Compositions comprising ackermania strains

Abstract

The present invention relates to a pharmaceutical composition, a food composition, a veterinary composition, and a cosmetic composition, which are effective in preventing or treating metabolic diseases, and which comprise an ackermania marxianus strain, a culture or a dried product thereof. The traditional probiotics generally have a very small treatment effect on metabolic diseases, and compared with the traditional probiotics, the new generation of medicinal probiotic strain disclosed by the invention has an excellent prevention or treatment effect on the metabolic diseases, and can be used as a novel prevention and treatment tool.

Description

Technical Field

[0001] The present invention relates to a composition comprising a strain of Akkermansia sp., and more specifically, to a composition comprising a strain of Akkermansia massiliensis. Background Technology

[0002] Metabolic diseases are caused by chronic metabolic disorders that lead to multiple conditions simultaneously, such as obesity, diabetes, hypertension, hyperlipidemia, coronary artery disease or arteriosclerosis, and non-alcoholic fatty liver disease. Most metabolic diseases are associated with overweight or obesity. The most serious problem with metabolic diseases is the development of chronic complications such as diabetic retinopathy, diabetic nephropathy, diabetic foot, diabetic neuropathy, hyperlipidemia, and cardiovascular disease. Most of these chronic complications, once they occur, undergo an irreversible process, and currently there is no way to completely stop this process. Without proper treatment, they can lead to severe symptoms, and are therefore considered one of the most serious threats to the health of modern people.

[0003] Currently, treating metabolic diseases with these complex symptoms requires the separate administration of medications such as hypoglycemic agents, antihypertensive drugs, and cholesterol-lowering agents. Therefore, there is a need to develop novel therapeutic agents that can simultaneously treat various symptoms to effectively manage and treat metabolic diseases with these complex symptoms.

[0004] Studies are currently underway to use probiotics alone or in combination with therapeutic agents to overcome the side effects caused by existing drugs or immunomodulators. With active research into the role of gut bacteria in promoting health, interest in probiotic formulations is also increasing.

[0005] As a treatment technology for metabolic diseases using probiotics, Korean Patent Publication No. 2011-0095929 discloses Lactobacillus reuteri, which hinders the absorption of lipids in the body; Korean Patent Publication No. 0996577 discloses Lactobacillus curvatus, which lowers blood cholesterol and inhibits obesity; and Korean Patent Publication No. 2010-0010015 discloses Lactobacillus johnsonii, which lowers blood cholesterol and inhibits obesity.

[0006] However, as mentioned above, probiotics have little effect on improving metabolic diseases, thus limiting their use as a new tool for prevention and treatment. Summary of the Invention

[0007] Technical issues

[0008] The present invention aims to overcome the limitations of the prior art. One object of the present invention is to provide a pharmaceutical composition that effectively prevents or treats metabolic diseases, comprising Akkermansia masiliforme strain.

[0009] Another object of the present invention is to provide a food composition that helps prevent or improve metabolic diseases, comprising a strain of Akkermansia masiliforme.

[0010] Another object of the present invention is to provide a veterinary drug composition or feed additive that helps prevent or improve metabolic diseases, comprising a strain of Akkermansia masiliforme.

[0011] Another object of the present invention is to provide a cosmetic composition that helps prevent or improve metabolic diseases, comprising a strain of Akkermansia masiliforme.

[0012] Technical solution

[0013] One embodiment of the present invention relates to a strain of Akkermansia massiliensis.

[0014] Another embodiment of the invention relates to a composition comprising a strain of Akkermansia masiliforme, a culture of the strain, or a dried product thereof.

[0015] Another embodiment of the present invention relates to a food composition that helps prevent or improve metabolic diseases, comprising a strain of Akkermansia masiliforme.

[0016] Another embodiment of the present invention relates to a veterinary composition or feed additive that helps prevent or improve metabolic diseases, comprising a strain of Akkermansia masiliforme.

[0017] Another embodiment of the present invention relates to a cosmetic composition that helps prevent or improve metabolic diseases, comprising a strain of Akkermansia masiliforme.

[0018] The effects of the invention

[0019] Pharmaceutical compositions containing the *Akermansia masiliforme* strain of the present invention can be used as pharmaceutical compositions for treating and / or preventing metabolic diseases, health food compositions, feed compositions, cosmetic compositions, etc.

[0020] Pharmaceutical compositions containing the *Akermansia masiliforme* strain of the present invention provide effective management and treatment of complex symptoms of metabolic diseases (metabolic syndrome) associated with these conditions by inhibiting weight gain and body fat gain, reducing insulin resistance, and lowering total cholesterol levels in the blood.

[0021] The *Akermansia masiliforme* strain of the present invention, as a new generation of medicinal probiotic strain, has excellent preventive or therapeutic effects on metabolic diseases and can be used as a new preventive and therapeutic tool. Attached Figure Description

[0022] Figure 1 The microscopic observation results of the *Ackermania massoris* strain (KCTC 13765BP) and the *Ackermania tumefaciens* strain ATCC BAA-835 of the present invention are shown.

[0023] Figure 2 The PCR analysis results of Akkermansia massoris strain EB-AMDK39 and Akkermansia ATCCBAA-835 of the present invention are shown.

[0024] Figures 3a to 3b The results of random amplified polymorphic DNA (RAPD) analysis of the genomic DNA of Akkermanella massoris strain EB-AMDK39 and Akkermanella ATCC BAA-835 strain of the present invention are shown.

[0025] Figure 4 The phylogenetic relationship between the *Ackermania massoris* strain of the present invention and other *Ackermania* strains is shown by comparison.

[0026] Figure 5 The results show the comparison of the presence of genes in the full-length genomes of Akkermansia masilionis strain EB-AMDK39 and Akkermansia ATCC BAA-835 strain based on the present invention.

[0027] Figure 6 The cytotoxicity test results of the *Ackermania massoris* strain EB-AMDK39 and the *Ackermania ts.* strain ATCC BAA-835 of the present invention are shown in comparison.

[0028] Figure 7 shows the results confirming whether the *Ackermania massoris* strain EB-AMDK39 and the *Ackermania ts.* strain ATCC BAA-835 of the present invention have hemolytic activity.

[0029] Figure 8 The results of analyzing the changes in body weight in the treatment groups of Akkermansia masiliforme EB-AMDK39, the negative control group (HFD), and the treatment group of Akkermansia ATCC BAA-835 strain of the present invention are shown.

[0030] Figure 9The results show the changes in subcutaneous fat, epididymal fat, and upper mesenteric fat in the treatment group of Akkermansia masiliforme EB-AMDK39 strain, the negative control group (HFD), and the treatment group of Akkermansia ATCC BAA-835 strain of the present invention.

[0031] Figure 10 The results show the changes in blood cholesterol and serum triglyceride (TG) concentrations in the treatment group, the negative control group (HFD), and the treatment group of Akkermansia massoris strain EB-AMDK39 of the present invention, as determined by ELISA.

[0032] Figure 11 Images and graphs are provided to illustrate the size of adipocytes and the degree of fat accumulation in the mesenteric adipose tissue of animals in each experimental group when treated with the Massiris Akkermansia EB-AMDK39 strain of the present invention. Detailed Implementation

[0033] The present invention will now be described in more detail with reference to the accompanying drawings.

[0034] In this invention, the "Akkermansia sp. strain" is a strain of the genus Akkermansia, belonging to the phylum Verrucomicrobiota. The Akkermansia sp. strain of this invention is not Akkermansia muciniphila or Akkermansia glycaniphila.

