EP4698198A1

EP4698198A1 - Composition comprising akkermansia sp.

Info

Publication number: EP4698198A1
Application number: EP25800696.4A
Authority: EP
Inventors: Jae-Gu SEO; Sang-Nam Lee; Dokyung Lee; Sungyoon Kim; Moon-Gi Hong; Dohak KIM; Da Yeon YOO; Seol-Ah YOON; Jiyeon Jung; Hye Rim Byeon
Original assignee: Enterobiome Inc
Current assignee: Enterobiome Inc
Priority date: 2024-06-21
Filing date: 2025-06-18
Publication date: 2026-02-25
Also published as: CN121604969A; AU2025270973A1; KR102732392B1; WO2025263981A1

Abstract

The present invention relates to a pharmaceutical composition, a food composition, a veterinary composition, and a cosmetic composition effective for preventing or treating metabolic diseases, which contain Akkermansia massiliensis, a culture or a dried product thereof. While traditional probiotics generally have an insignificant therapeutic effect on metabolic diseases, the next-generation pharmabiotic strain of the present invention is excellent in the prevention or treatment of metabolic diseases to the extent that it can be used as a new preventive and therapeutic tool.

Description

COMPOSITION COMPRISING AKKERMANSIA SP.

The present invention relates to a composition containing Akkermansia sp., and more particularly, to a pharmaceutical composition containing Akkermansia massiliensis.

A metabolic disease refers to a disease in which several diseases such as obesity, diabetes, hypertension, hyperlipidemia, coronary arteriosclerosis or arteriosclerosis, and non-alcoholic fatty liver disease occur simultaneously due to chronic metabolic disorders. Most metabolic diseases are accompanied by overweight or obesity. The most serious problem of a metabolic disease is the occurrence of chronic complications such as diabetic retinopathy, diabetic nephropathy, diabetic foot disease, diabetic neuropathy, hyperlipidemia, and cardiovascular disease. Most of these chronic complications proceed irreversibly once they occur, and there is still no way to completely block this process. Therefore, if appropriate treatment is not provided, they cause serious symptoms and are recognized as the most serious disease that threatens the health of modern people.

Until now, in order to treat metabolic diseases with these complex symptoms, hypoglycemic agents, antihypertensive agents, and cholesterol medications have been administered individually. Therefore, in order to efficiently manage and treat metabolic diseases with these complex symptoms, there is a need for the development of new treatments that can treat various symptoms simultaneously.

Studies have been conducted on the use of probiotics alone or in combination with therapeutic agents to overcome the side effects of conventional drugs or immunomodulators. As studies on the role of intestinal bacteria in promoting health are actively being conducted, there is a growing interest in probiotic preparations.

As a technology for treating metabolic diseases using probiotics, Korean Patent Application Publication No. 2011-0095929 discloses a Lactobacillus reuteri that inhibits lipid absorption in the body, Korean Patent No. 10-0996577 discloses Lactobacillus curvatus that lowers blood cholesterol and inhibits obesity, and Korean Patent Application Publication No. 2010-0010015 discloses Lactobacillus johnsonii which lowers blood cholesterol and inhibits obesity at the same time.

However, probiotics such as those mentioned above have limitations in their use as new preventive and therapeutic tools because their effects on improving metabolic diseases are minimal.

The present invention is intended to overcome the limitations of the above-described prior art, and an object of the present invention is to provide a pharmaceutical composition effective for preventing or treating metabolic diseases, containing Akkermansia massiliensis.

Another object of the present invention is to provide a food composition helpful in preventing or improving metabolic diseases, containing Akkermansia massiliensis.

Still another object of the present invention is to provide a veterinary composition or feed additive helpful in preventing or improving metabolic diseases, containing Akkermansia massiliensis.

Still another object of the present invention is to provide a cosmetic composition containing Akkermansia massiliensis.

An aspect of the present invention relates to Akkermansia massiliensis.

Another aspect of the present invention relates to a pharmaceutical composition containing Akkermansia massiliensis, a culture or a dried product of the strain.

Still another aspect of the present invention relates to a food composition helpful in preventing or improving metabolic diseases, containing Akkermansia massiliensis.

Still another aspect of the present invention relates to a veterinary composition or feed additive helpful in preventing or improving metabolic diseases, containing Akkermansia massiliensis.

Still another aspect of the present invention relates to a cosmetic composition helpful in preventing or improving metabolic diseases, containing Akkermansia massiliensis.

The pharmaceutical composition containing Akkermansia massiliensis of the present invention can be used as a pharmaceutical composition, a health functional food composition, a feed composition, a cosmetic composition, etc. for treating and/or preventing metabolic diseases.

The pharmaceutical composition containing Akkermansia massiliensis of the present invention provides the effects of effectively managing and treating complex symptoms of metabolic diseases (metabolic syndrome), such as diabetes, obesity, insulin resistance, and fatty liver, by inhibiting weight gain and body fat gain, reducing insulin resistance, and lowering total blood cholesterol concentration.

The Akkermansia massiliensis of the present invention is a next-generation pharmabiotic species that is excellent in preventing or treating metabolic diseases to the extent that it can be used as a new preventive and therapeutic tool.

FIG. 1 shows the results of microscopic observation of the Akkermansia massiliensis strain EB-AMDK39 of the present invention and Akkermansia muciniphila strain ATCC BAA-835.

FIG. 2 shows the results of PCR analysis of the Akkermansia massiliensis strain EB-AMDK39 of the present invention and Akkermansia muciniphila strain ATCC BAA-835.

FIGS. 3a and 3b show the results of Random Amplified Polymorphic DNA (RAPD) analysis of the genomic DNA of the Akkermansia massiliensis strain EB-AMDK39 of the present invention and Akkermansia muciniphila strain ATCC BAA-835.

FIG. 4 is a comparative diagram showing the phylogenetic relationship between Akkermansia massiliensis of the present invention and other Akkermansia sp. strains.

FIG. 5 shows the results of comparing the presence or absence of genes based on the genomes of the Akkermansia massiliensis strain EB-AMDK39 of the present invention and Akkermansia muciniphila strain ATCC BAA-835.

FIG. 6 shows a comparative illustration of the cytotoxicity test results of the Akkermansia massiliensis strain EB-AMDK39 of the present invention and Akkermansia muciniphila strain ATCC BAA-835.

FIG. 7 shows the results of confirming whether the Akkermansia massiliensis strain EB-AMDK39 of the present invention and Akkermansia muciniphila strain ATCC BAA-835possess hemolytic activity.

FIG. 8 shows the results of analyzing the body weight change in the group administered with the Akkermansia massiliensis strain EB-AMDK39 of the present invention administration group, the negative control group (HFD), and the Akkermansia muciniphila strain ATCC BAA-835 administration group.

FIG. 9 shows the results of analyzing the changes in the amount of subcutaneous fat, epididymal fat, and superior mesenteric fat in the Akkermansia massiliensis strain EB-AMDK39 administration group of the present invention, the negative control group (HFD), and the Akkermansia muciniphila strain ATCC BAA-835 administration group.

FIG. 10 shows the results of measuring, by ELISA, the changes in the concentrations of serum total cholesterol and serum triglycerides in the Akkermansia massiliensis strain EB-AMDK39 of the present invention administration group, the negative control group (HFD), and the Akkermansia muciniphila strain ATCC BAA-835 administration group.

FIG. 11 shows the images and a graph illustrating the size of fat cells formed in mesenteric fat tissue and the degree of fat accumulation in each experimental group when treated with the Akkermansia massiliensis strain EB-AMDK39 of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the attached drawings.

In the present application, "Akkermansia massiliensis strain is an Akkermansia sp." and belongs to the phylum of Verrucomicrobiota. The Akkermansia massiliensis of the present invention is distinct from Akkermansia muciniphila or Akkermansia glycaniphila.

In this specification, "metabolic disease" refers to the overall symptoms of diseases such as obesity, diabetes, hypertension, hyperlipidemia, coronary arteriosclerosis or arteriosclerosis, non-alcoholic fatty liver disease, etc. In this specification, "metabolic syndrome", "metabolic disease", "metabolic disorder", and "metabolic abnormality" are used interchangeably.

