Composition containing Akkermansia strains

Abstract

The present invention relates to pharmaceutical compositions, food compositions, veterinary compositions, and cosmetic compositions effective in preventing or treating metabolic diseases, comprising the Akkermansia massiliensis strain, its cultures, or dried products. While traditional probiotics generally have only minimal therapeutic effects on metabolic diseases, the next-generation pharmabiotic strains of the present invention exhibit such superior preventive or therapeutic effects on metabolic diseases that they can be used as new preventive and therapeutic tools.

Description

[Technical Field]

[0001] The present invention relates to a composition comprising a strain of the genus Akkermansia (Akkermansia sp.), and more specifically, to a composition comprising the strain Akkermansia massiliensis. [Background technology]

[0002] Metabolic diseases refer to a range of conditions that occur simultaneously due to chronic disorders, such as obesity, diabetes, hypertension, dyslipidemia, coronary artery disease, arteriosclerosis, and non-alcoholic steatohepatitis. Most metabolic diseases are accompanied by overweight or obesity. The most serious problem with metabolic diseases is the development of chronic complications such as diabetic retinopathy, diabetic nephropathy, diabetic foot complications, diabetic neuropathy, dyslipidemia, and cardiovascular disease.

[0003] Most of these chronic complications, once they occur, follow an irreversible progression path. Until now, there has been no way to completely interrupt this process, and if not treated appropriately, they can cause severe symptoms and are recognized as one of the most serious diseases threatening the health of modern people.

[0004] Until now, metabolic diseases with such complex symptoms have been treated by individually administering hypoglycemic agents, antihypertensive agents, cholesterol-lowering agents, and other medications. Therefore, in order to efficiently manage and treat metabolic diseases with such complex symptoms, there is a need to develop new therapeutic agents that can treat a variety of symptoms simultaneously.

[0005] To overcome the side effects of conventional drugs and immunomodulators, research is being conducted on using probiotics alone or in combination with therapeutic agents. As research into the role of gut bacteria in health promotion becomes more active, interest in probiotic preparations is increasing.

[0006] As a treatment technology for metabolic diseases using probiotics, Patent Document 1 discloses Lactobacillus reuteri that inhibits the absorption of lipids in the body, Patent Document 2 discloses Lactobacillus curvatus that suppresses the decrease in blood cholesterol and obesity, and Patent Document 3 discloses Lactobacillus johnsonii that suppresses obesity along with the decrease in blood cholesterol.

[0007] However, such probiotics have a negligible improvement effect on metabolic diseases, and thus there is a limitation in that it is difficult to use them as new preventive and therapeutic tools.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0009] The present invention is for overcoming the limitations of the above-described prior art, and an object of the present invention is to provide a pharmaceutical composition effective for a preventive or therapeutic agent for metabolic diseases containing Akkermansia muciniphila strain.

[0010] Another object of the present invention is to provide a food composition useful for the prevention or improvement of metabolic diseases containing Akkermansia muciniphila strain. [[ID=forty]]

[0011] Still another object of the present invention is to provide a veterinary composition or feed additive useful for the prevention or improvement of metabolic diseases containing Akkermansia muciniphila strain.

[0012] A further object of the present invention is to provide a cosmetic composition useful for preventing or improving metabolic diseases, including those caused by the Akkermansia massiliensis strain. [Means for solving the problem]

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

[0014] Another aspect of the present invention relates to a composition comprising the Ackermansia massiliensis strain, a culture or dried product of the strain.

[0015] Another aspect of the present invention relates to a food composition useful for preventing or improving metabolic diseases, including the strain of Ackermansia massiliensis.

[0016] A further object of the present invention relates to a veterinary composition or feed additive that is useful for preventing or improving metabolic diseases, including those caused by the Akkermansia massiliensis strain.

[0017] A further object of the present invention relates to cosmetic compositions useful for preventing or improving metabolic diseases, including strains of Ackermansia massiliensis. [Effects of the Invention]

[0018] The pharmaceutical composition containing the Akkermansia massiliensis strain of the present invention can be used in pharmaceutical compositions for the treatment and / or prevention of metabolic diseases, health functional food compositions, feed compositions, cosmetic compositions, and the like.

[0019] The pharmaceutical composition containing the Akkermansia massiliensis strain of the present invention suppresses weight gain and body fat increase, reduces insulin resistance, and lowers total cholesterol blood concentration, thereby effectively managing and treating complex symptoms of metabolic diseases (metabolic syndromes) such as diabetes, obesity, insulin resistance, and fatty liver.

[0020] The Ackermansia massiliensis strain of the present invention exhibits such excellent preventive or therapeutic effects against metabolic diseases that it can be used as a new preventive and therapeutic tool as a next-generation pharmaciotics strain. [Brief explanation of the drawing]