[0035] In this instruction manual, "metabolic disease" refers to the overall symptoms of conditions such as obesity, diabetes, hypertension, hyperlipidemia, coronary artery disease or atherosclerosis, and non-alcoholic fatty liver disease. In this instruction manual, "metabolic syndrome," "metabolic disease," or "metabolic disorder or abnormality" are used interchangeably.

[0036] The term "obesity" as used in this manual refers to a state of excessive body fat accumulation. Obesity is defined as body fat exceeding 25% of body weight in men and 30-35% of body weight in women. The Body Mass Index (BMI) is widely used as a standard measurement method. In Western cultures, a BMI greater than 30 kg / m² is considered obese. 2 Obesity is defined as a body mass index (BMI) of 25–30 kg / m². 2 Overweight is defined as a body mass index (BMI) greater than 28 kg / m² in the case of Asians.2 Obesity is defined as a body mass index (BMI) of 23–28 kg / m². 2 Overweight is defined as being overweight.

[0037] As used in this specification, the term "dyslipidemia" can refer to a state of increased lipid components in the blood, such as triglycerides, LDL cholesterol, phospholipids, and free fatty acids, or a state of decreased HDL cholesterol. For example, dyslipidemia can be any one of the following groups: hyperlipidemia, hyperLDL cholesterolemia, hypertriglyceridemia, and hypoHDL cholesterolemia.

[0038] As used in this specification, the term "subject" refers to all animals, including humans, that have or may have a metabolic disease. The term "animal" includes not only humans but also, but is not limited to, mammals such as dogs, cats, hamsters, rabbits, cattle, horses, sheep, pigs, goats, camels, and antelopes that require treatment for similar symptoms.

[0039] When “approximately” precedes a number, it indicates ±20%, preferably 10% of the value.

[0040] Akkermansia can also be substantially purified. As used herein, "substantially purified" means that a particular bacterial strain or two or more bacterial strains (e.g., Bacteroidetes, Firmicutes, Proteobacteria, or Verrucomicrobia) in a sample are substantially enriched. A sample can be substantially purified or concentrated to the bacterial strain or mixture of strains of interest. In this case, the proportion of that bacterial strain in the sample is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% or more, or the proportion of undesirable or other bacterial strains is about 40%, 30%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less.

[0041] In this specification, the *Akermansia masiliformes* strain may be included in the form of live bacteria, low-temperature sterilized strains, or inactivated bacteria. In this specification, "low-temperature sterilized strain" refers to a *Akermansia masiliformes* strain subjected to heat treatment at a low temperature. In one embodiment, a low-temperature sterilized *Akermansia masiliformes* strain refers to a *Akermansia masiliformes* strain heat-treated at a temperature of 50°C to 110°C for 5 minutes to 30 minutes. In one embodiment, low-temperature sterilization is performed within the range of approximately 50°C to approximately 110°C, approximately 50°C to approximately 100°C, preferably approximately 60°C to approximately 95°C, and more preferably approximately 70°C to approximately 90°C. Preferably, the bacteria to be sterilized at low temperatures are heat-treated at temperatures of approximately 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, or 70°C. In another embodiment, the low-temperature sterilization is performed at temperatures of approximately 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, or 100°C. In another embodiment, low-temperature sterilization involves heating at temperatures of 101°C, 102°C, 103°C, 104°C, 105°C, 106°C, 107°C, 108°C, 109°C, or 110°C.

[0042] In this invention, "inactivated bacterial cells" refers to a form of bacteria whose growth is inhibited by heat treatment or other methods. Inactivated bacterial cells may include antibacterial active substances such as cytoplasm, cell wall, and bacteriocin, as well as polysaccharides and / or organic acids.

[0043] In this specification, the term "culture" refers to a product obtained by culturing a strain of *Akermansia masiliformes*, which may include fermentation products. In one embodiment, the culture may be a fermentation product of culturing a strain of *Akermansia masiliformes* in a culture medium. The term "fermentation product" refers to a product obtained by enzymatic or metabolic decomposition of organic matter using microorganisms. In this invention, "fermentation" can refer to all activities or processes involving microorganisms on organic matter, including enzymatic or metabolic decomposition, other than putrefaction.

[0044] The culture (or fermentation product) may be a whole culture of *Akermansia masiliii* strain, its dilution, concentrate, dried product, freeze-dried product, pulverized product, and / or fractions. The concentrate can be obtained by centrifuging or evaporating the culture, and the dried product can be obtained by drying the culture using a dryer or similar method. Freeze-drying can be obtained by freeze-drying the culture using a freeze dryer or similar method, and the pulverized product can be obtained by physically or ultrasonically treating the strain or culture. The fractions can be obtained by applying centrifugation, chromatography, or other methods to the culture or pulverized product.

[0045] The culture or fermentation product can be in a solid (solid, such as dried material), liquid (liquid) or fluidized state, but is not necessarily limited to these.

[0046] In this specification, the terms “treatment,” “therapeutic,” “for treatment,” etc., refer to preventing, slowing, stopping, or reversing the development or severity of an existing condition, disease, disorder, or symptom.

[0047] In this specification, "functional food" is the same term as "food for special health use" (FoSHU), referring to a processed food composition with high medical or therapeutic effects, which effectively exerts biological regulatory functions in addition to providing nutrition. In this specification, "functional food" may be used interchangeably with terms such as "health supplement food" and "health food."

[0048] One embodiment of the present invention relates to a pharmaceutical composition for the prevention or treatment of metabolic diseases, comprising a strain of Akkermansia masiliforme with accession number KCTC 13765BP.

[0049] The *Akermansia masiliforme* strain of this invention was isolated from the feces of healthy Koreans. It is an oval-celled monococcus or diplococcus, 0.5-1 μm in size, and is an anaerobic, non-motile, Gram-negative, mucin-decomposing bacterium that does not form endospores. This *Akermansia masiliforme* strain produces a variety of mucin-decomposing enzymes, allowing it to use mucus as a carbon and nitrogen source. It can also metabolize some carbon sources by including N-acetylglucosamine and produce short-chain fatty acids such as propionic acid and acetic acid as its main metabolites.

[0050] The *Akermansia masilis* strain of the present invention possesses the 16S rRNA gene of SEQ ID NO:1.

[0051] In this invention, the Akkermansia massiri strain is not an Akkermansia muciniphila or Akkermansia glycaniphila strain. Genome-based phylogenetic analysis and mean nucleotide identity (ANI) value analysis definitively confirmed that the Akkermansia massiri strain of this invention is not an Akkermansia muciniphila or Akkermansia glycaniphila strain.

[0052] Compared to the ATCC BAA-835 strain, which serves as the standard Akkermansia strain, the *Akkermansia masilioni* strain of this invention has a genome-wide average nucleotide identity (gANI) of less than about 95%. In this invention, the gANI of the *Akkermansia masilioni* strain is less than 95% compared to the genome of *Akkermansia myxophilus*, for example, about 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, and 79%. In some embodiments, the gANI of the Akkermansia massoris strain (e.g., Akkermansia massoris strain KCTC 13765BP) of the composition of the present invention may be less than 95% compared to the genome of Akkermansia masiliophila, for example, about 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, or 71%.