The term "obesity" used herein may refer to a state in which body fat is excessively accumulated. The standard for obesity is that body fat accounts for 25% or more of body weight, and 30-35% or more for women. The body mass index (BMI) is widely used as a general measurement method. In the case of Westerners, when the BMI exceeds 30 kg/m², it is defined as obesity, whereas when it is 25-30 kg/m², it is defined as overweight. In the case of Asians, when it exceeds 28 kg/m², it is defined as obesity, whereas when it is 23-28 kg/m², it is defined as overweight.

The term "dyslipidemia" as used herein may refer to a state in which lipid components such as triglycerides, LDL cholesterol, phospholipids, and free fatty acids in the blood are increased, or a state in which HDL cholesterol is decreased. The dyslipidemia may be, for example, one or more selected from the group consisting of hyperlipidemia, hyper-LDL cholesterolemia, hyper-triglyceridemia, and hypo-HDL cholesterolemia.

The term "subject" as used herein refers to any animal, including humans, that has developed or is likely to develop a metabolic disease. The animal may be, but is not limited to, mammals such as dogs, cats, hamsters, rabbits, cows, horses, sheep, pigs, goats, camels, and antelopes that require treatment for symptoms similar to those of humans.

When "about" comes before a number, it means ±20%, preferably 10% of the numeric value.

The Akkermansia can also be substantially purified. The term "substantially purified" as used herein refers to a bacterial strain or a mixture of more than one bacterial strains (e.g., Bacteroidetes, Firmicutes, Proteobacteria, or Verrucomicrobia) that are substantially enriched in a sample. The sample can be substantially purified or enriched for the bacterial strain or mixture of strains of interest such that the sample is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or greater of the desired bacterial strain(s) or less than about 40%, 30%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the undesirable or other bacterial strains present.

In the present invention, the Akkermansia massiliensis may be included in the form of viable (live) bacteria, pasteurized bacteria, or inactivated bacteria (heat-killed). As used herein, the "pasteurized bacteria" means Akkermansia massiliensis that has been heat-treated at a low temperature. In an embodiment, the pasteurized Akkermansia massiliensis means Akkermansia massiliensis that has been heat-treated at a temperature of 50℃ to 110℃ for 5 minutes or more and less than 30 minutes. In an embodiment, the pasteurization is performed at a temperature ranging from about 50℃ to about 110℃, preferably from about 50℃ to about 100℃, more preferably from about 60℃ to about 95℃, and more preferably from about 70℃ to about 90℃. Preferably, the pasteurized bacteria are heat treated at about 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, or 70℃. In another embodiment, the pasteurization means heat treatment at a temperature of about 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃ or 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃, or 100℃. In still another embodiment, pasteurization means heat treatment at a temperature of 101℃, 102℃, 103℃, 104℃, 105℃, 106℃, 107℃, 108℃, 109℃, or 110℃.

In this application, the term "inactivated bacteria" means a form in which the growth of live bacteria is prevented by heat treatment, etc. The inactivated bacteria may include an antibacterial substance such as cytoplasm, cell membrane, cell wall, and bacteriocin; polysaccharides; and/or organic acids, etc.

In the present specification, the term "culture" refers to a product obtained by culturing Akkermansia massiliensis, and may include a fermentation product. In an embodiment, the culture may be a fermentation product obtained by culturing　Akkermansia massiliensis in a medium. The "fermentation product" refers to a result of enzymatic or metabolic decomposition of an organic substance using a microorganism. In the present application, "fermentation" may refer to any activity or process other than a putrefaction reaction involving enzymatic or metabolic decomposition of an organic substance using a microorganism.

The culture (or fermentation) may be a whole culture of Akkermansia massiliensis, a dilution thereof, a concentrate thereof, a dried product thereof, a lyophilized product thereof, an extract thereof, and/or a fraction thereof. The concentrate may be obtained by centrifuging or evaporating the culture, and the dried product may be obtained by drying the culture using a dryer or the like. The lyophilized product may be obtained by lyophilizing the culture using a lyophilizer or the like, and the extract may be obtained by physically or ultrasonically treating the strain or culture. The fraction may be obtained by subjecting the culture, the extract, etc. to a method such as centrifugation and chromatography.

The culture or fermentation product may be in a solid state (a solid, e.g., a dried product), liquid state (liquid), or fluidized state, but is not necessarily limited thereto.

As used herein, the terms such as "treat", "treating", and "to treat" mean to impede, slow, halt, or reverse the progression or severity of an existing condition, disease, disorder, or symptom.

In this specification, the term 'functional health food' is the same as food for special health use (FoSHU), and refers to a food composition with high medical and healthcare effects that is processed to effectively exhibit a bioregulatory function in addition to providing nutrition. In this specification, the term functional health food may be used interchangeably with terms such as health supplement food and health food.

An aspect of the present invention relates to a pharmaceutical composition for preventing or treating metabolic diseases, containing Akkermansia massiliensis having the Accession No. KCTC 13765BP.

The strain of Akkermansia massiliensis of the present invention, which was isolated from the feces of a healthy Korean person, is an oval-celled, monococcus or diplococcus with a size of 0.5-1 ㎛, which is an anaerobic, non-motile, gram-negative, non-endospore-forming, mucus-decomposing bacterium. The Akkermansia massiliensis produces several mucus-decomposing enzymes and is thus able to use mucus as a carbon and nitrogen source, metabolizes　some carbon sources by containing N-acetylglucosamine, and produces short-chain fatty acids such as propionic acid and acetic acid as main metabolites.

The strain of Akkermansia massiliensis of the present invention has a 16S rRNA gene of SEQ ID NO: 1.

The strain of Akkermansia massiliensis of the present invention is not Akkermansia muciniphila or Akkermansia glycaniphila. It was clearly confirmed that the Akkermansia massiliensis of the present invention is not Akkermansia muciniphila or Akkermansia glycaniphila by genome-based phylogenetic analysis and average nucleotide identity (ANI) value analysis.

The genome-wide average nucleotide identity (gANI), when compared between Akkermansia massiliensis of the present invention and ATCC BAA-835, which is an Akkermansia type strain, is less than about 95%. In the present invention, the strain of Akkermansia massiliensis has a gANI of less than 95%, such as about 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, and 79% compared to the genome of Akkermansia muciniphila. In some embodiments, in the composition of the present invention, Akkermansia massiliensis strain (e.g., an Akkermansia　massiliensis strain EB-AMDK39, KCTC 13765BP) may have a gANI of less than 95%, such as about any of 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% compared to the genome of Akkermansia glycaniphila.

The Akkermansia massiliensis of the present invention may be usefully used in the prevention or treatment of diabetes, obesity, obesity-related diseases, insulin resistance, fatty liver, hyperlipidemia, or metabolic diseases, by inhibiting weight gain and body fat gain, reducing insulin resistance, lowering blood total cholesterol concentration, and reducing the content of glutamic pyruvic transaminase (GPT), which is an indicator of blood hepatotoxicity.

The pharmaceutical composition of the present invention may contain a live Akkermansia massiliensis strain or a pasteurized bacterial strain thereof or inactivated bacteria thereof. The pasteurized Akkermansia massiliensis strain means heating at a temperature of 50℃ or higher and 110℃ or lower for 5 minutes or longer. For example, the pasteurization may be performed at 70℃ for 30 minutes. The pasteurized Akkermansia massiliensis strain EB-AMDK39 can reduce body fat accumulation more compared to the live bacteria. The reason why the pasteurized Akkermansia massiliensis is more effective has not been precisely identified, but it may be assumed that when pasteurization is performed, cell wall components such as Amuc_1100 or membrane proteins of Akkermansia massiliensis may enhance metabolic benefits in the host.