[0021] [Figure 1] This shows the results of microscopic observation of the Akkermansia massiliensis strain (KCTC 13765BP) and the Akkermansia muciniphila ATCC BAA-835 strain of the present invention. [Figure 2] These are the PCR analysis results for the Akkermansia massiliensis EB-AMDK39 strain and the Akkermansia muciniphila ATCC BAA-835 strain of the present invention. [Figure 3a] This shows the results of RAPD (Random Amplified Polymorphic DNA) analysis of the genomic DNA of the Akkermansia massiliensis EB-AMDK39 strain and the Akkermansia muciniphila ATCC BAA-835 strain of the present invention. [Figure 3b] This shows the results of RAPD (Random Amplified Polymorphic DNA) analysis of the genomic DNA of the Akkermansia massiliensis EB-AMDK39 strain and the Akkermansia muciniphila ATCC BAA-835 strain of the present invention. [Figure 4] This figure shows a comparison of the phylogenetic relationship between the Akkermansia massiliensis strain of the present invention and other Akkermansia strains. [Figure 5] This figure shows the results of comparing the presence or absence of genes in the full-length gene base of the Akkermansia massiliensis EB-AMDK39 strain and the Akkermansia muciniphila ATCC BAA-835 strain of the present invention. [Figure 6] This figure shows the results of cytotoxicity tests for the Akkermansia massiliensis EB-AMDK39 strain and the Akkermansia muciniphila ATCC BAA-835 strain of the present invention. [Figure 7]This figure shows the results of confirming the presence or absence of hemolytic activity in the Akkermansia massiliensis EB-AMDK39 strain and the Akkermansia muciniphila ATCC BAA-835 strain of the present invention. [Figure 8] This report presents the results of an analysis of body weight changes in the group treated with the Akkermansia massiliensis EB-AMDK39 strain, the negative control group (HFD), and the Akkermansia muciniphila ATCC BAA-835 strain according to the present invention. [Figure 9] This report presents the results of analyzing changes in subcutaneous fat, epididymal fat, and superior mesenteric fat volume in the group treated with the Akkermansia massiliensis EB-AMDK39 strain of the present invention, the negative control group (HFD), and the Akkermansia muciniphila ATCC BAA-835 strain. [Figure 10] These are the results of measuring changes in blood cholesterol and serum triglycerides concentrations by ELISA in the group treated with the Akkermansia massiliensis EB-AMDK39 strain of the present invention, the negative control group (HFD), and the Akkermansia muciniphila ATCC BAA-835 strain. [Figure 11] These are photographs and graphs showing the size of adipocytes and the degree of fat accumulation formed in the mesenteric adipose tissue of each experimental group of animals when treated with the Ackermansia massiliensis EB-AMDK39 strain of the present invention. [Modes for carrying out the invention]

[0022] The present invention will be described in detail below with reference to the attached drawings.

[0023] In this invention, the "Akkermansia massiliensis strain" is a strain of the genus Akkermansia sp. and belongs to the phylum Verrucomicrobiota. The Akkermansia massiliensis strain of this invention is not Akkermansia muciniphila or Akkermansia glycaniphila.

[0024] In this specification, “metabolic disease” refers to the overall symptoms of diseases such as obesity, diabetes, hypertension, dyslipidemia, coronary artery disease, arteriosclerosis, and non-alcoholic steatohepatitis. In this specification, “metabolic syndrome,” “metabolic disease,” “metabolic disorder,” or “metabolic abnormality” are used interchangeably.

[0025] As used herein, the term "obesity" can refer to a condition in which there is an excessive accumulation of body fat. Obesity is defined as body fat accounting for 25% or more of body weight, or 30-35% or more for women. However, the Body Mass Index (BMI) is the most commonly used measurement method. For Westerners, a BMI of 30 kg / m² is considered obese. 2 If you exceed this, you are considered obese (25-30 kg / m²). 2 In this case, it is defined as being overweight, and for East Asians, it is 28 kg / m². 2 If you exceed this range, you are considered obese (23-28 kg / m²). 2 This can be considered overweight.

[0026] As used herein, the term "dyslipidemia" may mean a condition in which lipid components in the blood, such as triglycerides, LDL cholesterol, phospholipids, and free fatty acids, are increased, or a condition in which HDL cholesterol is decreased. Such dyslipidemia may be one or more selected from the group consisting of, for example, dyslipidemia, hyper-LDL cholesterolemia, hypertriglyceridemia, and hypo-LDL cholesterolemia.

[0027] As used herein, the term "subject" means all animals, including humans, that have developed or are at risk of developing metabolic disease. Such animals may include, but are not limited to, humans, as well as mammals such as dogs, cats, hamsters, rabbits, cattle, horses, sheep, pigs, goats, camels, and antelopes that require treatment for similar symptoms.

[0028] When the number "approximately" precedes a number, it means ±20%, preferably 10% of the aforementioned numerical value.

[0029] Akkermansia can also be substantially purified. The term "substantially purified" as used here means that a sample is substantially enriched with a specific bacterial strain or two or more bacterial strains (e.g., Bacteroidetes, Firmicutes, Proteobacteria, or Verrucomicrobia). A sample may be substantially purified or enriched with the bacterial strain or strain mixture of interest.

[0030] In this case, the proportion of the relevant bacterial strain(s) in the sample must be at least approximately 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%, or the proportion of undesirable or other bacterial strains must be approximately 40%, 30%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less.

[0031] In this specification, the Akkermansia massiliensis strain may be live, pasteurized, or dead. In this specification, "pasteurized strain" means an Akkermansia massiliensis strain that has been heat-treated at a low temperature. In one example, a pasteurized Akkermansia massiliensis strain means an Akkermansia massiliensis strain that has been heat-treated at a temperature of 50°C to 110°C for 5 to 30 minutes.

[0032] According to one embodiment, pasteurization is performed by heating in the range of approximately 50°C to 110°C, approximately 50°C to 100°C, preferably approximately 60°C to 95°C, and more preferably approximately 70°C to 90°C. Preferably, pasteurized bacteria are heat-treated at approximately 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70°C.

[0033] In other embodiments, pasteurization is performed by heat treatment at temperatures of approximately 71, 72, 73, 74, 75°C, 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°C, or 100°C. In other embodiments, pasteurization is performed by heating at approximately 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110°C.

[0034] In this application, "dead bacteria agent" refers to a form in which live bacteria have been prevented from growing by heat treatment or other means. Dead bacteria agents may include cytoplasm, cell membranes, cell walls, antimicrobial active substances such as bacteriocins, polysaccharides, and / or organic acids.

[0035] In this specification, the term “culture” means the product obtained by culturing the Akkermansia massiliensis strain, and may include fermentates. In one example, the culture may be a fermentate obtained by culturing the Akkermansia massiliensis strain in a culture medium. The “fermentate” means the result of the enzymatic or metabolic decomposition of organic matter using microorganisms. In this application, “fermentation” may mean all activities or processes, including the enzymatic or metabolic decomposition of organic matter using microorganisms, excluding putrefaction reactions.