[0053] The *Akermansia masiliforme* strain of the present invention inhibits weight gain and body fat gain, reduces insulin resistance, lowers total cholesterol concentration in the blood, and reduces the level of glutamic pyruvic transaminase (GPT), an indicator of hepatotoxicity in the blood. Therefore, it can be effectively used to prevent or treat related diabetes, obesity, obesity-related diseases, insulin resistance, fatty liver, hyperlipidemia, or metabolic diseases.

[0054] The pharmaceutical compositions of the present invention may comprise live cells, cryogenically sterilized strains, or inactivated cells of the *Akkermansia masiliformes* strain. Cryogenic sterilization of *Akkermansia masiliformes* strains refers to heating at a temperature above 50°C and below 110°C for at least 5 minutes. For example, cryogenic sterilization can be performed at 70°C for 30 minutes. The cryogenically sterilized form of *Akkermansia masiliformes* strain EB-AMDK39 is more effective at reducing fat accumulation in the body than the live cells. The reason why the cryogenically sterilized form of *Akkermansia masiliformes* strain is more effective is not yet clear, but it can be speculated that cell wall components or membrane proteins of *Akkermansia masiliformes* strains, such as Amuc_1100, enhance the metabolic benefits to the host during cryogenic sterilization.

[0055] The beneficial effects of the strains of the present invention are presumed to be related to a gene cluster (Amuc_1098-Amuc_1102) including Amuc_1100, which is present on the surface of intestinal mucosal epithelial cells and immune cells located near the intestinal wall. This cluster is derived from a polypeptide that interacts with the Toll-like receptor 2 (TLR2) signaling pathway, which regulates intestinal homeostasis and host metabolism. For example, the Amuc_1100 polypeptide is expected to maintain the integrity of the intestinal mucosal barrier and regulate or promote the TRL2-signaling pathway by interacting with TLR2 present on the surface of immune cells, thereby promoting the secretion of cytokines (e.g., IL-6, IL-8, and IL-10) from immune cells. The prolipoprotein diglyceride transferase gene (Amuc_1104) is located very close to the gene cluster (Amuc_1098-Amuc_1102). Furthermore, Amuc_1100 remains stable under the temperature conditions used during low-temperature sterilization, thus potentially contributing to the effectiveness of low-temperature sterilized strains.

[0056] The *Akermansia masiliforme* strain of the present invention can be recovered after cultivation by separation processes such as centrifugation, and prepared into live bacterial form by drying processes such as freeze-drying for use.

[0057] The *Akermansia masilis* strain of the present invention is sensitive to oxygen, and therefore is preferably cultured under anaerobic conditions (80-90% nitrogen saturation, 0-5% hydrogen saturation, and 5-20% carbon dioxide saturation).

[0058] The composition of the liquid culture medium during cultivation can affect the growth of the strain and the formation of its active ingredients. Therefore, it is necessary to establish the optimal composition and content conditions of the liquid culture medium for culturing the Akkermansia masiliforme strain of this invention.

[0059] The liquid culture medium may contain one or more carbon sources selected from the group consisting of glucose, lactose, maltose, fructose, galactose, N-acetylglucosamine, mannose, 1-fucose, lactic acid, formic acid, acetic acid, propionic acid, 1,2-propanediol, and butyric acid, but is not limited thereto. Preferably, it may contain glucose and N-acetylglucosamine. The liquid culture medium may contain one or more nitrogen sources selected from the group consisting of tryptone, peptone, soy peptone, L-glutamic acid, and ammonium, but is not limited thereto.

[0060] The liquid culture medium may contain one or more trace elements 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 thereto.

[0061] The pH of the liquid culture medium can be 6.8–7.2. Preferably, the pH is 7.0. pH can affect protein activity by altering the charge of the amino or carboxyl groups of amino acids, which are the units of enzyme proteins important for cellular metabolism. Furthermore, under external environmental conditions, changes in pH affect the ionization of microbial nutrients, thereby influencing the absorption of nutrients by microorganisms.

[0062] Preferably, the liquid culture medium is cultured in a medium containing glucose, N-acetylglucosamine, threonine, soybean peptone, or any combination thereof.

[0063] Drug formulation can be carried out by known methods, preferably in pharmaceutically acceptable oral, topical, transdermal, transmucosal, and injectable dosage forms, and more preferably in oral dosage forms.

[0064] In addition to the active ingredients, the compositions of the present invention may also contain pharmaceutically acceptable carriers and / or excipients, and may be formulated with various pharmaceutically commonly used additives such as binders, disintegrants, coating agents, and lubricants.

[0065] The pharmaceutical compositions of the present invention can be formulated into powder, granules, tablets, capsules, or liquid forms by mixing the *Akermansia masiliforme* strain of the present invention with suitable carriers, excipients, and auxiliary active ingredients. The compositions of the present invention can be formulated into products for oral or enteral administration. Furthermore, the compositions of the present invention can be prepared into enteric-coated products using known methods, allowing the composition to pass through the gastrointestinal tract and reach the small intestine, rapidly releasing the microorganisms as active ingredients into the intestine.

[0066] The excipients that can be used in this invention include sugars, such as sucrose, lactose, mannitol, and glucose; and starches, such as corn starch, potato starch, rice starch, and partially gelatinized starch. The binders include polysaccharides, such as dextrin, sodium alginate, carrageenan, guar gum, gum arabic, and agar; naturally occurring macromolecules, such as tragacanth gum, gelatin, and gluten; cellulose derivatives, such as hydroxypropyl cellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, hydroxypropyl ethylcellulose, and sodium carboxymethyl cellulose; and polymers, such as polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, polyethylene glycol, polyacrylic acid, polymethacrylic acid, and vinyl acetate resin.

[0067] Cellulose derivatives such as carboxymethyl cellulose, calcium carboxymethyl cellulose, and low-substituted hydroxypropyl cellulose, as well as starches such as sodium carboxymethyl starch, hydroxypropyl starch, corn starch, potato starch, rice starch, and partially gelatinized starch can be used as disintegrants.

[0068] Examples of lubricants that can be used in this invention include talc, stearic acid, calcium stearate, magnesium stearate, colloidal silica, hydrogenated silica, various waxes and hydrogenated oils.

[0069] Coating agents include water-insoluble polymers, such as dimethylaminoethyl methacrylate-methacrylic acid copolymer, polyvinyl acetal diethylaminoacetate, ethyl acrylate-methacrylic acid copolymer, ethyl acrylate-methyl methacrylate-trimethylammonium chloromethacrylate ethyl acrylate copolymer, ethyl cellulose, etc.; enteric polymers, such as methacrylic acid-ethyl acrylate copolymer, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate, etc.; and water-soluble polymers, such as methylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, polyethylene glycol, etc., but are not necessarily limited to these.

[0070] In the compositions of the present invention for the prevention or treatment of metabolic diseases, the dosage of the strain as the active ingredient can be determined taking into account factors including the type of disease, age, weight, sex, patient's condition, severity of condition, and route of administration. Therefore, the dosage therapy can vary considerably, but can be routinely determined using standard methods.

[0071] The pharmaceutical composition of the present invention may contain 10, relative to the total weight of the composition. 2 CFU to 10 15 CFU contains either a strain of *Akermansia masiliforme* as the active ingredient or a culture containing an equivalent amount of live bacteria.

[0072] In the case of adult patients, 1×10 can usually be administered as needed, either once or in multiple divided doses. 2 The above-mentioned live bacteria, low-temperature sterilized strains, or inactivated bacteria are preferably 1×10⁶. 2 Up to 1×10 15 The bacteria may be live, low-temperature sterilized, or inactivated. The specific dosage form, route of administration, and dosage of the pharmaceutical compositions disclosed in this invention can be determined by medical personnel considering the patient's condition.