The beneficial effects of the strain of the present invention are presumed to be derived from polypeptides that are associated with a gene cluster (Amuc_1098-Amuc_1102) including Amuc_1100 and that interact with the signaling pathway of toll-like receptor 2 ("TLR2"), which is present on the surface of intestinal epithelial cells and immune cells located near the intestinal wall, and regulates intestinal homeostasis and host metabolism. For example, the Amuc_1100 polypeptide is expected to maintain the integrity of the intestinal wall of intestinal mucous membranes, and interact with the toll-like receptor 2(TRL2) present on the surface of immune cells so as to regulate or promote TRL2-signaling pathway thereby promoting the secretion of cytokines (e.g., IL-6, IL-8, and IL-10) from immune cells. The prolipoprotein idasylglyceryl transferase gene (Amuc_1104) is located in close proximity to the gene cluster (Amuc_1098-Amuc_1102). In addition, Amuc_1100 remains stable under temperature conditions used during pasteurization, which may contribute to the effect of low-temperature sterilized strains.

The Akkermansia massiliensis strain of the present invention can be cultured, recovered through a separation process such as centrifugation, and prepared into a probiotic form by drying, for example, freeze-drying, and used.

Since the Akkermansia massiliensis of the present invention is sensitive to oxygen, it is preferable to culture the strain under anaerobic conditions (80-90% nitrogen, 0-5% hydrogen, and 5-20% carbon dioxide).

The components of the liquid medium during cultivation may affect the growth of the strain and the production of active ingredients. Therefore, it is necessary to establish the components and content conditions of the liquid medium optimized for the cultivation of the Akkermansia massiliensis strain of the present invention.

The liquid medium may include, but is not limited to, one or more selected from the group consisting of glucose, lactose, maltose, fructose, galactose, N-acetylglucosamine, mannose, 1-fucose, lactate, formate, acetate, propionate, 1,2-propanediol, and butyrate as a carbon source. Preferably, the liquid medium may include glucose and N-acetylglucosamine. The liquid medium may include, but is not limited to, one or more selected from the group consisting of tryptone, peptone, soy peptone, L-glutamic acid, and ammonium as a nitrogen source.

The liquid medium may contain, as trace elements, 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 necessarily limited thereto.

The liquid medium may have a pH of 6.8 to 7.2. Preferably, the liquid medium may have a pH of 7.0. The pH may change the charge of the amine group or carboxyl group of an amino acid, which is a unit of an enzyme protein important for cell metabolism, thereby affecting the activity of protein. In addition, a change in pH in the external environment may affect the ionization of microbial nutrients, thereby affecting the uptake of nutrients by microorganisms.

It is preferable that the above liquid medium be cultured in a medium containing glucose, N-acetylglucosamine, threonine, soy peptone, or any combination thereof.

The pharmaceutical formulation may be performed by a known method, and preferably, it may be in the form of pharmaceutically acceptable oral, topical, transdermal, transmucosal, and injectable formulations, and more preferably, it may be an oral formulation.

The composition of the present invention may further contain, in addition to the above-mentioned active ingredients, a pharmaceutically acceptable carrier and/or an excipient, and may be formulated and prepared together with various additives commonly used pharmaceutically, such as binders, disintegrants, coating agents, and lubricants.

The pharmaceutical composition of the present invention may be formulated in the form of a powder, granule, tablet, capsule, or liquid by mixing the Akkermansia massiliensis of the present invention with a suitable carrier, excipient, auxiliary active ingredient, etc. The composition of the present invention may be formulated as a product for enteral or oral administration. In addition, the composition of the present invention may be prepared into a product by an enteric coating using a known method so that the composition can pass through the stomach and reach the small intestine, and thereby the microorganism, as an active ingredient, can be rapidly released into the intestine.

Excipients that can be used in the present invention include sugars such as sucrose, lactose, mannitol, and glucose; and starches such as corn starch, potato starch, rice starch, and partially pregellantinized starch. As binders, polysaccharides such as dextrin, sodium alginate, carrageenan, guar gum, acacia, and agar; naturally-occurring macromolecular substances such as tragacanth, gelatin, and gluten; cellulose derivatives such as hydroxypropylcellulose, methylcellulose, hydroxypropylmethyl cellulose, ethylcellulose, hydroxypropylethylcellulose, and sodium carboxymethylcellulose; and polymers such as polyvinylpyrrolidone, polyvinyl alcohol, polyvinylacetate, polyethylene glycol, polyacrylic acid, polymethacrylic acid, and vinyl acetate resin.

The disintegrants to be used in the present invention may include cellulose derivatives such as carboxymethyl cellulose, calcium carboxymethyl cellulose, and low-substituted hydroxypropyl cellulose, and starches such as sodium carboxymethyl starch, hydroxypropyl starch, corn starch, potato starch, rice starch, and partially gelatinized starch.

Examples of the lubricants to be used in the present invention include talc, stearic acid, calcium stearate, magnesium stearate, colloidal silica, hydrosilicon dioxide, various types of waxes, hydrogenated oils, etc.

Coating agents include water-insoluble polymers such as a dimethylaminoethyl methacrylate-methacrylic acid copolymer, polyvinylacetal diethylaminoacetate, an ethyl acrylate-methacrylic acid copolymer, an ethyl acrylate-methyl methacrylate-chlorotrimethylammonium ethyl methacrylate copolymer, and ethyl cellulose; enteric polymers such as a methacrylic acid-ethyl acrylate copolymer, hydroxypropyl methyl cellulose phthalate, and hydroxypropyl methyl cellulose acetate succinate; and water-soluble polymers such as methyl cellulose, hydroxy propyl methyl cellulose, polyvinyl pyrrolidone, and polyethylene glycol, but are not necessarily limited thereto.

The administration dose of the strains, which are active ingredients in the composition for preventing or treating metabolic diseases of the present invention, may be determined by considering factors including the type of various diseases, age, weight, sex, medical condition of the patient, severity of the condition, and route of administration. Accordingly, although the dose regimen may vary widely, it may be routinely determined using standard methods.

The pharmaceutical composition of the present invention contains Akkermansia massiliensis as an active ingredient in an amount of 10² CFU to 10¹⁵ CFU based on the total weight of the composition, or contains a culture having an equivalent number of viable cells.

Generally, for adult patients, 1Х10² or more live, low-temperature sterilized, or inactivated bacteria, preferably 1Х10² to 1Х10¹⁵ of live, low-temperature sterilized, or inactivated bacteria may be administered once or in multiple divided doses as needed. The exact formulation, route of administration, and dose of the pharmaceutical composition disclosed herein may be determined by a physician in consideration of the patient's conditions.

In an embodiment of the present invention, the composition contains Akkermansia massiliensis in the range of about 1Х10² to about 1Х10¹⁵ cells/g composition, about 1Х10³ to about 1Х10¹⁴ cells/g composition, preferably about 1Х10⁴ to about 1Х10¹³ cells/g composition, more preferably about 1Х10⁵ to about 1Х10¹² cells/g composition, even more preferably about 1Х10⁶ to about 1Х10¹¹ cells/g composition, about 1Х10⁷ to about 1Х10¹⁰ cells/g composition, about 1Х10⁸ to about 1Х10¹² cells/g composition.

In an embodiment of the present invention, the composition contains Akkermansia massiliensis in the range of about 1Х10² to about 1Х10¹⁵ cells/mL composition, about 1Х10³ to about 1Х10¹⁴ cells/mL composition, preferably about 1Х10⁴ to about 1Х10¹³ cells/mL composition, more preferably about 1Х10⁵ to about 1Х10¹² cells/mL composition, even more preferably about 1Х10⁶ to about 1Х10¹¹ cells/mL composition, about 1Х10⁷ to about 1Х10¹⁰ cells/mL composition, about 1Х10⁸ to about 1Х10¹² cells/mL composition.

In another embodiment of the present invention, the composition contains Akkermansia massiliensis in the range of about 1Х10⁶ to about 1Х10¹⁰ cells/g or cells/mL composition, preferably about 1Х10⁸ to about 1Х10¹⁰ cells/g or cells/mL composition, more preferably about 1Х10⁹ to about 1Х10¹⁰ cells/g or cells/mL composition.

In an embodiment of the present invention, the composition of the present invention may further contain other probiotic strains or one or more prebiotics in addition to Akkermansia massiliensis.