[0036] The culture (or fermented product) may be the whole culture of the Akkermansia massiliensis strain, its dilution, concentrate, dried product, freeze-dried product, crushed product, and / or fractions. The concentrate can be obtained by centrifuging or evaporating the culture, the dried product can be obtained by drying the culture using a dryer or the like, the freeze-dried product can be obtained by freeze-drying the culture using a freeze-dryer or the like, and the crushed product can be obtained by physically or ultrasonically treating the strain or culture. The fractions can be obtained by applying methods such as centrifugation and chromatography to the culture, crushed product, etc.

[0037] The cultured or fermented product may be in a solid phase (solid, e.g., a dry product), a liquid phase (liquid), or a fluid phase, but is not necessarily limited to these.

[0038] In this specification, terms such as “to treat,” “to treat,” and “to treat” mean to prevent, slow, halt, or reverse the progression or severity of an existing condition, disease, disorder, or symptom.

[0039] In this specification, "functional food" is the same term as "food for special health use (FoSHU)," and refers to a food composition with high medical and therapeutic effects that has been processed to efficiently exhibit biological regulatory functions in addition to nutritional supply. In this specification, "functional food" may be used interchangeably with terms such as "health supplement food" and "health food."

[0040] One aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of metabolic diseases, comprising the Akkermansia massiliensis strain with deposit number KCTC 13765BP.

[0041] The Akkermansia massiliensis strain of the present invention was isolated from the feces of healthy Koreans, and is a monococcal or diplococcal bacterium with oval-shaped cells measuring 0.5-1 μm in size. It is an anaerobic bacterium, non-motile, Gram-negative, and does not form resistant spores, and is a mucin-degrading bacterium. The Akkermansia massiliensis strain produces several mycogenic enzymes that allow it to use mucus as a source of carbon and nitrogen, and it can metabolize some carbon sources, including N-acetylglucosamine, producing short-chain fatty acids such as propionic acid and acetic acid as its main metabolites.

[0042] The Ackermansia massiliensis strain of the present invention possesses the 16s rRNA gene of Sequence ID No. 1.

[0043] In the present invention, the Akkermansia massiliensis strain is not an Akkermansia muciniphila or Akkermansia glycaniphila strain. Phylogenetic analysis and mean nucleotide identity (ANI) analysis of the Akkermansia massiliensis strain of the present invention have clearly confirmed that it is not an Akkermansia muciniphila or Akkermansia glycaniphila strain.

[0044] The Akkermansia massiliensis strain of the present invention has a whole-genome mean nucleotide identity (gANI) of less than approximately 95% compared to the standard Akkermansia strain, ATCC BAA-835.

[0045] The Akkermansia massiliensis strains of the present invention have gANI of approximately 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, and 79% of the genome of Akkermansia muciniphila, respectively, with less than 95% gANI.

[0046] In some embodiments, the Akkermansia massiliensis strain of the present invention (for example, the Akkermansia massiliensis strain KCTC 13765BP of the present invention) may have less than 95% gANI compared to the genome of Akkermansia glycanifila, such as approximately 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%.

[0047] The Akkermansia massiliensis strain of the present invention can be usefully used to prevent or treat diabetes, obesity, obesity-related diseases, insulin resistance, fatty liver, dyslipidemia, or metabolic diseases by suppressing weight gain and increased body fat, reducing insulin resistance, lowering total cholesterol blood levels, and decreasing the content of GPT (glutamic pyruvic transaminase), a marker of hepatotoxicity in the blood.

[0048] The pharmaceutical composition of the present invention may contain a live strain of Akkermansia massiliensis, or a pasteurized strain or a dead strain. Pasteurization of the Akkermansia massiliensis strain means heating at a temperature of 50°C or higher but less than 110°C for 5 minutes or more. For example, it can be pasteurized at 70°C for 30 minutes. The pasteurized form of the Akkermansia massiliensis EB-AMDK39 strain may reduce body fat accumulation more significantly than the live strain.

[0049] The exact reasons why pasteurized forms of Akkermansia massiliensis strains are more effective are not fully understood, but it can be hypothesized that pasteurization enhances the metabolic advantages of Akkermansia massiliensis strains such as Amuc_1100 in the host.

[0050] The advantageous effects of the strain of the present invention are thought to be related to a gene cluster (Amuc_1098–Amuc_1102) containing Amuc_1100, and to originate from polypeptides that interact with the signaling pathway of Toll-like receptor 2 (TLR2), which is present on the surface of intestinal mucosal epithelial cells and immune cells located near the intestinal barrier, and regulates intestinal homeostasis and host metabolism.

[0051] For example, the Amuc_1100 polypeptide is expected to maintain the integrity of the intestinal mucosal barrier and promote the secretion of cytokines (e.g., IL-6, IL-8, and IL-10) from immune cells by interacting with TLR2 present on the surface of immune cells to regulate or enhance the TLR-2 signaling pathway.

[0052] The prolipoprotein idasylglyceryltransferase gene (Amuc_1104) is located in very close proximity to the gene cluster (Amuc_1098–Amuc_1102). Furthermore, Amuc_1100 is stably maintained under the temperature conditions used during pasteurization and may contribute to the influence of pasteurized strains.

[0053] The Ackermansia massiliensis strain of the present invention can be cultured and recovered through a separation process such as centrifugation, and then produced and used in the form of a viable fungal agent by drying, for example, freeze-drying.

[0054] The Akkermansia massiliensis strain of the present invention is sensitive to oxygen and is preferably cultured under anaerobic conditions (nitrogen saturation of 80-90%, hydrogen saturation of 0-5%, and carbon dioxide saturation of 5-20%).