[0073] In one embodiment of the present invention, the composition of the present invention contains approximately 1 × 10⁻⁶ Akkermansia strains. 2 To approximately 1×10 15 Cells / g composition, approximately 1×10 3To approximately 1×10 14 Cells / g composition, preferably about 1×10⁻⁶ 4 To approximately 1×10 13 Cells / g composition, more preferably about 1×10⁻⁶ cells / g composition, 5 To approximately 1×10 12 More preferably about 1 × 10⁻⁶ cells / g composition, 6 To approximately 1×10 11 Cells / g composition, approximately 1×10 7 To approximately 1×10 10 Cells / g composition, approximately 1×10 8 To approximately 1×10 12 Cells / g composition.

[0074] In one embodiment of the present invention, the composition of the present invention contains approximately 1 × 10⁻⁶ Akkermansia strains. 2 To approximately 1×10 15 Cells / mL composition, approximately 1×10 3 To approximately 1×10 14 The composition of cells / mL is preferably about 1×10⁻⁶. 4 To approximately 1×10 13 Cells / mL composition, more preferably about 1×10⁻⁶ cells / mL composition, 5 To approximately 1×10 12 A composition of cells / mL is more preferably about 1×10⁻⁶ cells / mL. 6 To approximately 1×10 11 Cells / mL composition, approximately 1×10 7 To approximately 1×10 10 Cells / mL composition, approximately 1×10 8 To approximately 1×10 12 Cells / mL composition.

[0075] In another embodiment of the present invention, the content of *Akermansia masiliforme* strain in the composition of the present invention is about 1 × 10⁻⁶. 6 To approximately 1×10 10 The composition is cells / g or cells / mL, preferably about 1×10⁻⁶. 8 To approximately 1×10 10 The composition is expressed as cells / g or cells / mL, more preferably about 1×10⁻⁶. 9 To approximately 1×10 10 Composition of cells / g or cells / mL.

[0076] In one embodiment of the present invention, in addition to the *Masiliacobacterium masiliacus* strain, the composition of the present invention may also contain other probiotic strains or one or more prebiotics.

[0077] Other probiotic strains may include probiotics belonging to the phyla Bacteroidetes, Firmicutes, Actinobacteria, or Proteobacteria. In one embodiment, other probiotics may be Ruminococcus, Clostridium, Bacteroides, Neglecta, Bifidobacterium, Egerthella, Clostridiaceae, Parabacteroides, Bilophila, Dorea, Collinsella, and Faecalibacterium.

[0078] Examples of prebiotics that may be used in this invention may include, but are not limited to, inulin and inulin-like fructans, fructooligosaccharides, β-glucan, xylose, arabinose, arabinoxylan, ribose, galactose, rhamnose, cellobiose, fructose, lactose, salicin, sucrose, glucose, aescin, trehalose, maltose, mannose, molasses, mucin or mucin, raffinose, fructooligosaccharides, galactooligosaccharides, amino acids, alcohols, fermentable carbohydrates, and any combination thereof.

[0079] Another embodiment of the present invention relates to a food composition comprising a strain of Akkermansia masiliforme, a culture thereof, or a dried product thereof. The food composition of the present invention may contain a strain of Akkermansia masiliforme, a culture thereof, or a dried product of the strain as an active ingredient.

[0080] The food composition of the present invention can be prepared into functional beverages and other health-functional foods, health-aid foods, and special nutritional supplements. The food forms may include beverages, such as tea, fruit juice, carbonated drinks, electrolyte drinks, etc.; processed dairy products, such as milk, yogurt, etc.; food products, such as chewing gum, rice cakes, Korean sweets, bread, biscuits, noodles, etc.; and health-functional food preparations, such as powders, tablets, capsules, etc.

[0081] In addition to the active ingredient, the food composition of the present invention may contain sweeteners, flavoring agents, physiologically active ingredients, minerals, etc.

[0082] Sweeteners can be natural or synthetic. Natural sweeteners can include sugar sweeteners, such as corn syrup solids, honey, sucrose, fructose, lactose, maltose, etc.

[0083] Flavorings can be natural or synthetic. Preferably, natural flavorings are used. Natural flavorings can be derived from apples, lemons, citrus fruits, grapes, strawberries, peaches, or from green tea leaves, Solomon's seal rhizome, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. Furthermore, flavorings derived from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo, etc., can also be used. Synthetic flavorings can be esters, alcohols, aldehydes, and terpenes, etc.

[0084] Physiologically active substances can include catechins, such as catechin, epicatechin, gallocatechin, and epigallocatechin; or vitamins, such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, and riboflavin.

[0085] Minerals that can be used include calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc, etc.

[0086] In addition to the sweeteners and the like, the food compositions of the present invention may include preservatives, emulsifiers, acidulants, thickeners, etc., as needed. These preservatives, emulsifiers, etc., are known in the art, and any known in the art may be used.

[0087] Another embodiment of the present invention can be a cosmetic composition comprising the Akkermansia massoris strain as an active ingredient, preferably Akkermansia massoris strain EB-AMDK39 (accession number KCTC13765BP), a culture of the strain, or a dried product of the strain.

[0088] The cosmetic compositions of the present invention can be prepared in various forms according to conventional cosmetic composition preparation methods. Specifically, the cosmetic compositions of the present invention can be prepared into dosage forms selected from the group consisting of solutions, topical ointments, creams, soothing gels, foams, nourishing lotions, moisturizing lotions, masks, moisturizing lotions, shower gels, lotions, makeup primers, serums, soaps, liquid detergents, bath products, sunscreens, sunscreen oils, suspensions, emulsions, creams, gels, moisturizing lotions, powders, soaps, foaming cleansers, oils, powder foundations, lotion foundations, wax foundations, patches, and sprays, but are not necessarily limited thereto.

[0089] Furthermore, the cosmetic composition may contain conventional additives commonly used in the field of cosmetic compositions, such as stabilizers, solubilizers, vitamins, pigments, and fragrances, and may contain a cosmetically acceptable carrier.

[0090] Acceptable carriers for cosmetics include, but are not limited to, purified water, oils, waxes, fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickeners, viscosity stabilizers, chelating agents, buffers, preservatives, and lower alcohols. Depending on the requirements, they may also include moisturizers, anti-inflammatory agents, antibacterial agents, antifungal agents, vitamins, UV shielding agents, antibiotics, perfumes, and dyes.

[0091] Another embodiment of the present invention provides a veterinary drug composition or feed additive for the prevention or treatment of metabolic diseases, comprising a strain of Akkermansia masiliformes (accession number: KCTC 13765BP) or a culture thereof. The Akkermansia masiliformes strain (accession number: KCTC 13765BP) is as described above.

[0092] The veterinary drug composition or feed additive for the prevention or treatment of metabolic diseases can be prepared by adding Akkermansia masiliforme strain (accession number: KCTC 13765BP) within an appropriate effective concentration range according to various feed preparation methods known in the art.

[0093] The present invention will now be described in detail through embodiments. However, the following embodiments are merely examples of the present invention, and the scope of the present invention is not limited to the following embodiments.

