermansia muciniphila EB-AMDK19 group was significantly reduced compared with the Garcinia cambogia and Akkermansia muciniphila BAA-835 groups (P < 0.01). For epididymal fat, compared with the high-fat diet group, body fat was significantly reduced only in the Akkermansia muciniphila EB-AMDK19 group (P < 0.05).

[0174] From these results, it can be seen that the administration of Akkermansia muciniphila EB-AMDK19 strain has a more excellent inhibitory effect on obesity induced by a high-fat diet than the administration of Garcinia cambogia and Akkermansia muciniphila BAA-835 strain.

[0175] In particular, it can be seen from these results that the Akkermansia muciniphila EB-AMDK19 strain has a strong effect on reducing body fat, which is known to cause complications such as cardiovascular diseases, rather than a simple effect on reducing obesity, and therefore has greater significance as an obesity therapeutic agent.

[0176] Example 5: Confirmation of therapeutic or preventive effects on metabolic diseases

[0177] 5.1. Oral glucose tolerance test (OGTT)

[0178] In order to confirm the effect of administration of Akkermansia muciniphila EB-AMDK19 strain on glucose tolerance, 12 weeks after the experiment, glucose (glucose 2g / kg) was orally administered to mice in a state of 18 hours of fasting. Blood was collected from the tail vein 30, 60, 90 and 120 minutes before and after the administration of glucose, and blood glucose was measured using a blood glucose meter. The results are as follows: Fig.11 shown.

[0179] according to Fig.11 , blood glucose did not decrease significantly in the group administered with Akkermansia muciniphila EB-AMDK19 strain before glucose administration, but blood glucose decreased the most in the administration group. 30 minutes after glucose administration, blood glucose decreased in all administration groups compared with the high-fat diet group (P < 0.001), and blood glucose in the Akkermansia muciniphila EB-AMDK19 strain administration group decreased significantly compared with other administration groups to a level that was not significant compared with the normal group. This research result indicates that oral administration of Akkermansia muciniphila EB-AMDK19 strain can alleviate obesity-induced hyperglycemia and improve glucose tolerance.

[0180] 5.2. Analysis of lipid biochemical indicators in blood

[0181] After fasting for 18 hours, blood was collected from each experimental animal, and the separated plasma was used to measure insulin, insulin resistance (HOMA-IR) index, total cholesterol (TC) as an indicator of lipid content, and glutamic pyruvic transaminase (GPT) as an indicator of liver function.

[0182] Specifically, the insulin concentration was measured using an insulin ELISA kit (Morinaga, Japan), and the insulin resistance index (HOMA-IR index) was calculated using the following formula.

[0183] Fasting insulin concentration (mU / L) × fasting blood glucose concentration (mmol / L) / 22.5

[0184] The total cholesterol (TC) and glutamic pyruvic transminase (GPT) concentrations as lipid composition indicators were quantified using the corresponding detection kits purchased from Asan Pharmaceutical Co., Ltd. (Korea). The results are shown in Fig.12 shown.

[0185] By confirming the results of insulin concentration, it was confirmed that the insulin and insulin resistance index (HOMA-IR) increased in the high-fat diet group were significantly reduced in all the treatment groups. For the cholesterol increased in the high-fat diet group, it was observed that the insulin resistance index was significantly reduced only in the group treated with the Akkermansia muciniphila EB-AMDK19 strain (P < 0.05). The GPT concentration, which indicates the degree of liver cell damage, was significantly reduced in all the treatment groups. The reduced concentrations of insulin, cholesterol, and GPT in the blood due to the administration of the Akkermansia muciniphila EB-AMDK19 strain are closely related to the weight loss effect.