[0055] The components of the liquid culture medium during cultivation can affect the growth of the bacterial strain and the production of active ingredients. Therefore, it is necessary to establish the components and content conditions of the liquid culture medium optimized for culturing the Ackermansia massiliensis strain of the present invention.

[0056] The liquid culture medium may contain, but is not limited to, one or more substances 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-propenediol, and butyric acid as a carbon source. Preferably, it may contain glucose and N-acetylglucosamine. The liquid culture medium may, but is not limited to, one or more substances selected from the group consisting of tryptone, peptone, soy peptone, L-glutamic acid, and ammonium as a nitrogen source.

[0057] The liquid culture medium may contain, but is not limited to, 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.

[0058] The aforementioned liquid culture medium may have a pH of 6.8 to 7.2. Preferably, the pH is 7.0. pH can change the charge of the amine group and carboxylic acid of amino acids, which are the units of enzyme proteins important for cellular metabolism, and can affect protein activity. In addition, changes in pH in the external environment can affect the ionization of microbial nutrients, and thus affect how microorganisms take in nutrients.

[0059] The liquid culture medium is preferably cultured in a medium containing glucose, N-acetylglucosamine, threonine, soy peptone, or any combination thereof.

[0060] Pharmaceutical formulation can be carried out by known methods, and is preferably in the form of pharmacochemically feasible oral, topical, transdermal, transmucosal, and injectable formulations, and more preferably in the form of an oral formulation.

[0061] The compositions of the present invention may further contain pharmaceutically acceptable carriers and / or excipients in addition to the active ingredients, and may be formulated in dosage forms with a variety of other commonly used pharmaceutically appropriate additives such as binders, decomposing agents, coating agents, and lubricants.

[0062] The pharmaceutical composition of the present invention can be formulated into powder, granule, tablet, capsule, or liquid form by mixing the Akkermansia massiliensis strain of the present invention with a suitable carrier, excipient, auxiliary active ingredient, etc. The composition of the present invention can be formulated as a product for intra-intestinal or oral administration. Furthermore, the composition of the present invention can be formulated as a product by enteric coating using known methods so that it passes through the gastrointestinal tract and reaches the small intestine, and the active ingredient microorganism is rapidly released into the intestines.

[0063] Excipients usable 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 pre-gelanthinized starch.

[0064] The binder contains 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 hydroxypropyl cellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, hydroxypropyl ethylcellulose, and sodium carboxymethylcellulose; and polymers such as polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, polyethylene glycol, polyacrylic acid, polymethacrylic acid, and vinyl acetate resin.

[0065] As decomposition agents, cellulose derivatives such as carboxymethylcellulose, carboxymethylcellulose calcium, and low-substituted hydroxypropylcellulose, as well as starches such as sodium carboxymethyl starch, hydroxypropyl starch, corn starch, potato starch, rice starch, and partially pre-gelatinized starch may be used.

[0066] Examples of lubricants that can be used in the present invention include talc, stearic acid, calcium stearate, magnesium stearate, colloidal silica, hydrated silicon dioxide, various types of waxes, and hydrogenated oils.

[0067] Examples of coating agents include dimethylaminoethyl methacrylate-methacrylic acid copolymer, polyvinyl acetal diethylaminoacetic acid, ethyl acrylate-methacrylic acid copolymer, ethyl acrylate-methyl methacrylate-chlorotrimethylammonium ethyl methacrylate copolymer, water-insoluble polymers such as ethyl cellulose, enteric polymers such as methacrylic acid-ethyl acrylate copolymer, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, and water-soluble polymers such as methylcellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, and polyethylene glycol, but are not necessarily limited thereto.

[0068] In the composition for preventing or treating metabolic diseases of the present invention, the dosage of the strain as an active ingredient can be determined in consideration of factors including various disease types, age, weight, gender, the medical condition of the patient, the severity of the condition, and the administration route. Therefore, the volume therapy can vary widely but can be routinely determined using standard methods.

[0069] The pharmaceutical composition of the present invention contains the Akkermansia muciniphila strain as an active ingredient at a content of 10 2 to 10 15 CFU, or contains a culture having an equivalent number of viable bacteria.

[0070] Generally, in the case of adult patients, 1×10 2 or more viable bacteria, pasteurized bacteria, or dead bacteria, preferably 1×10 2 to 1×10 15Live bacteria, pasteurized bacteria, or dead bacteria may be administered in one or several doses as needed. The exact dosage form, route of administration, and dose of the pharmaceutical compositions disclosed herein may be determined by a physician in consideration of the patient's condition.

[0071] In one embodiment of the present invention, the composition of the present invention is approximately 1 × 10 2 〜1×10 15 Composition of cells / g, approximately 1 × 10 3 〜1×10 14 A composition of cells / g, preferably about 1 × 10⁻⁶ 4 〜1×10 13 A composition of cells / g, more preferably about 1 × 10 5 〜1×10 12 A composition of cells / g, more preferably about 1 × 10⁻⁶ 6 〜1×10 11 Composition of cells / g, approximately 1 × 10 7 〜1×10 10 Composition of cells / g, approximately 1 × 10 8 〜1×10 12 Contains Akkermansia massiliensis strains in the cell / g composition range.

[0072] In one embodiment of the present invention, the composition of the present invention is approximately 1 × 10 2 〜1×10 15 Composition of cells / mL, approximately 1 × 10⁶ 3 〜1×10 14 A composition with cells / mL, preferably about 1 × 10⁶ 4 〜1×10 13 A composition with cells / mL, more preferably about 1 × 10⁶ 5 〜1×10 12 A composition of cells / mL, more preferably about 1 × 10⁶ 6 〜1×10 11 Composition of cells / mL, approximately 1 × 10⁶ 7 〜1×10 10 Composition of cells / mL, approximately 1 × 10⁶ 8 〜1×10 12 Contains Akkermansia massiliensis strains in a composition range of cells / mL.