[0094] Example

[0095] Example 1: Isolation and identification of Akkermansia masiliii strain EB-AMDK39

[0096] 1.1. Isolation and Identification of Strains

[0097] To isolate *Akermansia masiliii* strains from the feces of a healthy Korean woman (7 years old, BMI 19.9), the strains were selectively cultured in an anaerobic chamber under strictly anaerobic conditions (5% H2, 15% CO2, and 80% N2) using the mucin medium recommended by Derrien (0.4 g / L potassium dihydrogen phosphate, 0.53 g / L sodium dichlorophosphate, 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 anhydrous sodium sulfide) before isolation. (Derrien et al., Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium, Int. J. Syst. Evol. Microbiol., 2004 Sep;54(Pt 5):1469-1476).

[0098] 1.2. Microscopic observation

[0099] To confirm whether the isolated strain was *Akermansia masiliii*, the isolated strain was observed under a microscope, and the results are shown below. Figure 1 middle. Figure 1 These are microscopic images at 1000x magnification of the *Ackermania marcescens* ATCC BAA-835 standard strain (part A) and the *Ackermania massoris* EB-AMDK39 strain (part B). Figure 1 As shown, the results of observing Akkermansia myxophilus ATCC BAA-835 standard strain (part A) and Akkermansia massoris strain EB-AMDK39 strain (part B) at a magnification of 1000x confirmed that the strains have similar shapes, both being straight or curved rod-shaped cells.

[0100] 1.3. PCR Analysis

[0101] To confirm whether the isolated strain was *Akermansia masiliii*, PCR analysis was performed on the isolated strain using the AM-specific primers (SEQ ID NO:2 and SEQ ID NO:3) listed in Table 1 below, and the results are shown below. Figure 2 middle. Figure 2In the diagram, lane M represents the DNA size marker, lane 1 represents the Akkermansia myxophilus strain (ATCCBAA-835) as the positive control, lane 2 represents the Akkermansia massoris strain EB-AMDK39, and lane 3 represents the results of the Ginkgo control group (distilled water).

[0102] like Figure 2 As shown, it can be confirmed that the *Ackermania massoris* strain EB-AMDK39 of the present invention exhibits banding results similar to those of the *Ackermania glutinis* ATCC BAA-835 standard strain.

[0103] [Table 1]

[0104]

[0105] 1.4. Random Amplified Polymorphic DNA (RAPD) Analysis

[0106] To verify whether the *Akermansia massoris* strain EB-AMDK39 isolated above is identical to previously reported strains of the same genus, RAPD, a molecular typing method, was performed. For this purpose, genomic DNA extracted from the bacterial cells was amplified using the universal primers listed in Table 2 below, followed by electrophoresis on a 1% agarose gel for 1 hour and 30 minutes. DNA fragmentation patterns were compared using a UV perforator, and the results are shown in Figure 3.

[0107] [Table 2]

[0108]

[0109] As shown in Figure 3, the *Ackermania massoris* strain EB-AMDK39 of this invention exhibits a different RAPD banding pattern compared to the *Ackermania massoris* ATCC BAA-835 standard strain. Since the RAPD banding pattern of *Ackermania massoris* is known to differ across species, it is confirmed that the *Ackermania massoris* strain EB-AMDK39 of this invention is a different species from the *Ackermania massoris* ATCC BAA-835 standard strain.

[0110] 1.5. Whole genome sequencing

[0111] To analyze the genomic similarity between the above-isolated Akkermansia massoris strain EB-AMDK39 and Akkermansia ATCC BAA-835, the whole genome base sequence was analyzed using PacBio technology and compared with the standard strain, as shown in Table 3 below.

[0112] [Table 3]

[0113]

[0114] As shown in Table 3, there are differences in the whole genome statistical values ​​when comparing the Akkermansia massiliensis EB-AMDK39 strain of the present invention with the Akkermansia ATCC BAA-835 strain, and the results of measuring the average nucleotide identity (ANI) value confirm that the value is below the limit for defining species boundaries (95-96%).

[0115] The genomic information of *Ackermania massoris* strain EB-AMDK39 obtained in the above examples was functionally classified. Protein-coding genes were functionally classified according to the definition of a Cluster of Orthologous Group (COG), as shown in Table 4 below. COG is a search-based comparative database used to find orthologous genes. Based on COG, the functions of each gene in the full-length genome of *Ackermania massoris* strain EB-AMDK39 were confirmed and compared with those of *Ackermania tumefaciens* strain ATCC BAA-835, as shown in Table 4.

[0116] [Table 4]

[0117]

[0118] As shown in Table 4, 22 out of 25 COG functional codes matched by the *Ackermania massoris* EB-AMDK39 strain and *Ackermania myxotropica* ATCC BAA-835 strain of this invention were identified, excluding "Nuclear structure (Y)", "RNA processing and modification (A)", and "Chromatin structure and dynamics (B)". Overall, 2028 and 1799 genes, respectively, were identified as COG codes in *Ackermania massoris* EB-AMDK39 strain and *Ackermania myxotropica* ATCC BAA-835 strain, respectively, but 494 and 309 genes, respectively, did not match. Specifically, it was confirmed that the *Ackermania massoris* strain EB-AMDK39 of this invention possesses more genes than *Ackermania marcescens* strain ATCC BAA-835 in 16 COG codes, including "Intracellular trafficking, secretion, and vesicular transport" (U), "Cell wall / membrane / envelope biogenesis" (M), "Carbohydrate transport and metabolism" (G), and "Signal transduction mechanisms" (T). In summary, it can be confirmed that the *Ackermania massoris* strain EB-AMDK39 of this invention differs significantly from *Ackermania marcescens* strain ATCC BAA-835 in its full-length genome.

[0119] 1.6. Phylogenetic tree analysis using the full-length 16S rRNA gene base sequence.

[0120] To analyze the full-length 16S rRNA gene sequence of the *Akkermansia masilionis* strain EB-AMDK39 isolated above, the 16S rRNA gene was amplified using 27F and 1541R primers as shown in Table 5 below, and the sequence was then determined using a 3730xl DNA analyzer. A phylogenetic tree was constructed based on the 16S rRNA gene sequence of the *Akkermansia masilionis* strain EB-AMDK39 obtained above and the 16S rRNA gene sequences of other species of the same genus that have been published, and is shown below. Figure 4 middle.

[0121] Phylogenetic analysis was performed using MEGA-X. A phylogenetic tree was constructed by the neighbor-joining method with 1000 bootstraps and is shown in Figure 4 . The average nucleotide identity (ANI) value evolutionary distance was evaluated using the pyani v0.2.7 program with the -m ANIb setting. The complete or draft genome sequences of Akkermansia muciniphila strain ATCC BAA-835 (accession number: GCF_000020225.1), Akkermansia muciniphila strain CBA5201 (accession number: GCF_004104435.1), Akkermansia muciniphila strain JCM30893 (accession number: GCF_009731575.1), Akkermansia muciniphila strain EB-AMDK19 (accession number: GCF_004015105.1), Akkermansia muciniphila strain EB-AMDK27 (accession number: GCF_010223015.1), and Akkennansia glycaniphila Pyt strain (accession number: GCF_900097105.1) were downloaded from the NCBI genome database (https: / / www.ncbi.nlm.nih.gov / genome / ) and used. A phylogenetic tree was constructed using the 16S rRNA gene base sequences of other strains of the same species and is shown in Figure 4 a.