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

Examples of prebiotics that may be used in the present invention include, but are not limited to, inulin and inulin-type fructans, oligofructose, beta-glucans, xylose, arabinose, arabinoxylan, ribose, galactose, rhamnose, cellobiose, fructose, lactose, salicin, sucrose, glucose, esculin, trehalose, maltose, mannose, melibiose, mucus or mucin, raffinose, fructooligosaccharides, galacto-oligosaccharides, amino acids, alcohols, fermentable carbohydrates, and any combination thereof.

Another aspect of the present invention relates to a food composition containing Akkermansia massiliensis, a culture thereof, or a dried product thereof. The food composition of the present invention may contain Akkermansia massiliensis, a culture thereof, or a dried product thereof as an active ingredient.

The food composition of the present invention may be prepared into health functional foods such as functional beverages, health supplementary foods, and special nutritional supplementary foods, and the food types may include beverages such as teas, juices, carbonated beverages, and ionic beverages, processed dairy products such as milk and yogurt, foods such as gums, rice cakes, Korean traditional sweet snacks, breads, confectioneries, and noodles, and health functional food preparations such as powders, tablets, and capsules.

The food composition of the present invention may contain, in addition to the active ingredients, a sweetener, a flavoring agent, a physiologically active ingredient, minerals, etc.

Sweeteners may be natural or synthetic. Natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Both natural and synthetic flavoring agents may be used. Preferably, natural flavoring agents are used. Natural flavoring agents may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or from green tea leaves, leaves of Solomon's Seal, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo nuts, etc. may be used. Synthetic flavoring agents may be used from esters, alcohols, aldehydes, terpenes, etc.

As physiologically active substances, catechins such as catechin, epicatechin, gallocatechin, and epigallocatechin, and vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, and riboflavin may be used.

Minerals that may 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.

In addition, the food composition of the present invention may contain, in addition to the sweetener, etc., a preservative, an emulsifier, an acidulant, a thickener, etc., as needed. These preservative, emulsifier, etc. are known in the art, and any of those known in the art may be used.

Another aspect of the present invention may be a cosmetic composition, and the cosmetic composition contains, as an active ingredient, the Akkermansia massiliensis, preferably the Akkermansia massiliensis strain EB-AMDK39(Accession No. KCTC 13765BP), a culture of the strain, or a dried product of the strain.

The cosmetic composition of the present invention may be prepared in various forms according to a conventional method for preparing a cosmetic composition. Specifically, the cosmetic composition of the present invention may be prepared in a formulation selected from the group consisting of a solution, an ointment for external use, a cream, a soothing gel, a foam, a nourishing toner, an emollient toner, a pack, an emollient, a body wash, an emulsion, a makeup base, an essence, a soap, a liquid cleanser, a bath agent, a sun screen cream, a sun oil, a suspension, an emulsified suspension, a paste, a gel, a lotion, a powder, a soap, a foam cleansing, an oil, a powder foundation, an emulsified suspension foundation, a wax foundation, a patch, and a spray, but is not necessarily limited thereto.

In addition, the cosmetic composition may contain conventional excipients such as stabilizers, solubilizers, vitamins, pigments, and fragrances commonly used in the field of cosmetic compositions, and may contain a carrier acceptable for cosmetic use.

Acceptable carriers for cosmetic use include, but are not limited to, purified water, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, surfactants, absorbents, thickeners, viscosity stabilizers, chelating agents, buffers, preservatives, and lower alcohols. Humectants, anti-inflammatory agents, antibactericides, antifungal agents, vitamins, sunscreens, antibiotics, perfumes, and dyes may also be included as needed.

Still another aspect of the present invention provides a veterinary composition or feed additive for preventing or treating metabolic diseases, containing Akkermansia massiliensis strain EB-AMDK39 (Accession No. KCTC 13765BP) or a culture thereof. The strain of Akkermansia massiliensis strain EB-AMDK39 (Accession No. KCTC 13765BP) is the same as described above.

The veterinary composition or feed additive for preventing or treating metabolic diseases may be prepared by adding the Akkermansia massiliensis strain EB-AMDK39 (Accession No. KCTC 13765BP) at an appropriate effective concentration range according to various feed preparation methods known in the art.

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

Examples

Example 1: Isolation and identification of Akkermansia massiliensis strain EB-AMDK39

1.1. Isolation and identification of the strain

In order to isolate Akkermansia sp. from the feces of a healthy Korean (female, 7 years old, BMI 19.9), the strain was isolated after selective cultivation using a mucin medium suggested by Derrien (0.4 g/L　monopotassium 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 an acidic trace element solution, 1 mL an alkaline trace element solution, 1 mL a vitamin solution, 2.5 g/L porcine gastric mucus (Type III), and 0.25 g/L anhydrous sodium sulfide) under strict anaerobic conditions (5% H₂, 15% CO₂, and 80% N₂) using an anaerobic chamber. (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).

1.2. Microscopic observation

In order to confirm whether the isolate is Akkermansia massiliensis, the isolate was observed under a microscope, and the results are shown in FIG. 1. In FIG. 1, A indicates Akkermansia muciniphila　strain ATCC BAA-835 (type strain), and B indicates Akkermansia massiliensis strain EB-AMDK39 at a magnification of 1,000x. As shown in FIG. 1, as a result of observing Akkermansia muciniphila strain ATCC BAA-835 (A) and the Akkermansia massiliensis strain EB-AMDK39 (B) at a magnification of 1,000x, it was confirmed that the shapes of the strains were similar, with oval or rod-shaped cells.

1.3. PCR analysis

In order to confirm whether the isolate was Akkermansia massiliensis, the isolate was subjected to PCR analysis using the AM-specific primers (SEQ ID NO: 2 and SEQ ID NO: 3) shown in Table 1 below, and the results are shown in FIG. 2. In FIG. 2, Lane M indicates a DNA size marker, Lane 1 indicates Akkermansia muciniphila (ATCC BAA-835), which is a positive control, Lane 2 indicates Akkermansia massiliensis strain(EB-AMDK39), and Lane 3 indicates the result of the negative control (distilled water).

As shown in FIG. 2, it was confirmed that the Akkermansia massiliensis strain EB-AMDK39 of the present invention showed a band with a result value similar to that of　the Akkermansia muciniphila strain ATCC BAA-835 (type strain).

Designation	Direction	Sequence (5'→3')	Amplicon size	SEQ ID NO:
AM1	Forward	CAG CAC GTG AAG GTG GGG AC	327 bp	SEQ ID NO: 2
AM2	Reverse	CCT TGC GGT TGG CTT CAG AT	327 bp	SEQ ID NO: 3

1.4. Random Amplified Polymorphic DNA (RAPD) analysis

In order to confirm whether the Akkermansia massiliensis strain EB-AMDK39 isolated as above is identical to the previously reported strains of the same Akkermansia sp., RAPD, which is a type of molecular typing, was performed. To this end, genomic DNA extracted from the bacteria was amplified using the universal primers shown in Table 2 below, electrophoresed on a 1% agarose gel for 1 hour and 30 minutes, and DNA fragmentation patterns were compared on a UV perforator, and the results are shown in FIG. 3.

Designation	Direction	Sequence (5'→3')	SEQ ID NO:
ERIC-1	Forward	ATG TAA GCT CCT GGG GAT TCA C	SEQ ID NO: 4
ERIC-2	Reverse	AAG TAA GTG ACT GGG GTG AGC G	SEQ ID NO: 5
(GTG)₅	Forward/Reverse	GTG GTG GTG GTG GTG	SEQ ID NO: 6

As confirmed through FIG. 3, the Akkermansia massiliensis strain EB-AMDK39 of the present invention showed a different RAPD band pattern when compared to the Akkermansia muciniphila strain ATCC BAA-835 (type strain). Since it is known that the RAPD band pattern of Akkermansia muciniphila　is different when the species are different, it was confirmed that the Akkermansia massiliensis strain EB-AMDK39 of the present invention is a species different from the Akkermansia muciniphila strain ATCC BAA-835.