[0073] In another embodiment of the present invention, the composition of the present invention is approximately 1 × 10 6 〜1×10 10 A composition containing cells / g or cells / mL, preferably about 1 × 10⁻⁶ 8 〜1×10 10 A composition containing cells / g or cells / mL, more preferably about 1 × 10 9 〜1×10 10 Contains Akkermansia massiliensis strains in a composition range of cells / g or cells / mL.

[0074] In one 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 the Ackermansia massiliensis strain.

[0075] Other probiotic strains may include probiotics from Bacteroidetes, Firmicutes, Actinobacteria, and the phylum Proteobacteria.

[0076] In one example, other probiotics may include Ruminococcus, Clostridium, Bacteroides, Neglecta, Bifidobacterium, Egerthella, Clostridiaceae, Parabacteroides, Bilophila, Dorea, Collinsella, and Faecalibacterium.

[0077] Examples of prebiotics that may be used in the present invention include, but are not limited to, inulin, inulin-type fructans, oligofructose, β-glucan, xylose, arabinose, arabinoxylan, ribose, galactose, rhamnose, cellobiose, fructose, lactose, salicin, sucrose, glucose, esculin, trehalose, maltose, mannose, melibiose, mucus or mucin, raffinose, fructooligosaccharides, galactooligosaccharides, amino acids, alcohols, fermented carbohydrates, and any combination thereof.

[0078] Another aspect of the present invention relates to a food composition comprising the Akkermansia massiliensis strain, a culture or dried product thereof. The food composition of the present invention may contain the Akkermansia massiliensis strain, a culture or dried product of the strain as an active ingredient.

[0079] The food composition of the present invention can be manufactured as a functional food, health supplement, or special nutritional supplement, such as a functional beverage. The food may take the form of beverages such as tea, juice, carbonated drinks, or ion drinks; processed dairy products such as milk or yogurt; foods such as gum, mochi, traditional sweets, bread, confectionery, or noodles; or functional food preparations such as powders, tablets, or capsules.

[0080] In addition to its active ingredient, the food composition of the present invention may contain sweeteners, flavoring agents, physiologically active ingredients, minerals, and the like.

[0081] Sweeteners can be natural or synthetic. Examples of natural sweeteners include corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

[0082] Both natural and synthetic flavorings can be used. Natural flavorings are preferred. Natural flavorings may be derived from apples, lemons, citrus fruits, grapes, strawberries, peaches, etc., or from tea leaves, Polygonatum odoratum, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. In addition, flavorings derived from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo nuts, etc., can be used. Synthetic flavorings may include esters, alcohols, aldehydes, terpenes, etc.

[0083] As physiologically active substances, catechins such as catechin, epicatechin, gallocatechin, and epigallocatechin, as well as vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, and riboflavin may be used.

[0084] Minerals that can be used include calcium, magnesium, chromium, cobalt, copper, fluorides, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, and zinc.

[0085] Furthermore, the food composition of the present invention may contain, as necessary, preservatives, emulsifiers, acidulants, thickeners, etc., in addition to the sweeteners mentioned above. Such preservatives, emulsifiers, etc., are well known in the industry, and any of those known in the industry may be used.

[0086] Another embodiment of the present invention may be a cosmetic composition, which contains, as an active ingredient, the Akkermansia massiliensis strain, preferably the Akkermansia massiliensis EB-AMDK39 strain (accession number KCTC 13765BP), a culture of the strain, or a dried product of the strain.

[0087] The cosmetic composition of the present invention can be manufactured in a variety of forms by conventional cosmetic composition manufacturing methods. Specifically, the cosmetic composition of the present invention can be manufactured in dosage forms selected from the group consisting of, but is not limited to, solutions, topical ointments, creams, soothing gels, foams, nourishing lotions, softening lotions, packs, softening waters, body washes, emulsions, makeup bases, essences, soaps, liquid detergents, bath additives, sunscreen creams, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, cleansing foams, oils, powder foundations, emulsion foundations, wax foundations, patches, and sprays.

[0088] Furthermore, the cosmetic composition may contain common auxiliary agents such as stabilizers, solubilizers, vitamins, pigments, and fragrances that are commonly used in the field of cosmetic compositions, and may contain carriers that are acceptable for cosmetic use.

[0089] Acceptable carriers for cosmetic use include, but are not limited to, purified water, oils, waxes, fatty acids, fatty acid alcohols, fatty acid esters, surfactants, hygroscopic agents, thickeners, viscosity stabilizers, chelating agents, buffers, preservatives, and lower alcohols. Moisturizers, anti-inflammatory agents, antibacterial agents, antifungal agents, vitamins, sunscreens, antibiotics, perfumes, and dyes may also be included as needed.

[0090] A further embodiment of the present invention provides a veterinary composition or feed additive for the prevention or treatment of metabolic diseases, comprising the Akkermansia massiliensis strain (deposit number: KCTC 13765BP) or a culture thereof. The Akkermansia massiliensis strain (deposit number: KCTC 13765BP) is as described above.

[0091] The aforementioned veterinary compositions or feed additives for the prevention or treatment of metabolic diseases can be manufactured by adding Akkermansia massiliensis strain (deposit number: KCTC 13765BP) in an appropriate effective concentration range using a variety of feed manufacturing methods known in the industry.

[0092] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0093] Examples Example 1: Isolation and identification of Ackermansia massiliensis strain EB-AMDK39 1.1. Isolation and identification of bacterial strains To isolate Akkermansia massiliensis strains from the feces of a healthy Korean female (7 years old, BMI 19.9), strains were selected, cultured, and isolated using Derrien's mucin medium (0.4 g / L monopotassium monophosphate, 0.53 g / L sodium dichloride phosphate, 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 trace acid solution, 1 mL trace alkaline solution, 1 mL vitamin solution, 2.5 g / L porcine gastric mucus (Type III)), and 0.25 g / L sodium sulfide notahydrate) in an anaerobic chamber under severe anaerobic conditions (5% H2, 15% CO2, and 80% N2). (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).