[0122] [Table 5]

[0123]

[0124] As Figure 4 shown, as a result of phylogenetic tree analysis of evolutionary relationships by 16S rRNA gene base sequence analysis, Akkermansia massiliensis strain EB-AMDK39 is genetically within the genus Akkermansia but is a different species from Akkermansia muciniphila. Its full-length 16S rRNA gene base sequence is 100% identical to Akkermansia sp. Marseille-P6666, and thus it was confirmed to be Akkermansia massiliensis.

[0125] 1.7. Gene Presence Comparison and Functional Gene Exploration Based on Full-Length Genome

[0126] The presence or deletion of genes in the full-length genomes of *Ackermania massoris* strain EB-AMDK39 and *Ackermania marcescens* strain ATCBAA-835, based on the present invention, was compared. A compatiblecontig database was constructed using Anvi'o version 7.1 (https: / / merenlab.org / 2016 / 02 / 27 / the-anvio-interactive-interface / ), and information analysis was performed on the constructed database based on COG and the Kyoto Encyclopedia of Genes and Genomes (KEGG). A genome library was then established, and pan-genome analysis was performed using the -minbit 0.5, -mcl-inflation 10, and -use-ncbi-blast options. The results of comparing gene presence / deletion based on the analysis and the genes specifically present in *Ackermania massoris* strain EB-AMDK39 are shown below. Figure 5 middle. Figure 5 In this context, SCG clusters represent single-copy core gene clusters. The CbiE, CbiL, CbiK, CbiC, CbiG, and CbiD genes marked in the attached figure are those that play a direct role in vitamin B12 production. They were specifically observed in the full-length genome of Akkermansia masiliii EB-AMDK39 and represent, respectively, the synthesis of vitamin B12 adenosyl cobalamide, cobalt-precorrin-2C(20)-methyltransferase, sirohydrochlorin cobaltochelatase, cobalt-precorrin-8-methylmutase, cobalt-precorrin-4-methyltransferase, and cobalt-precorrin-5BC(1)-methyltransferase. C(1)-methyltransferase).

[0127] like Figure 5As shown, since both *Ackermania massoris* strain EB-AMDK39 and *Ackermania myxophilus* strain ATCBAA-835 belong to the *Ackermania* genus, the shared proportion of the SCG cluster is high, and the presence / deletion patterns of strain-specific genes were identified. Among the strain-specific genes of *Ackermania massoris* EB-AMDK39, gene compositions (CbiE, CbiL, CbiK, CbiC, CbiG, and CbiD) that play a direct role in vitamin B12 synthesis were identified in a prominent pattern.

[0128] 1.8. Confirm the ability of Akkermansia masiliii strains to produce short-chain fatty acids (SCFAs).

[0129] Short-chain fatty acids (SCFAs), such as butyrate, acetate, and propionate, are metabolites produced by intestinal bacteria. They play an important role in the host's energy metabolism and participate in energy balance as signal transduction carriers acting on G protein-coupled receptors (GPR41 and GPR43).

[0130] Short-chain fatty acids (SCFAs) reduce intestinal motility and increase intestinal transit rate in enteroendocrine cells via GPR41. This induces the secretion of peptide YY (PYY), thereby reducing energy intake and preventing obesity. Furthermore, SCFAs increase insulin sensitivity by inducing glucagon-like peptide-1 (GLP-1) via GPR43, thus increasing satiety. GPR43 activity also inhibits insulin signaling in adipose tissue, preventing fat accumulation. SCFAs can enhance glucose metabolism and activate intestinal gluconeogenesis (IGN), thereby reducing food intake through enterobacterial neural circuitry. Further, vitamin B12 affects the strain's ability to produce SCFAs. Specifically, it can act as a coenzyme catalyzing the conversion of succinate to propionic acid. The *Akermansia masiliforme* strain of the present invention possesses methylmalonyl-coA mutase, which catalyzes the conversion of succinic acid to propionic acid with vitamin B12 as a cofactor.

[0131] In the described embodiment, a gene composition specifically present in *Akermansia masiliii* strain EB-AMDK39 that directly plays a role in vitamin B12 synthesis was identified. Therefore, to confirm the changes in functional metabolites with the presence or absence of vitamin B12 during culture, the strain was cultured in test tubes, and the content of short-chain fatty acids (SCFAs) in the culture medium was analyzed by gas chromatography (GC). For this purpose, the culture medium was centrifuged at 12000 x g for 5 minutes to recover the supernatant, and the supernatant was filtered using a 0.2 μm syringe filter before analysis. A gas chromatograph (Agilent 7890N) equipped with an FFAP column (30 m × 0.320 mm, 0.25 μm phase) was used, with the conditions shown in Table 6. The analytical results are shown in Table 7 below.

[0132] [Table 6]

[0133]

[0134] [Table 7]

[0135]

[0136] As shown in Table 7, the *Ackermania massoris* strain EB-AMDK39 of the present invention exhibits a different short-chain fatty acid production capacity compared to *Ackermania myxophilus* ATCC BAA-835. Specifically, when vitamin B12 is not added to the culture medium, the main short-chain fatty acids of *Ackermania myxophilus* ATCC BAA-835 are acetate and succinate; conversely, regardless of the presence or absence of vitamin B12, the main short-chain fatty acids of the *Ackermania massoris* strain EB-AMDK39 of the present invention are acetate and propionate, with propionate production being approximately 40 times higher. These results demonstrate that the short-chain fatty acid production capacity of the *Ackermania massoris* strain EB-AMDK39 of the present invention differs significantly from that of *Ackermania myxophilus* ATCC BAA-835 depending on the presence or absence of vitamin B12.

[0137] Example 2: Bacterial characteristics and safety analysis of Akkermansia masiliii strain EB-AMDK39

[0138] 2.1. Confirm the sugar utilization of the isolated Akkermansia masiliii strain EB-AMDK39

[0139] To confirm the sugar utilization of the *Ackermania massoris* strain EB-AMDK39 isolated above, it was cultured using the API50CH kit (Biomerieux, France), and then compared with the standard strain of *Ackermania mucilaginosa* (ATCC BAA-835) to determine whether growth was achieved using each sugar. The results are shown in Table 8 below.

[0140] [Table 8]

[0141]

[0142]

[0143] As shown in Table 8, it was confirmed that the Akkermansia masilionis strain EB-AMDK39 of the present invention differed from the standard Akkermansia muciniphila strain (ATCC BAA-835) in its ability to utilize ribose, D-galactose, D-fructose, D-mannose, D-lactose and L-fucose.

[0144] 2.2. Confirm the antimicrobial susceptibility of Akkermansia masiliform strains.

[0145] To confirm the antimicrobial susceptibility of the *Akermansia masilionis* EB-AMDK39 strain isolated above, the minimum inhibitory concentrations (MICs) of 16 antimicrobial agents, including piperacillin-tazobactam (PTZ), cefazolin (CTZ), chloramphenicol (CHL), clindamycin (CLI), meropenem (MEM), moxifloxacin (MXF), metronidazole (MTZ), tetracycline (TET), ampicillin (AMP), gentamicin (GEN), kanamycin (KAN), streptomycin (STR), nalidixic acid (NAL), sulfamethoxazole (SMZ), trimethoprim (TMP), and apramycin (APR), were determined according to the broth microdilution method as per the Clinical & Laboratory Standards Institute (CLSI) guidelines. The results are shown in Table 9 below.