[0186] 5.3. Relief of colon mucosal damage

[0187] 5.3.1. Histological evaluation of colonic mucosa

[0188] In order to confirm whether the Akkermansia muciniphila EB-AMDK19 strain of the present invention has a therapeutic effect on colon mucosal damage, the colon tissues of the high-fat diet group (HFD), the Garcinia cambogia administration group (GC), the Akkermansia muciniphila ATCC BAA-835 strain administration group (BAA-835) and the Akkermansia muciniphila EB-AMDK19 strain administration group (EB-AMDK19) of the present invention were stained with Alcian blue and photographed ( Fig.13 ). Based on these results, the results of numerically calculating the positive area (%) in each group are shown in a bar graph.

[0189] pass Fig.13 It was confirmed that the goblet cells and superficial epithelial tissue in the colon tissue of the high-fat diet group (HFD) disappeared, and a large number of immune cells also infiltrated into the colon tissue. In contrast, the structure of the mucosa layer in the colon tissue of the Akkermansia muciniphila EB-AMDK19 strain-administered group of the present invention was maintained, and a structure similar to that of the colon tissue of the normal group was observed. It was confirmed that the goblet cells of the colon mucosa that were reduced in the high-fat diet group (HFD) were significantly increased in the Akkermansia muciniphila EB-AMDK19 strain-administered group (p < 0.05).

[0190] 5.3.2. Evaluation of intestinal mucosal protein expression

[0191] In order to evaluate the effect of the strain of the present invention on the expression of immune-related hormones and tight junction intestinal mucosal proteins in the colon, real-time PCR was performed using the primers in Table 10 below and the same method as in Example 2.2. The results are shown in FIG. Fig.14 shown.

[0192] Table 10

[0193]

[0194] And, if Fig.14 As shown, compared with the increased expression of Toll-like receptor 2 (TLR2, p < 0.05), TLR4 (p < 0.05), IL-6 (p < 0.05), TNF-α (p < 0.01), and monocyte chemoattractant protein-1 (MCP-1, p < 0.05) as inflammatory mediators in the high-fat diet group, the expression of TLR2 (p < 0.05), TLR4 (p < 0.05), IL-6 (p < 0.05), TNF-α (p < 0.01), and MCP-1 (p < 0.05) was significantly reduced in the Akkermansia muciniphila EB-AMDK19 strain administration group. It was also observed that TLR2, TLR4, and TNF-α were significantly reduced in the Akkermansia muciniphila BAA-835 administration group. Compared with the high-fat diet group, IL-10 (p<0.05), an anti-inflammatory cytokine, was significantly increased only in the Akkermansia muciniphila EB-AMDK19 strain-administered group.

[0195] On the other hand, the expression of glucagon like peptide-1 (GLP-1), an appetite suppressant hormone, was significantly decreased in the high-fat diet group (p < 0.01), but significantly increased in the EB-AMDK19 strain-administered group compared with the high-fat diet group (p < 0.01). The expression of peptide YY (peptideYY, PYY), another appetite suppressant hormone, was significantly decreased in the high-fat diet group (p < 0.05), but significantly increased in the Garcinia cambogia (p < 0.05), Akkermansia muciniphila BAA-835 (p < 0.01) strain-administered group, and EB-AMDK19 (p < 0.01) strain-administered group compared with the high-fat diet group. The expression of zonular occludens-1 (ZO-1, p < 0.01) and occludin (p < 0.01), which are intestinal mucosal proteins, was significantly reduced in the high-fat diet group. In contrast, in the group administered with the Akkermansia muciniphila EB-AMDK19 strain of the present invention, the expression of ZO-1 (p < 0.05) was significantly increased, and the expression of tight junction-associated protein, which is a tight junction intestinal mucosal protein expressed in the colon mucosa and maintains intestinal permeability at an appropriate level, was increased. That is, it can be confirmed that the administration of the Akkermansia muciniphila EB-AMDK19 strain of the present invention provides the effect of alleviating inflammation of the colon mucosa caused by obesity and improving intestinal permeability.