1.5. Whole genome sequencing

In order to analyze the similarity between the Akkermansia massiliensis strain EB-AMDK39 isolated as above and Akkermansia muciniphila strain ATCC BAA-835 at the genome level, the whole genome sequence was analyzed using the PacBio technique and compared with that of the type strain, as shown in Table 3 below.

Genome Statistics	Strain
Genome Statistics	ATCC BAA-835	EB-AMDK39
Accession No.	CP001071	CP025834
Assembly Level	Complete	Complete
Seq. Category	Chromosome	Chromosome
Total Size (Mb)	2.6641	2.7242
GC (%)	55.8	55.3
Protein	2,246	2,140
Gene	2,321	2,357
CDS	2,257	2,293
Coding	2,246	2,250
rRNA	9	9
tRNA	52	52
Other RNA	3	3
Pseudogene	11	44
ANI Identity (%)*	-	90.1368
*ANI identity means similarity to ATCC BAA-835.

As can be seen in Table 3 above, it was confirmed that there was a difference when comparing the whole genome statistics between the Akkermansia massiliensis strain EB-AMDK39 of the present invention and the Akkermansia muciniphila strain ATCC BAA-835, and as a result of measuring the average nucleotide identity (ANI) values, it was confirmed that the values were below the cut-off line (95-96%) that determines the species boundary.

The genome information of the Akkermansia massiliensis strain EB-AMDK39 obtained in Examples above was functionally classified. The protein coding genes were functionally classified according to the cluster of orthologous groups (COG) definition, and the results are shown in Table 4 below. Based on COG, which is a search-oriented database for comparison to find orthologous genes, the function of each gene in the genome of the Akkermansia massiliensis strain EB-AMDK39 was confirmed, and was compared with the Akkermansia muciniphila strain ATCC BAA-835, and the results are shown in Table 4.

CODE	COG functional description	BAA-835	EB-AMDK39
J	Translation, ribosomal structure and biogenesis	185	198
A	RNA processing and modification	0	0
K	Transcription	74	80
L	Replication, recombination and repair	96	114
B	Chromatin structure and dynamics	0	0
D	Cell cycle control, cell division, chromosome partitioning	24	24
Y	Nuclear structure	0	0
V	Defense mechanisms	50	46
T	Signal transduction mechanisms	65	88
M	Cell wall/membrane/envelope biogenesis	194	238
N	Cell motility	12	14
Z	Cytoskeleton	3	3
W	Extracellular structures	10	10
U	Intracellular trafficking, secretion, and vesicular transport	30	40
O	Posttranslational modification, protein turnover, chaperones	109	114
C	Energy production and conversion	94	99
G	Carbohydrate transport and metabolism	133	156
E	Amino acid transport and metabolism	150	154
F	Nucleotide transport and metabolism	56	59
H	Coenzyme transport and metabolism	91	102
I	Lipid transport and metabolism	56	54
P	Inorganic ion transport and metabolism	96	96
Q	Secondary metabolites biosynthesis, transport and catabolism	13	18
R	General function prediction only	168	206
S	Function unknown	90	115
-	Not assigned	309	494

As can be seen in Table 4, it was confirmed that the Akkermansia massiliensis strain EB-AMDK39 of the present invention and the Akkermansia muciniphila strain ATCC BAA-835 match in 22 out of 25 COG functional codes, excluding "Nuclear structure (Y)", "RNA processing and modification (A)", and "Chromatin structure and dynamics (B)". In total, 2,028 and 1,799 genes in the Akkermansia massiliensis strain EB-AMDK39 and the Akkermansia muciniphila strain ATCC BAA-835, respectively, were classified into COG codes, but 494 and 309 genes, respectively, were not classified. Specifically, it was confirmed that the Akkermansia massiliensis strain EB-AMDK39 of the present invention has more genes than the Akkermansia muciniphila 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 Akkermansia massiliensis strain EB-AMDK39 of the present invention has clear differences from the Akkermansia muciniphila strain ATCC BAA-835 over the genome.

1.6. Phylogenetic tree analysis using full-length 16S rRNA gene sequences

In order to analyze the nucleotide sequence of the full-length 16S rRNA gene of the Akkermansia massiliensis strain EB-AMDK39 isolated as described above, the 16S rRNA gene was amplified using the 27F and 1541R primers shown in Table 5 below, and the sequence was determined using a 3730xl DNA analyzer. Based on the 16S rRNA gene sequence of the Akkermansia massiliensis strain EB-AMDK39 of the present invention obtained in this manner and other previously published strains of the same genus, a phylogenetic tree was created and the result is shown in FIG. 4.

The phylogenetic analysis was performed using MEGA-X, and the phylogenetic tree was constructed through the neighbor-joining method using 1000 bootstraps, which is shown in FIG. 4. The average nucleotide identity (ANI) value was used to evaluate the evolutionary distance using the pyani v0.2.7 program with the -m ANIb setting. Complete or draft genome sequences of the Akkermansia muciniphila strain ATCC BAA-835 (Accession No. GCF_000020225.1), the Akkermansia muciniphila strain CBA5201 (Accession No. GCF_004104435.1), the Akkermansia muciniphila strain JCM30893 (Accession No. GCF_009731575.1), the Akkermansia muciniphila strain EB-AMDK19 (Accession No. GCF_004015105.1), the Akkermansia muciniphila strain EB-AMDK27 (Accession No. GCF_010223015.1), and the Akkermansia glycaniphila Pyt strain (Accession No. GCF_900097105.1) were downloaded from the from NCBI Genome database (https:/www.ncbi.nlm.nih.gov/genome/) and used. A phylogenetic tree was created using the 16S rRNA gene sequences of other strains of the same species, and the results are shown in FIG. 4a.

Designation	Direction	Sequence (5'→3')	Amplicon size	SEQ ID NO:
27F	Forward	AGA GTT TGA TCM TGG CTC AG	1,505 bp	SEQ ID NO: 7
1541R	Reverse	AAG GAG GTG ATC CAG CCG CA	1,505 bp	SEQ ID NO: 8

As shown in FIG. 4, the phylogenetic tree analysis of the evolutionary relationship using 16S rRNA gene sequence analysis confirmed that although the Akkermansia massiliensis strain EB-AMDK39 strain genetically belongs to the genus Akkermansia, it is a species different from Akkermansia muciniphila, and its full-length 16S rRNA gene sequence was 100% identical to that of the 16S rRNA gene of Akkermansia massiliensis (Akkermansia sp. Marseille-P6666), thus confirming that it is Akkermansia massiliensis.

1.7. Comparison of presence or absence of genes based on genome and screening of functional genes

The presence or absence of genes based on the genomes of the Akkermansia massiliensis strain EB-AMDK39 and the Akkermansia muciniphila strain ATCC BAA-835 were compared. A compatible contig database was constructed by applying the Anvi'o version 7.1 (https:/merenlab.org/2016/02/27/the-anvio-interactive-interface/) program, and information analysis was performed on the constructed database based on the COG and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases. Based on this, a genome repository was created, and pan genome analysis was performed with the -minbit 0.5, -mcl-inflation 10, and -use-ncbi-blast options. The results of gene presence/absence comparison based on the above analysis and genes specifically present in the Akkermansia massiliensis strain EB-AMDK39 are shown in FIG. 5. In FIG. 5, SCG Clusters represent single-copy core gene clusters. CbiE, CbiL, CbiK, CbiC, CbiG, and CbiD, which are indicated in the figure, are genes that play a direct role in vitamin B12 production and were specifically observed in the genome of Akkermansia massiliensis strain EB-AMDK39, and represent Synthesis of vitamin b12 adenosyl cobalamide, Cobalt-precorrin-2 C(20)-methyltransferase, Sirohydrochlorin cobaltochelatase, Cobalt-precorrin-8 methylmutase, cobalt-precorrin-4 methyltransferase, and Cobalt-precorrin-5B C(1)-methyltransferase, respectively.