[0146] [Table 9]

[0147]

[0148] As shown in Table 9, the *Akermansia masiliii* strain EB-AMDK39 of this invention was confirmed to exhibit resistance to cefazolin, clindamycin, moxifloxacin, ciprofloxacin, and nalidixic acid (quinolone antibiotics), and gentamicin, kanamycin, streptomycin, and apramycin (aminoglycoside antibiotics), while showing sensitivity to other antimicrobial agents. Furthermore, its antimicrobial resistance pattern differed slightly when compared to *Akermansia myxophilus* ATCC BAA-835.

[0149] Resistance to quinolones and aminoglycosides is also present in the same Akkermansia genus, therefore it is considered an intrinsic characteristic (intrinsic resistance). In most cases, this resistance is due to the drug failing to effectively reach its site of action, thus suggesting that each antibiotic has a different spectrum of action (antimicrobial spectrum) and is effective only against specific types of microorganisms. Furthermore, Filardi et al.'s report confirmed that all Akkermansia myxophilus strains, regardless of genotype, showed low sensitivity to ciprofloxacin and aminoglycosides, and the efflux pump system present in the cells has been reported to potentially contribute to this resistance.

[0150] Furthermore, the bioinformatics-based programs PlasmidFinder (https: / / cge.cbs.dtu.dk / services / PlasmidFinder / ), PHASTER (https: / / phaster.ca / ), and MobileElement Finder (cge.cbs.dtu.dk / services / MobileElementFinder) were applied to the complete full-length genome of the *Akermansia massoris* EB-AMDK39 strain of this invention to confirm its availability or endogenous nature. As a result, no obvious sequences were found in mobile genetic elements such as plasmids, mobile genetic elements (MGEs), and prophages in the *Akermansia massoris* EB-AMDK39 strain, and no phenotype-related resistance genes were detected.

[0151] 2.3. Analysis of hemolytic activity and virulence factors of the isolated strains

[0152] To verify the safety of the *Akermansia masiliii* strain EB-AMDK39 isolated above, its hemolytic activity was evaluated. Therefore, the strain was cultured on blood agar medium prepared by adding 5% w / v defibrinated sheep blood to trypsin-soy agar (17.0 g / L casein trypsin digest, 3.0 g / L soybean trypsin digest, 2.5 g / L glucose, 5.0 g / L sodium chloride, 2.5 g / L potassium phosphate, 15 g / L agar). The results are shown below. Figure 6 In the middle. For example Figure 6 As shown, the Massiris Akkermansia EB-AMDK39 strain of the present invention did not exhibit β-hemolysis (a completely transparent portion around the colony) associated with pathogenicity.

[0153] To confirm the presence of possible toxigenic genes in *Akermansia masiliii* strain EB-AMDK39, the VFDB (http: / / www.mgc.ac.cn / VFs / ) and Virulence Finder 2.0 (https: / / cge.cbs.dtu.dk / services / VirulenceFinder / ) platforms were used. Virulence genes include bacterial toxins, cell surface proteins that mediate bacterial attachment, cell surface carbohydrates and proteins that protect bacteria, and hydrolytic enzymes that may promote bacterial pathogenicity. The analytical tools used were those specified in the "Guidelines for Functional Raw Materials of Probiotics in Health Foods," with basic analytical conditions of similarity > 60%, coverage > 60%, amino acid length > 40, and E-value of 0.01.

[0154] The presence of virulence factors was confirmed by comparing Akkermansia massoris strain EB-AMDK39 with known pathogens (E. coli, Enterococcus, Listeria, and S. aureus) using the Virulence Finder tool. No virulence factors were detected.

[0155] Based on VFDB analysis, six possible virulence factors were identified in the full-length genome of *Akermansia masilionis* strain EB-AMDK39. However, the detected genes are associated with cell wall / membrane / envelope biogenesis, adhesion, or other functions, and are essential for cell structure, function, and adaptation. These genes also participate in the adaptation or survival of pathogenic bacteria in hostile / host environments, and therefore were confirmed as virulence factors in the database (Table 10). Thus, it was confirmed that these genes are essential for bacterial survival in the absence of other pathogenic mechanisms, and that these genes are not virulence-causing genes.

[0156] [Table 10]

[0157]

[0158] 2.4. Confirm the cytotoxicity of the isolated strain.

[0159] To confirm the cytotoxicity of the *Akkermansia masiliforme* EB-AMDK39 strain isolated above, lactate dehydrogenase (LDH) analysis was performed according to the toxin production evaluation method described in the "Guidelines for Safety Evaluation of Probiotics as Functional Raw Materials in Health Foods". The cell lines used in the experiment were coliform epithelial cell lines (HT-29 and Caco-2), and *Akkermansia masiliforme* EB-AMDK39 was used at a concentration of 10... 3 ~10 7 The experiment was conducted using CFU / mL treatment. Pseudomonas aeruginosa ATCC 17831 strain was used as a positive control, treated with the same concentration as EB-AMDK39. Cytotoxicity was calculated using the following formula and converted to cell viability, as shown in Figure 7.

[0160] [Formula 1]

[0161]

[0162] As shown in Figure 7, the *Akermansia masilionis* EB-AMDK39 strain of the present invention was confirmed to have no cytotoxicity at all tested concentrations.

[0163] Using *Ackermania marcescens* ATCC BAA-835 as a control group, *Ackermania marcescens* strain EB-AMDK39 isolated from human feces was identified using biochemical (API) and molecular biological methods (16S rRNA sequence analysis, RAPD, and full-length genome analysis, etc.). Furthermore, by confirming antibiotic resistance tests, hemolytic activity, virulence factors, and cytotoxicity, the isolated strain was confirmed to be a safe strain capable of functioning as probiotics. Based on these results, the isolated *Ackermania marcescens* strain EB-AMDK39 was deposited at the Korea Center for Microbial Resources (KCTC), Korea Institute of Biotechnology, with accession number KCTC 13765BP.

[0164] Example 3: Confirmation of the effects of live Akkermansia masiliii EB-AMDK39 in an obesity-induced mouse model Anti-obesity effects

[0165] 3.1. strains sample

[0166] In this embodiment, the *Ackermania mutans* ATCC BAA-835 (control group) and *Ackermania marcescens* EB-AMDK39 strains were prepared to a concentration of 1×10⁻⁶ live bacteria. 8 CFU / 100μl PBS (25% glycerol, 0.05% cysteine / PBS) was used for animal studies.

[0167] 3.2. Animal Experiments

[0168] Animal experiments were conducted in accordance with the Animal Use and Care Protocol of the Institutional Animal Care and Use Committee (IACUC). Seven-week-old male C57BL / 6 mice were purchased and, after a one-week acclimatization period, were housed for eight weeks. The housing environment was maintained at the specified temperature (22°C) and relative humidity (40–60%), with a regulated 12-hour light-dark cycle. To induce obesity, mice were fed a high-fat diet (60 kcal% fat; Research Diets Inc., NJ, USA), while the normal control group was fed a 10 kcal% fat diet. Drinking water was provided freely. As shown in Table 11, the experimental groups were divided into five groups. Each group was divided into Normal (normal diet control group), HFD (high-fat diet for obesity induction group), ORL (high-fat diet for obesity induction + orlistat administration group), ATCC BAA-835 (high-fat diet for obesity induction + Akkermansia myxophilus ATCC BAA-835 strain administration group), and Akkermansia massoris EB-AMDK39 (high-fat diet for obesity induction + Akkermansia massoris EB-AMDK39 strain administration group). ATCC BAA-835 strain (live bacteria) and orlistat (ORL; Xenical, 10 mg / kg) as an oral obesity treatment agent were used as positive control groups.