[0094] 1.2. Microscopic observation To confirm whether the isolated strain was an Akkermansia massiliensis strain, the isolated strain was observed under a microscope, and the results are shown in Figure 1. In Figure 1, A is the standard Akkermansia muciniphila ATCC BAA-835 strain, and B is a micrograph of the Akkermansia massiliensis EB-AMDK39 strain magnified at 1,000x. As shown in Figure 1, the standard Akkermansia muciniphila ATCC BAA-835 strain (A) and the Akkermansia massiliensis EB-AMDK39 strain (B) were observed under 1,000x magnification, and it was confirmed that the patterns of the strains were similar in shape, with both being straight or curved rod-shaped cells.

[0095] 1.3. PCR analysis To confirm whether the isolated strain was an Akkermansia massiliensis strain, the isolated strain 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 Figure 2. In Figure 2, lane M is the DNA size marker, lane 1 is the positive control group Akkermansia muciniphila strain (ATCC BAA-835), lane 2 is the Akkermansia massiliensis EB-AMDK39 strain, and lane 3 is the negative control group (distilled water).

[0096] As shown in Figure 2, it was confirmed that the Akkermansia massiliensis EB-AMDK39 strain of the present invention produced result values ​​in a band similar to that of the Akkermansia muciniphila ATCC BAA-835 standard strain.

[0097] [Table 1]

[0098] 1.4.Random Amplified Polymorphic DNA (RAPD) analysis To verify whether the isolated Akkermansia massiliensis EB-AMDK39 strain is the same as previously reported strains of the same genus Akkermansia massiliensis, RAPD, a type of molecular typing, was performed. For this purpose, genomic DNA extracted from the bacterial cells was amplified using the general-purpose primers shown in Table 2 below, followed by electrophoresis on a 1% agarose gel for 1 hour and 30 minutes. The DNA segmentation patterns were then compared on a UV perforation machine, and the results are shown in Figure 3.

[0099] [Table 2]

[0100] As can be seen in Figure 3, the Akkermansia massiliensis EB-AMDK39 strain of the present invention showed a different RAPD band pattern compared to the Akkermansia muciniphila ATCC BAA-835 standard strain. It is known that the RAPD band pattern of Akkermansia muciniphila differs depending on the species, and it was confirmed that the Akkermansia massiliensis EB-AMDK39 strain of the present invention is a different species from the Akkermansia muciniphila ATCC BAA-835 standard strain.

[0101] 1.5. Whole genome sequencing To analyze the similarity between the isolated Akkermansia massiliensis EB-AMDK39 strain and the Akkermansia muciniphila ATCC BAA-835 strain at the genetic level, the entire nucleotide sequence of the genes was analyzed using PacBio techniques and compared with a standard strain, as shown in Table 3 below.

[0102] [Table 3]

[0103] As can be seen from Table 3 above, when comparing the overall genetic statistics of the Akkermansia massiliensis EB-AMDK39 strain and the Akkermansia muciniphila ATCC BAA-835 strain of the present invention, a difference was found. The ANI (Average Nucleotide Identity) value was measured and confirmed to be below the cut line (95-96%) that determines the species boundary.

[0104] The genomic information of the Akkermansia massiliensis EB-AMDK39 strain secured in the above example was functionally classified. Protein-coding genes were functionally classified according to the COG (Cluster of orthologous group) definition and are shown in Table 4 below. To find heterologous genes, the function of each gene on the full-length Akkermansia massiliensis EB-AMDK39 strain was confirmed based on the COG, a database of search centers used for comparison, and compared with the Akkermansia muciniphila ATCC BAA-835 strain, as shown in Table 4.

[0105] [Table 4]

[0106] As can be seen from Table 4, it was confirmed that the Akkermansia massiliensis EB-AMDK39 strain and Akkermansia muciniphila ATCC BAA-835 strain of the present invention correspond to 22 of the 25 COG functional codes, excluding "nuclear structure (Y)", "RNA processing and modification (A)", and "Chromatin structure and dynamics (B)".

[0107] Overall, 2,028 genes were isolated from the Akkermansia massiliensis EB-AMDK39 strain and 1,799 genes from the Akkermansia muciniphila ATCC BAA-835 strain using COG codes, but 494 genes and 309 genes from the Akkermansia muciniphila ATCC BAA-835 strain were found to be missing. More specifically, the Akkermansia massiliensis EB-AMDK39 strain of the present invention was found to have more genes than the Akkermansia muciniphila ATCC BAA-835 strain 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)".

[0108] Overall, it can be confirmed that the Akkermansia massiliensis EB-AMDK39 strain of the present invention has clear differences from the Akkermansia muciniphila ATCC BAA-835 strain in terms of full-length genetic material.

[0109] 1.6. Analysis of phylogenetic trees using the full-length 16S rRNA gene sequence To analyze the full-length 16S rRNA gene sequence of the Akkermansia massiliensis EB-AMDK39 strain isolated as described above, the 16S rRNA gene was amplified using the 27F and 1541R primers shown in Table 5 below, and then the sequence was determined using a 3730xl DNA analyzer. Based on the 16S rRNA gene sequences of the Akkermansia massiliensis EB-AMDK39 strain obtained in this way and those of other strains of the same genus that have been previously published, a phylogenetic tree was constructed and is shown in Figure 4.

[0110] The phylogenetic analysis was performed using MEGA-X, and the phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstraps, as shown in Figure 4.

[0111] The mean nucleotide identity (ANI) value was used to evaluate evolutionary distance by applying the pyani v0.2.7 program with the -m ANIb setting.