As confirmed through FIG. 5, the Akkermansia massiliensis strain EB-AMDK39 and Akkermansia muciniphila　strain ATCC BAA-835 share a high proportion of SCG clusters as they are strains belonging to　the genus Akkermansia, and the presence/absence pattern of genes specific to each strain was confirmed. Among the genes specific to the Akkermansia massiliensis strain EB-AMDK39, the gene constitution (CbiE, CbiL, CbiK, CbiC, CbiG, and CbiD) that plays a direct role in vitamin B12 synthesis was confirmed as a prominent pattern.

1.8. Confirmation of ability of Akkermansia massiliensis to produce short-chain fatty acids (SCFAs)

Short chain fatty acids (SCFAs), such as butyrate, acetate, and propionate, are metabolites produced by intestinal bacteria and play an important role in the host's energy metabolism, and they are involved in energy balance as signaling mediators that act on G protein-coupled receptors (GPR41 and GPR43).

Short-chain fatty acids (SCFAs) decrease intestinal motility and increase intestinal transit speed through GPR41 in enteroendocrine cells. This induces peptide YY (PYY) secretion, thereby reducing energy intake and preventing obesity. In addition, GPR43 by short-chain fatty acids induces glucagon-like peptide 1 (GLP-1) to increase satiety through increased insulin sensitivity, and the activation of GPR43 inhibits insulin signaling in fat tissue, thereby preventing fat accumulation. Short-chain fatty acids (SCFAs) can improve glucose metabolism and activate intestinal gluconeogenesis (IGN), which can reduce food intake through the gut-brain neural circuit. In addition, vitamin B12 affects the short-chain fatty acid-producing ability of the strain. Specifically, it can act as a coenzyme that catalyzes the conversion of succinic acid to propionic acid. The Akkermansia massiliensis strain EB-AMDK39 of the present invention has methylmalonyl-coA mutase that catalyzes the conversion of succinic acid into propionic acid using vitamin B12 as a cofactor.

In Example above, the genetic constitution that plays a direct role in vitamin B12 synthesis specifically present in the Akkermansia massiliensis strain EB-AMDK39 was confirmed, and thus, in order to confirm the changes in functional metabolites according to the presence or absence of vitamin B12 during culture, the strain was cultured in a test tube, and the content of short-chain fatty acids (SCFAs) contained in the culture medium was analyzed by gas chromatography (GC). To this end, the culture was centrifuged at 12,000 xg for 5 minutes, and the supernatant was recovered, and the supernatant was filtered using a 0.2 ㎛ syringe filter and used for analysis. A gas chromatograph (Agilent 7890N) equipped with an FFAP column (30 m Х 0.320 mm, 0.25 ㎛ phase) was used, and the conditions were set as shown in Table 6, and the analysis results are shown in Table 7 below.

Flow	H₂: 40 mL/min, Air: 350 mL/min
Injector Temp.	240℃
Detector Temp.	250℃
Oven Temp.	40℃ (hold 2 min)→65℃/10 min (hold 2 min)→240℃/10 min (hold 5 min)
Injection Vol.	2 ㎕
Split Ratio	20:1

Strain	Addition of Vitamin B12	Amount of short-chain fatty acid Production (㎍/mL)
Strain	Addition of Vitamin B12	Acetate	Propionate	Succinate
ATCC BAA-835	None	280.64 ± 32.21	32.44 ± 14.25	785.09 ± 32.41
ATCC BAA-835	0.1 mg/L	653.4 ± 78.84	1415.97 ± 145.01	7.23 ± 6.98
EB-AMDK39	None	618.14 ± 79.15	1433.01 ± 211.19	20.84 ± 28.41
EB-AMDK39	0.1 mg/L	606.09 ± 75.30	1429.03 ± 165.42	10.20 ± 28.50

As can be seen in Table 7, the Akkermansia massiliensis strain EB-AMDK39 of the present invention exhibited a short-chain fatty acid-producing ability that is different from that of Akkermansia muciniphila strain ATCC BAA-835. Specifically, when vitamin B12 was not added to the culture medium, the major short-chain fatty acids of Akkermansia muciniphila strain ATCC BAA-835 were acetate and succinate, whereas the Akkermansia massiliensis strain EB-AMDK39 of the present invention produced acetate and propionate as major short-chain fatty acids regardless of the presence or absence of vitamin B12, and in particular, it was confirmed that the amount of propionate production was about 40 times higher. Through the results above, it can be seen that the Akkermansia massiliensis strain EB-AMDK39 of the present invention has a clear difference in short-chain fatty acid-producing ability compared to Akkermansia muciniphila strain ATCC BAA-835 depending on the presence or absence of vitamin B12.

Example 2: Microbiological characteristics and safety analysis of Akkermansia massiliensis strain EB-AMDK39

2.1. Confirmation of sugar utilization of Akkermansia massiliensis strain EB-AMDK39

In order to determine the sugar utilization of　the Akkermansia massiliensis strain EB-AMDK39 of the present invention isolated above, the strain was cultured using the API50CH kit (Biomerieux, France), and the growth by utilizing each sugar was compared with that of the Akkermansia muciniphila type strain (ATCC BAA-835), and the results are shown in Table 8 below.

No	Carbohydrates	BAA-835	EB-AMDK39	No	Carbohydrates	BAA-835	EB-AMDK39
0	Negative control	-	-	25	Esculine	-	-
1	Glycerol	-	-	26	Salicine	-	-
2	Erythritol	-	-	27	D-Cellobiose	-	-
3	D-Arabinose	w	w	28	D-Maltose	-	-
4	L-Arabinose	w	w	29	*D-Lactose* *(bovine origin)*	+	-
5	*Ribose*	+	w	30	D-Melibiose	-	-
6	D-Xylose	w	w	31	D-Saccharose (sucrose)	-	-
7	L-Xylose	w	w	32	D-Trehalose	-	-
8	Adonitol	-	-	33	Inuline	-	-
9	β-Methyl-xyloside	-	-	34	D-Melezitose	-	-
10	*D-Galactose*	w	-	35	D-Raffinose	-	-
11	D-Glucose	+	+	36	Amidon (starch)	-	-
12	*D-Fructose*	w	-	37	Glycogene	-	-
13	*D-Mannose*	+	-	38	Xylitol	-	-
14	L-Sorbose	-	-	39	Gentiobiose	-	-
15	L-Rhamnose	-	-	40	D-Turanose	-	-
16	Dulcitol	-	-	41	D-Lyxose	w	w
17	Inositol	-	-	42	D-Tagatose	-	-
18	D-Mannitol	-	-	43	D-Fucose	-	-
19	D-Sorbitol	-	-	44	*L-Fucose*	+	w
20	Methyl-α D-mannopyranoside	-	-	45	D-Arabitol	-	-
21	Methyl-α D-glucopyranoside	-	-	46	L-Arabitol	-	-
22	N-Acetylglucosamine	+	+	47	Potassium Gluconate	-	-
23	Amygdaline	-	-	48	Potassium 2-Ketogluconate	-	-
24	Arbutine	-	-	49	Potassium 5-Ketogluconate	w	w
+ : growth, w : weak growth, - : no growth,

As can be seen in Table 8 above, it was confirmed that the Akkermansia massiliensis strain EB-AMDK39 of the present invention has differences in the ability of utilizing ribose, D-galactose, D-fructose, D-mannose, D-lactose, and L-fucose, compared to the Akkermansia muciniphila type strain (ATCC BAA-835).

2.2. Confirmation of antimicrobial susceptibility of A. massiliensis strain

In order to determine the antimicrobial susceptibility of the Akkermansia massiliensis strain EB-AMDK39, the minimum inhibitory concentration (MIC) for a total of 16 antimicrobial agents, including piperacillin-tazobactam (PTZ), ceftizoxime (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), was determined according to the broth microdilution method of the Clinical & Laboratory Standard Institute (CLSI) guidelines, and the results are shown in Table 9 below.