[0169] [Table 11]

[0170]

[0171] 3.3. Confirm body weight and weight gain.

[0172] During the 8-week experiment, the weight of each experimental group was measured weekly and the weight gain was calculated. The results are presented below. Figure 8 (Chinese) Reference Figure 8 Compared with the obesity-induced group fed only a high-fat diet, the body weight and weight gain were significantly reduced in the Akkermansia muciniphila ATCCBAA-835 and Akkermansia masseri EB-AMDK39 treatment groups. These results confirm that Akkermansia masseri EB-AMDK39 induces weight loss.

[0173] 3.4. Confirm the weight changes of adipose tissue and liver tissue.

[0174] At the end of the experiment, mice were anesthetized with CO2 and sacrificed. The weights of the removed subcutaneous fat, epididymal fat, mesenteric fat, and liver tissue were measured and displayed. Figure 9 middle.

[0175] refer to Figure 9 Compared to the normal diet group, the obesity-induced group showed a significant increase in the weight of subcutaneous fat, epididymal fat, and mesenteric adipose tissue. Conversely, the group that consumed the same high-fat diet as the obesity-induced group, but was treated with Akkermansia masiliforme EB-AMDK39, showed a decrease in adipose tissue weight, with statistical significance confirmed in subcutaneous fat and epididymal fat.

[0176] Furthermore, compared to the normal diet group, the obesity-induced group (HFD) showed a significant increase in liver tissue weight, which was significantly reduced after administration of *Akermansia masilionis* EB-AMDK39 strain. These results indicate that administration of the *Akermansia masilionis* EB-AMDK39 strain of the present invention inhibits obesity by reducing body fat.

[0177] 3.5. Analysis of Blood Lipid Biochemical Indicators

[0178] Blood was collected from laboratory animals that were sacrificed after an 18-hour fast, and serum was obtained by centrifugation. Serum levels were measured as lipid indicators, including total cholesterol (TC) and triglycerides (TG), and the results are presented graphically. Figure 10 In this case, Wako's LabAssay is used. TM Cholesterol and LabAssay TM Triglyceride products measure total cholesterol and triglyceride concentrations.

[0179] refer to Figure 10 Analysis of blood total cholesterol levels showed that the obesity-induced group (HFD) had significantly increased total cholesterol levels compared to the normal diet group, and these levels were further reduced by oral administration of Akkermansia masiliforme EB-AMDK39 strain.

[0180] Compared with the normal diet group, the obesity-induced group (HFD) had significantly increased levels of triglycerides (TG) in the blood. Furthermore, compared with the obesity-induced group (HFD), the group treated with *Akermansia masiliforme* EB-AMDK39 strain had significantly reduced levels of triglycerides in the blood.

[0181] 3.6. Compare the size of fat cells

[0182] To determine and compare the diameter of adipocytes, subcutaneous fat was harvested from sacrificed mice and fixed in formalin. The subcutaneous fat was embedded in paraffin and then prepared into tissue sections with a thickness of 4 μm. For microscopic observation of adipocytes, the tissue sections were stained with hematoxylin and eosin, and the diameter of adipocytes was measured using ImageJ. (The results are shown below.) Figure 11 middle.

[0183] refer to Figure 11 Because the high-fat diet induced obesity, the adipocyte diameter was significantly increased in the HFD group. Conversely, compared with the HFD group, the adipocyte diameter was significantly reduced in the groups that were orally administered Akkermansia muciniphila ATCC BAA-835, Akkermansia massoris EB-AMDK39, and orlistat (ORL).

[0184] It has been confirmed that oral administration of Akkermansia masiliforme EB-AMDK39 reduces adipocyte size and fat accumulation.

[0185] It will be apparent to those skilled in the art that the present invention can be modified and varied in many ways without departing from its spirit and scope. The specific embodiments described in this specification are merely illustrative of preferred embodiments of the invention and should not be construed as limiting the invention. The scope of protection of the present invention should be defined by the appended claims, and the various modifications and variations described above should be included within the scope of protection of the present invention.

[0186] [Collection Number]

[0187] Name of the depository: Korea Center for the Preservation of Typical Microorganisms

[0188] Accession number: KCTC 13765BP

[0189] Date of preservation: December 5, 2018.

[0190] The Budapest Treaty on the International Recognition of Microbial Deposits for Use in Patent Proceedings

[0191] International format

[0192] Regarding the original preservation certificate

[0193] Issued according to 7.1

[0194] Depositor: AnTai Microbial Technology Co., Ltd.

[0195] Address: 32 Dongguk-ro, Ilsan-gu, Goyang-si, Gyeonggi-do, South Korea

[0196]

[0197] Style BP / 4 (KCTC Style 17) Single Page (Reissued)

[0198] The Budapest Treaty on the International Recognition of Microbial Deposits for Use in Patent Proceedings

[0199] Proof of revisions to subsequent instructions or scientific descriptions and / or classification nomenclature

[0200] Issued according to 8.2

[0201] Depositor: AnTai Microbial Technology Co., Ltd.

[0202] Address: 32 Dongguk-ro, Ilsan-gu, Goyang-si, Gyeonggi-do, South Korea

[0203]

[0204] appendix : Communicate subsequent instructions or modify scientific descriptions and classification names in accordance with Rule 8.1.

[0205] Style BP / 8 (KCTC Style 17) Single Page.

Claims

1. A composition, characterized in that, Contains strains of Akkermansia masiliforme, or cultures or dried products of said strains.

2. The composition according to claim 1, characterized in that, The *Masiri* strain is neither *Ackermania mucilaginosa* nor *Ackermania polysaccharide*.

3. The composition according to claim 2, characterized in that, Compared with Akkermansia muciniphila or Akkermansia polysaccharide-loving bacteria, the Akkermansia masiliii strain has less than 95% average nucleotide identity across the entire genome.

4. The composition according to claim 1, characterized in that, The *Massilia klerman* strain mentioned is the *Massilia klerman* strain with accession number KCTC 13765BP.

5. The composition according to claim 1, characterized in that, The composition comprises live, low-temperature sterilized, or inactivated strains of the *Massilyakermania* strain.

6. The composition according to claim 1, characterized in that, The composition is for the prevention or treatment of metabolic diseases, including obesity, diabetes, insulin resistance, hypertension, hyperlipidemia, dyslipidemia, coronary artery disease, arteriosclerosis, and non-alcoholic fatty liver disease.

7. The composition according to claim 1, characterized in that, The composition contains 1 × 10⁻⁶ units relative to the total weight of the composition. 2 CFU to 1×10 15 CFU, 1×10 3 CFU to 1×10 14 CFU, 1×10 4 CFU to 1×10 13 CFU, 1×10 5 CFU to 1×10 12 CFU, 1×10 6 CFU to 1CFU×10 11 CFU, 1×10 7 CFU to 1×10 10 CFU or 10 8 CFU to 10 12 CFU of *Akermansia masiliforme* strains, or cultures containing equal amounts of live, low-temperature sterilized, or inactivated cells.

8. The composition according to claim 1, characterized in that, The Massiri Akkermansia strain is a substantially purified or freeze-dried strain.

9. The composition according to claim 1, characterized in that, The composition also contains a pharmaceutically acceptable carrier or excipient.

10. The composition according to claim 1, characterized in that, The composition may also contain other probiotic strains or one or more prebiotics.

11. The composition according to claim 1, characterized in that, The composition is a veterinary drug composition, feed additive, or cosmetic composition.

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