[0112] Akkermansia muciniphila ATCC BAA-835 strain (accession:GCF_000020225.1), Akkermansia muciniphila CBA5201 strain (accession:GCF_004104435.1), Akkermansia muciniphila JCM30893 strain (accession:GCF_009731575.1), Akkermansia muciniphila EB-AMDK19 strain (accession:GCF_00401510 5.1) The complete or draft genome sequences of the Akkermansia muciniphila EB-AMDK27 strain (accession:GCF_010223015.1) and the Akkermansia glycaniphila Pyt strain (accession:GCF_900097105.1) were downloaded from the NCBI genome database (https: / / www.ncbi.nlm.nih.gov / genome / ). Phylogenetic trees were constructed using the nucleotide sequences of the 16S rRNA gene of other strains of the same species and are shown in Figure 4a.

[0113] [Table 5]

[0114] As shown in Figure 4, sequencing analysis of the 16S rRNA gene revealed that the Akkermansia massiliensis EB-AMDK39 strain is genetically classified as belonging to the genus Akkermansia, but is a different species from Akkermansia muciniphila. The full-length 16S rRNA gene sequence is 100% identical to that of Akkermansia sp. Marseille-P6666, confirming that it is indeed Akkermansia massiliensis.

[0115] 1.7. Comparison of gene presence / absence in full-length genotypes and search for functional genes The presence or absence of genes in the full-length genotype of the Akkermansia massiliensis EB-AMDK39 strain and the Akkermansia muciniphila ATCC BAA-835 strain of the present invention was compared. A compatible contig database was constructed using the Anvi'o version 7.1 program (https: / / merenlab.org / 2016 / 02 / 27 / the-anvio-interactive-interface / ), and information analysis based on the COG and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases was performed on the constructed database.

[0116] Based on this, a genome repository was generated, and pan-genome analysis was performed with the -minbit0.5, -mcl-inflation10, and -use-ncbi-blast options. The results of comparing the presence / absence of genes based on the above analysis, and the genes specifically present in the Ackermansia massiliensis EB-AMDK39 strain are shown in Figure 5.

[0117] In Figure 5, SCG Clusters represent single-copy core gene clusters. CbiE, CbiL, CbiK, CbiC, CbiG, and CbiD shown in the figure are genes that play a direct role in vitamin B12 synthesis and have been specifically observed in the full-length gene of Ackermansia massiliensis EB-AMDK39. They 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.

[0118] As confirmed in Figure 5, the Akkermansia massiliensis EB-AMDK39 strain and the Akkermansia muciniphila ATCC BAA-835 strain belong to the genus Akkermansia, and while they share a high proportion of SCG clusters, the presence / absence of specific genes in each strain was confirmed. Among the genes specific to the Akkermansia massiliensis EB-AMDK39 strain, the gene configuration that plays a direct role in vitamin B12 synthesis (CbiE, CbiL, CbiK, CbiC, CbiG, and CbiD) was identified as a prominent feature.

[0119] 1.8. Confirmation of the short-chain fatty acid (SCFA) production ability of the Akkermansia massiliensis strain. Short-chain fatty acids (SCFAs), such as butyrate, acetate, and propionate, are metabolites produced by gut bacteria and play a crucial role in host energy metabolism. They act as signaling mediators on G protein-coupled receptors (GPR41 and GPR43), contributing to energy equilibrium.

[0120] Short-chain fatty acids (SCFAs) are mediated by enteroendocrine cells (GPR41), which reduces intestinal motility and increases intestinal movement speed. This induces the secretion of PYY (peptide YY), which reduces energy intake and prevents obesity.

[0121] Furthermore, GPR43 produced by short-chain fatty acids induces GLP-1 (Glucagon-like peptide 1), increasing insulin sensitivity and thus increasing satiety. GPR43 activity also suppresses insulin signaling in adipose tissue, preventing fat accumulation. Short-chain fatty acids (SCFAs) improve glucose metabolism and can activate IGN (intestinal gluconeogenesis), which can reduce food intake through the gut-brain neural circuit. In addition, vitamin B12 affects the short-chain fatty acid production ability of bacterial strains.

[0122] More specifically, it can act as a coenzyme that catalyzes the conversion of succinic acid to propionic acid. In the case of the Akkermansia massiliensis strain of the present invention, it has a methylmalonyl-coA mutase that catalyzes the conversion of succinic acid to propionic acid with vitamin B12 as a cofactor.

[0123] In the above example, to confirm the gene configuration that plays a direct role in the synthesis of vitamin B12, which is specifically present in the Akkermansia massiliensis EB-AMDK39 strain, and to confirm the changes in functional metabolites depending on the presence or absence of vitamin B12 during culture, the content of short-chain fatty acids (SCFAs) contained in the culture medium was analyzed by gas chromatography (GC) after culturing in test tubes.

[0124] For this purpose, the culture medium was centrifuged at 12,000xg for 5 minutes to collect the upper layer, which was then filtered using a 0.2 μm syringe filter before being used for 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 set as shown in Table 6, and the analysis results are shown in Table 7 below.

[0125] [Table 6]

[0126] [Table 7]

[0127] As can be seen from Table 7, the Akkermansia massiliensis EB-AMDK39 strain of the present invention showed a different short-chain fatty acid production ability compared to Akkermansia muciniphila ATCC BAA-835.

[0128] In detail, when vitamin B12 is not added to the culture medium, the main short-chain fatty acids of the Akkermansia muciniphila ATCC BAA-835 strain are acetic acid and succinate. In contrast, the Akkermansia massiliensis EB-AMDK39 strain of the present invention was found to have acetic acid and propionic acid as its main short-chain fatty acids, regardless of the presence or absence of vitamin B12, with particularly high production of propionic acid, approximately 40 times more.

[0129] Based on the results described above, it was confirmed that the Akkermansia massiliensis EB-AMDK39 strain of the present invention has a clear difference in short-chain fatty acid production ability compared to the Akkermansia muciniphila ATCC BAA-835 strain, depending on the presence or absence of vitamin B12.