Cut-off values (mg/L)	Antibiotics	PTZ	CTZ	CHL	CLI	MEM	MXF	MTZ	TET
	CLSI (Anaerobes)	≥128/4	≥128	≥32	≥8	≥16	≥8	≥32	≥16
	EFSA (E.coli, G-)	N/A	N/A	>16	N/A	N/A	N/A	N/A	>8
ATCC BAA-835		≤0.5/4 (S)	2 (S)	4 (S)	≤0.125 (S)	1 (S)	>32 (R)	≤0.25 (S)	2 (S)
EB-AMDK39		≤0.5/4 (S)	256 (R)	8 (S)	>64 (R)	4 (S)	>32 (R)	1 (S)	0.5 (S)
Cut-off values (mg/L)	Antibiotics	AMP	GEN	KAN	STR	NAL	SMZ	TMP	APR
	CLSI (Anaerobes)	≥2	N/A	N/A	N/A	N/A	N/A	N/A	N/A
	EFSA (E.coli, G-)	>8	>2	>8	>16	>16	>256	>2	>8
ATCC BAA-835		≤0.5 (S)	64 (R)	>256 (R)	256 (R)	64 (R)	8 (S)	1 (S)	>128 (R)
EB-AMDK39		4 (S)	64 (R)	>256 (R)	512 (R)	>128 (R)	8 (S)	1 (S)	>128 (R)
PTZ: piperacillin-tazobactam, CTZ: ceftizoxime, CHL: chloramphenicol, CLI: clindamycin, MEM: meropenem, MXF: moxifloxacin, MTZ: metronidazole, TET: tetracycline, AMP: ampicillin, GEN: gentamicin, KAN: kanamycin, STR: streptomycin, NAL: nalidixic acid, SMZ: sulfamethoxazole, TMP: trimethoprim, APR: apramycin

As can be seen in Table 9, the Akkermansia massiliensis strain EB-AMDK39 of the present invention was confirmed to be resistant to ceftizoxime, clindamycin, quinolone-based antibiotics such as moxifloxacin, ciprofloxacin, and nalidixic acid, and aminoglycoside-based antibiotics such as gentamicin, kanamycin, streptomycin, and apramycin, and to be susceptible to antibiotics except these. In addition, there were some differences in the antibiotic resistance pattern when compared to Akkermansia muciniphila　strain ATCC BAA-835.

Resistance to quinolone-based and aminoglycoside-based antibiotics is considered to be an intrinsic resistance that exists equally in the same Akkermansia sp. In most cases, this resistance is caused by the ineffective delivery of the drug to the site of action, and it seems to apply to cases where each antibiotic has a different range of action (antibacterial spectrum) and is effective only against specific types of microorganisms. In addition, in the report by Filardi et al., it was confirmed that all Akkermansia muciniphila strains showed low sensitivity to ciprofloxacin and aminoglycosides regardless of their genotypes, and it was reported that the intracellular efflux pump system is potentially involved in this resistance.

Additionally, bioinformatics-based PlasmidFinder (https:/cge.cbs.dtu.dk/services/PlasmidFinder/), PHASTER(https:/phaster.ca/), Mobile Element Finder(cge.cbs.dtu.dk/services/MobileElementFinder) programs were applied to the complete genome of the Akkermansia massiliensis strain EB-AMDK39 of the present invention to confirm whether it was obtainable or endogenous. As a result, no significant sequences were present in mobile genetic factors such as plasmids, mobile genetic elements (MGEs), and prophage in the Akkermansia massiliensis strain EB-AMDK39, and no phenotype-related resistance genes were detected.

2.3. Analysis of hemolytic activity and virulence factors of A. massiliensis strain

In order to verify the safety of the Akkermansia massiliensis strain EB-AMDK39 isolated as above, whether it possessed hemolytic activity was evaluated. 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 casein pancreatic digest, 3.0 g/L soybean pancreatic digest, 2.5 g/L dextrose, 5.0 g/L sodium chloride, 2.5 g/L potassium phosphate, 15 g/L agar), and the results are shown in FIG. 6. As can be seen from FIG. 6, the Akkermansia massiliensis strain EB-AMDK39 of the present invention did not exhibit beta-hemolysis (a completely transparent area around the colony) associated with pathogenicity.

In order to confirm the presence or absence of possible toxigenic genes in the Akkermansia massiliensis strain EB-AMDK39, VFDB (http:/www.mgc.ac.cn/VFs/) and Virulence Finder 2.0 (https:/cge.cbs.dtu. dk/services/VirulenceFinder/) platforms were utilized. Virulence factors include bacterial toxins, cell surface proteins mediating bacterial attachment, cell surface carbohydrates and proteins that protect bacteria, and hydrolytic enzymes that may contribute to bacterial pathogenicity. The analysis tool used above is specified in the 'Guidelines for Functional Raw Materials of Probiotics for Health Functional Foods', and > 60% similarity, > 60% coverage, > 40 amino acids in length, and E-value 0.01 were applied as the basic analysis conditions.

Using the Virulence Finder analysis tool, the presence of virulence factor genes was confirmed by comparative analysis of the Akkermansia massiliensis strain EB-AMDK39 with well-known virulence bacteria (E. coli, Enterococcus, Listeria, and S. aureus), and as a result, no virulence factors were detected in both strains.

As a result of performing virulence factor analysis based on VFDB, six possible virulence factors were identified in the genome of the Akkermansia massiliensis strain EB-AMDK39, respectively. However, the genes detected are related to cell wall/membrane/envelope biogenesis, adherence or other functions, which are essential elements for cell structure, function, and adaptation. These genes are also involved in pathogenic bacterial adaptation or survival in the hostile/host environment, and thus were confirmed as virulence factors in the database (Table 10). Therefore, it was confirmed that these genes are essential for bacterial survival in the absence of other pathogenic mechanisms, and these genes are not genes that cause virulence.

Match	Function	Accession (VFDB)	Locus_Tag, Identity (%)
Match	Function	Accession (VFDB)	BAA-835	EB-AMDK39
Glucose-1-Phosphate Thymidylyltransferase	Capsule	VFG047039	60.137	60.481
GDP-Mannose 4,6-Dehydratase	Capsule	VFG002225	65.607	65.607
Chaperonin GroEL	Adherence	VFG012095	61.466	61.616
Elongation Factor Tu	Adherence	VFG046465	72.843	72.589
Catalase katA	Stress survival	VFG037028	-	73.629
Acyl Carrier Protein	Other	VFG011430	-	61.039

2.4. Confirmation of cytotoxicity of A. massiliensis strain

To evaluate the cytotoxicity of the Akkermansia massiliensis EB-AMDK39 strain isolated as described above, a lactate dehydrogenase (LDH) assay was performed with reference to the toxin production evaluation method among the safety evaluation test methods notified in the Guide to Safety Evaluation of Functional Raw Materials of Health Functional Foods and Probiotics. The cell lines used in the test were colon epithelial cell lines (HT-29 and Caco-2), and the test was performed by treating the Akkermansia massiliensis strain EB-AMDK39 at a concentration of 10³ CFU/mL to 10⁷ CFU/mL. Pseudomonas aeruginosa　ATCC 17831 was used as a positive control and treated at the same concentration as EB-AMDK39. As a result, the cytotoxicity was calculated using the equation below, converted into cell viability, and the results are shown in FIG. 7.

[Equation 1]

As shown in FIG. 7, the Akkermansia massiliensis strain EB-AMDK39 of the present invention was confirmed to be non-cytotoxic at all concentrations tested.

The Akkermansia massiliensis strain EB-AMDK39, isolated from human feces, was identified by biochemical (API) and molecular biological methods (16S rRNA sequence analysis, RAPD, and genome analysis, etc.) using Akkermansia muciniphila strain ATCC BAA-835 as a reference strain. It was confirmed to be a safe strain by showing no antibiotic resistance genes, hemolytic activity, virulence factors, and cytotoxicity. The Akkermansia massiliensis strain EB-AMDK39 isolated based on these results was deposited with Korean Collection for Type Cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology and assigned Accession No. KCTC 13765BP.