[0196] 5.4. Improvement of liver damage and fatty disease

[0197] The visceral adipose tissue of the mice in each experimental group was taken out and weighed. A portion of the visceral adipose tissue was cut and immersed in 10% buffered formalin. Then, after fixing in paraffin, the adipose tissue was sliced ​​into 4 mm thick slices using a microtome (Reichert-Jung 2050, ALT, United States (USA)), placed on a slide, and then stained with H&E (Hematoxylin&Eosin) and ORO (Oil Red O) staining similar to Example 3.1, and the shape and state of the hepatocytes and lipid droplets were observed under an optical microscope at a magnification of 200 to 400 times. The results are as follows. Figure 15-16 shown.

[0198] Reference Figure 15-16, the worsened steatosis (p < 0.05) and increased lipid droplets (p < 0.001) in the liver of the high-fat diet group were significantly reduced in the Akkermansia muciniphila EB-AMDK19 strain-administered group (p < 0.01, p < 0.001), respectively. It was also observed that the reduction rate of lipid droplets was more significant compared with the Garcinia cambogia and Akkermansia muciniphila BAA-835 strain-administered groups, and therefore, the improvement phenomenon was the most obvious.

[0199] 5.5. Changes in adipocyte size

[0200] Mesenteric adipose tissue of mice in each experimental group was isolated and analyzed by H&E staining. Fig.17 See Fig.17 , the size of adipocytes in the high-fat diet administration group increased compared with the normal group (p < 0.001). In contrast, a decrease in size was observed in the Garcinia cambogia, Akkermansia muciniphila BAA-835, and Akkermansia muciniphila EB-AMDK19 strain administration groups compared with the high-fat diet administration group (p < 0.001). In particular, the size of adipocytes in the Akkermansia muciniphila EB-AMDK19 strain administration group of the present invention was more significantly reduced compared with the Garcinia cambogia and Akkermansia muciniphila BAA-835 strain administration groups as the standard strain.

[0201] Example 6: Analysis of microbial flora in feces

[0202] 6.1. Changes in microbial diversity

[0203] In order to analyze intestinal microorganisms, the feces of mice in each experimental group were taken, and genomic DNA was isolated using QIAamp DNA stool mini kit (QIAamp DNA stool mini kit, Qiagen, USA). In order to confirm the genomic DNA results of the extracted microorganisms, the position of the band was confirmed by electrophoresis at 100V for 25 minutes in 1XTAE buffer and 1.5% agarose gel (QA-Agarose, Q-biogene, USA). The purity of DNA was measured at A260 / A280 using a spectrophotometer.

[0204] In order to amplify specific parts of variable region 3 of 16S rDNA using microbial DNA isolated from feces, specially prepared barcode primers were used and amplified by PCR process. The amplified PCR products were sequenced using the Ion Torrent Next-Generation sequencing platform (Ion PGM, Life Technologies, USA), which is a next-generation base sequence analysis method (NGS), and the microbial flora was analyzed. The result values ​​with low quality or too small readings in the obtained microbial sequences were removed, entered into the Greengenes database, and assigned OUT (operational taxonomic units).

[0205] Next, the microbial flora between clusters was analyzed using Quantitative insights into microbial ecology (QIIME) 1.9.0, a tool for integrated analysis of microbiome data. In order to compare the α-diversity of each microbial administration group, PCA analysis was performed based on the results of the normal group, high-fat diet group, Akkermansia muciniphila BAA-835 administration group, and Akkermansia muciniphila EB-AMDK19 administration group. α-Diversity was presented by OTUs and Chao1 analysis, and β-diversity was confirmed by UniFracbased principal coordinates analysis (PCoA) analysis. Linear discriminant analysis effect size (LEfSe) analysis was performed using an online program (http: / / huttenhower.sph.harvard.edu / galaxy).

[0206] In the analysis results, there was a difference in α-diversity between the normal group and the high-fat diet group ( Fig.18 There were some differences in diversity between the group treated with Akkermansia muciniphila EB-AMDK19 and the normal group, the high-fat diet group, and the group treated with Akkermansia muciniphila BAA-835.

[0207] 6.2. Linear discriminant analysis (LDA) score

[0208] In order to compare the β-diversity between the experimental groups, the LDA scores of the microorganisms with more differences in each group were analyzed at the order and family levels. Fig.18 , Fig.19). As a result, compared with other groups, Muribaculaceae, Anaeroplasmataceae, Clostridiaceae, etc. were abundant in the normal group. Compared with other groups, Lactobacillales, Prevotellaceae, Streptococcaceae, and Lactobacillaceae increased significantly in the high-fat diet group, and Marinifilaceae, etc. increased significantly in the Akkermansia muciniphila BAA-835 strain group compared with other groups. Clostridiales, Saccharimonadales, Mycoplasmatales, Ruminococcaceae, Saccharimonadaceae, and Mycoplasmataceae increased relatively significantly in the Akkermansia muciniphila EB-AMDK19 strain group. The results of relative abundance analysis of microorganisms showed that compared with the normal group, changes occurred in the high-fat diet group. At the order level, Lactobacillales recovered from the EB-AMDK19 strain-treated group to the level of normal group microorganisms, and at the family level, Rikenellaceae, Prevotellaceae, and Streptococcaceae recovered from the EB-AMDK19 strain-treated group to the level of normal group microorganisms.

[0209] Based on the above results, it can be confirmed that the strain and pharmaceutical composition of the present invention can be effectively used to improve carbohydrate and lipid metabolism to treat obesity.

[0210] The specific embodiments described in this specification are only used to illustrate the preferred embodiments of the present invention and should not be construed as limiting the present invention. It is obvious to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit and scope of the present invention. The protection scope of the present invention shall be defined by the protection scope of the attached invention claims, and the above-mentioned various modifications and variations shall be included in the protection scope of the present invention.

[0211] Accession number

[0212] Name of depository institution: Korea Institute of Biotechnology

[0213] Accession number: KCTC13761BP

[0214] Collection date: 20181205

[0215]

Claims

1. A muciniphilic Akkermansia Akkermansia muciniphila ) EB-AMDK19 strain, characterized in that The deposit number is KCTC13761BP.

2. A pharmaceutical composition for preventing or treating inflammatory diseases or metabolic diseases, characterized in that: Comprising the strain described in claim 1.

3. The pharmaceutical composition for preventing or treating inflammatory diseases or metabolic diseases according to claim 2, characterized in that: The composition comprises the probiotics of the strain or comprises the strain sterilized at low temperature.

4. The pharmaceutical composition for preventing or treating inflammatory diseases or metabolic diseases according to claim 2, characterized in that: The pharmaceutical composition is used for preventing or treating inflammatory bowel disease, insulin resistance, dyslipidemia, or for reducing cholesterol or body weight.

5. The pharmaceutical composition for preventing or treating inflammatory diseases or metabolic diseases according to claim 2, characterized in that: Each 1g of the above composition is 10 8 CFU to 10 12 The CFU content contains the Akkermansia muciniphila EB-AMDK19 strain as an effective component, or contains a culture having an equivalent amount of live bacteria or low-temperature sterilized bodies.

6. The pharmaceutical composition for preventing or treating inflammatory diseases or metabolic diseases according to claim 2, characterized in that: The above composition further comprises a pharmaceutically acceptable carrier or excipient.

7. A composition for suppressing appetite, characterized in that The invention comprises the Akkermansia muciniphila EB-AMDK19 strain with the deposit number KCTC13761BP as described in claim 1.

8. A composition for increasing the secretion of glucagon-like peptide-1 and peptide YY as appetite suppressant hormones, characterized in that: The invention comprises the Akkermansia muciniphila EB-AMDK19 strain with the deposit number KCTC13761BP as described in claim 1.

9. A food, characterized in that The invention comprises the Akkermansia muciniphila EB-AMDK19 strain with the deposit number KCTC13761BP as described in claim 1.

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