Example 3: Confirmation of anti-obesity efficacy by administering live Akkermansia massiliensis strain EB-AMDK39 in an obesity-induced mouse model

3.1. Strain samples

The Akkermansia muciniphila strain ATCC BAA-835 (control) and the Akkermansia massiliensis strain EB-AMDK39 used in this Example were prepared at a concentration of 1Х10⁸ CFU/100 ㎕ PBS (25% glycerol, 0.05% cysteine in PBS) for use in animal testing.

3.2. Animal Testing

Animal experiments were performed in compliance with the Animal Use and Care Protocol of the Institutional Animal Care and Use Committee (IACUC). The experimental animals were purchased as 7-week-old male C57BL/6 mice, which were housed for 8 weeks after an acclimation period of one week. The housing environment was maintained at a constant temperature (22℃) and relative humidity (40-60%) with a 12-hour light/dark cycle. In order to induce obesity, high-fat feed (60 kcal% fat; Research Diets Inc., NJ, USA) was fed, and the normal control group (Normal) was fed a 10 kcal% fat feed. Drinking water was provided ad libitum. The experimental groups were divided into five groups as shown in Table 11 below. Each of the groups was Normal (normal diet normal control), HFD (high-fat diet fed obesity-induced group), ORL (high-fat diet fed obesity-induced group + orlistat administration group), ATCC BAA-835 (high-fat diet fed obesity-induced group + Akkermansia muciniphila strain ATCC BAA-835 administration group), and Akkermansia massiliensis strain EB-AMDK39 (high-fat diet fed obesity-induced group + Akkermansia massiliensis strain EB-AMDK39 administration group), and orlistat (ORL; Xenical, 10 mg/kg), which is an oral obesity treatment drug, were used as positive controls.

	Group name	Feed for Feeding	Oral Administration Substances
1	Normal	Normal diet (10 Kcal% fat)	PBS (25% glycerol)
2	HFD	High-fat diet (60 Kcal% fat)	PBS (25% glycerol)
3	BAA-835	High-fat diet (60 Kcal% fat)	Akkermansia muciniphila BAA-835
4	EB-AMDK39	High-fat diet (60 Kcal% fat)	Akkermansia massiliensis EB-AMDK39
5	ORL	High-fat diet (60 Kcal% fat)	Orlistat 10 mg/kg

3.3. Confirmation of body weight and weight gain

During the 8-week experiment, the body weight of each experimental group was measured weekly, and the body weight gain was calculated. The results are shown in the graph in FIG. 8. Referring to FIG. 8, compared to the obesity-induced group that only consumed a high-fat diet, the body weight and body weight gain significantly decreased in the group administered with the Akkermansia muciniphila strain ATCC BAA-835 and the group administered with the Akkermansia massiliensis strain EB-AMDK39. Through these results, it can be confirmed that the Akkermansia massiliensis strain EB-AMDK39 induces weight loss.

3.4. Confirmation of weight changes in fat tissue and liver tissue

Upon completion of the experiment, the mice were anesthetized with CO₂, sacrificed, and the weights of the extracted subcutaneous fat, epididymal fat, mesenteric fat, and liver tissues were measured. The results are shown as a graph in FIG. 9.

Referring to FIG. 9, the weights of subcutaneous fat, epididymal fat, and mesenteric fat tissues in the obesity-induced group (HFD) significantly increased compared to the normal diet group. In contrast, in the EB-AMDK39 strain administration group, the weight of fat tissue decreased compared to the obesity-induced group, and statistical significance was confirmed in subcutaneous fat and epididymal fat.

In addition, the liver tissue weight of the obesity-induced group (HFD) was significantly increased compared to the normal diet group (Normal) but significantly decreased following administration of the Akkermansia massiliensis strain EB-AMDK39. These results suggest that the administration of the Akkermansia massiliensis strain EB-AMDK39 of the present invention inhibits obesity by reducing body fat.

3.5. Analysis of blood lipid biochemical indicators

After fasting the experimental animals in each group for 18 hours, they were sacrificed and the blood was collected and centrifuged to obtain serum. The total cholesterol (TC) and triglyceride (TG) levels in the serum, which are lipid content indicators, were measured, and the results are shown as a graph in FIG. 10. In particular, total cholesterol and triglyceride concentrations were measured using LabAssay™ Cholesterol and LabAssay™ Triglyceride products from Wako Chemicals.

Referring to FIG. 10, the results of analyzing the total blood cholesterol level showed that the level significantly increased in the obesity-induced group (HFD) compared to the normal diet group (Normal) and decreased again by oral administration of the Akkermansia massiliensis strain EB-AMDK39.

Blood triglyceride (TG) levels were significantly increased in the obesity-induced group (HFD) compared to the normal diet group. However, the blood triglyceride levels in the group administered with the Akkermansia massiliensis strain EB-AMDK39 were significantly reduced compared to those of the obesity-induced group (HFD).

3.6. Comparison of size of fat cells

In order to measure and compare the size of adipocytes, subcutaneous fat obtained after sacrificing the mice was formalin-fixed and embedded in paraffin. Paraffin-embedded specimens were sectioned at 4 ㎛ and were stained with hematoxylin and eosin. The observed microscopic images were then analyzed to measure the diameter of adipocytes using the ImageJ program, and the results are shown in FIG. 11.

Referring to FIG. 11, the diameter of adipocytes in the HFD group significantly increased as obesity was induced by feeding a high-fat diet. In contrast, the group orally administered with the Akkermansia muciniphila strain ATCC BAA-835, Akkermansia massiliensis strain EB-AMDK39, or orlistat (ORL) showed a significant decrease compared to the HFD group.

It can be confirmed that oral administration of Akkermansia　massiliensis strain EB-AMDK39 decreased the adipocyte size and fat accumulation.

The present invention can be variously modified and changed without departing from the spirit and scope thereof, and this fact will be obvious to those skilled in the art. The specific embodiments described in this specification are only for illustrating preferred embodiments of the present invention, and should not be construed as limiting the present invention.

The protection scope of the present invention should be determined by the appended claims, and the various modifications and changes described above are intended to be included in the protection scope of the present invention.

[Accession No.]

Depository Institution: Korean Collection for Type Cultures

Accession No.: KCTC 13765BP

Deposition Date: December 5, 2018

Claims

A composition comprising an Akkermansia massiliensis strain, and a culture or dried product of the strain.
The composition of claim 1, wherein the Akkermansia massiliensis strain is not an Akkermansia muciniphila strain or an Akkermansia glycaniphila strain.
The composition of claim 2, wherein the Akkermansia massiliensis strain has less than 95% genome-wide average nucleotide identity (gANI) compared to Akkermansia muciniphila or Akkermansia glycaniphila.
The composition of claim 1, wherein the Akkermansia massiliensis strain is an Akkermansia massiliensis strain EB-AMDK39 with Accession No. KCTC 13765BP.
The composition of claim 1, wherein the composition comprises a live bacteria, a pasteurized bacteria, or an inactivated bacteria of the Akkermansia massiliensis strain.
The composition of claim 1, wherein the composition is for preventing or treating metabolic diseases which are obesity, diabetes, insulin resistance, hypertension, hyperlipidemia, dyslipidemia, coronary arteriosclerosis, arteriosclerosis, and non-alcoholic fatty liver disease.
The composition of claim 1, wherein the composition comprises the Akkermansia massiliensis strain in an amount of 1Х10² CFU to 1Х10¹⁵ CFU, 1Х10³ CFU to 1Х10¹⁴ CFU, 1Х10⁴ CFU to 1Х10¹³ CFU, or 1Х10⁵ CFU to 1Х10¹² CFU, or 1Х10⁶ CFU to 1Х10¹¹ CFU, 1Х10⁷ CFU to 1Х10¹⁰ CFU, or 10⁸ CFU to 10¹² CFU based on the total weight of the composition, or comprises a culture having an equivalent number of a live bacteria, a pasteurized bacteria, or an inactivated bacteria.
The composition of claim 1, wherein the Akkermansia massiliensis is a substantially purified and freeze-dried strain.
The composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.
The composition of claim 1, wherein the composition further comprises a different probiotic strain or one or more prebiotics.
The composition of claim 1, wherein the composition is a veterinary composition, a feed additive, or a cosmetic composition.