[0130] Example 2: Analysis of the mycological characteristics and safety of the Ackermansia massiliensis EB-AMDK39 strain. 2.1 Confirmation of sugar utilization of isolated Akkermansia massiliensis EB-AMDK39 To understand the sugar utilization capabilities of the Akkermansia massiliensis EB-AMDK39 strain of the present invention isolated as described above, the strain was cultured using the API50CH kit (Biomerieux, France), and the ability to grow using each sugar was compared with the standard Akkermansia muciniphila strain (ATCC BAA-835). The results are shown in Table 8 below.

[0131] [Table 8]

[0132] As can be seen from Table 8 above, the Akkermansia massiliensis EB-AMDK39 strain of the present invention was found to differ from the Akkermansia muciniphila standard strain (ATCC BAA-835) in its ability to utilize ribose, D-galactose, D-fructose, D-mannose, D-lactose, and L-fucose.

[0133] 2.2 Confirmation of antimicrobial susceptibility of Akkermansia massiliensis strains To determine the susceptibility of the isolated Akkermansia massiliensis EB-AMDK39 strain to antimicrobial agents, the minimum inhibitory concentrations (MICs) 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), were determined according to the broth microdilution method of the Clinical & Laboratory Standard Institute (CLSI) guidelines. The results are shown in Table 9 below.

[0134] [Table 9]

[0135] As can be seen from Table 9, the Ackermansia massiliensis EB-AMDK39 strain of the present invention was found to be resistant to ceftizoxime, clindamycin, the quinolone antibiotics moxifloxacin, ciprofloxacin and nalidixic acid, and the aminoglycoside antibiotics gentamicin, kanamycin, streptomycin and apramycin, but susceptible to all other antimicrobial agents.

[0136] Furthermore, there were some differences in the antimicrobial resistance patterns when compared to Ackermansia muciniphila ATCC BAA-835.

[0137] Resistance to quinolone and aminoglycoside antimicrobial agents is a common characteristic of the Akkermansia genus and is considered an intrinsic resistance. In most cases, such resistance arises because the drug cannot effectively reach its target site, and is considered to be the case where each antibiotic has a different range of action (antimicrobial spectrum) and is effective only against specific types of microorganisms.

[0138] Furthermore, Filardi et al. reported that all Akkermansia muciniphila strains, regardless of genotype, exhibit low sensitivity to ciprofloxacin and aminoglycosides, suggesting that the intracellular efflux pump system is potentially involved in this resistance.

[0139] Furthermore, the complete, full-length genetic material of the Ackermansia massiliensis EB-AMDK39 strain of the present invention was subjected to bioinformatics-based programs PlasmidFinder (https: / / cge.cbs.dtu.dk / services / PlasmidFinder / ), PHASTER (https: / / phaster.ca / ), and Mobile Element Finder (cge.cbs.dtu.dk / services / MobileElementFinder) to confirm its acquired / endogenous properties.

[0140] As a result, no significant sequences were found in the Ackermansia massiliensis EB-AMDK39 strain, such as plasmids, MGEs (Mobile Genetic Elements), and mobile genetic elements like prophages, and no resistance genes related to the phenotype were detected.

[0141] 2.3. Analysis of hemolytic activity and pathogenic factors of isolated bacterial strains To verify the safety of the Akkermansia massiliensis EB-AMDK39 strain isolated as described above, we evaluated whether it possessed hemolytic activity. For this purpose, the strain was cultured using 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 Figure 6. As can be seen in Figure 6, the Akkermansia massiliensis EB-AMDK39 strain of the present invention did not exhibit β-hemolysis (completely transparent areas around the colony), which is related to pathogenicity.

[0142] To identify the presence or absence of possible toxigenic genes in the Akkermansia massiliensis EB-AMDK39 strain, we utilize the VFDB (http: / / www.mgc.ac.cn / VFs / ) and Virulence Finder 2.0 (https: / / cge.cbs.dtu.dk / services / VirulenceFinder / ) platforms. These toxic factors include bacterial toxins, cell surface proteins that mediate bacterial adhesion, cell surface carbohydrates and proteins that protect bacteria, and hydrolytic enzymes that can contribute to bacterial pathogenicity.

[0143] The analytical tools used as described above are those specified in the "Guidelines for Functional Probiotics in Health Functional Foods," and the following basic analytical conditions were applied: >60% similarity, >60% coverage, >40 amino acids in length, and E-value 0.01.

[0144] By applying the Virulence Finder analysis tool to the Ackermansia massiliensis EB-AMDK39 strain, and conducting a comparative analysis with well-known pathogenic bacteria (E. coli, Enterococcus, Listeria, and S. aureus), the presence or absence of pathogenic factor genes was confirmed, and no pathogenic factors were detected.

[0145] Analysis of pathogenic factors based on the VFDB revealed six possible virulence factors in the full-length gene of the Ackermansia massiliensis EB-AMDK39 strain. The detected genes are related to cell wall / membrane / envelope biogenesis, adherence, or other functions, and are essential elements for cellular structure, function, and adaptation. These genes are also involved in the adaptation or survival of pathogenic bacteria in a hostile / host environment, and were therefore identified as virulence factors from the database (Table 10). Thus, unless there are other pathogenic mechanisms, these genes are essential for bacterial survival, and it was confirmed that these genes do not induce pathogenicity.

[0146] [Table 10]

[0147] 2.4. Confirmation of cytotoxicity of isolated bacterial strains To confirm the cytotoxicity of the Akkermansia massiliensis EB-AMDK39 strain isolated as described above, lactate dehydrogenase (LDH) analysis was performed, referring to the toxin production evaluation method among the safety evaluation test methods specified in the "Safety Evaluation Guide for Probiotics, Functional Ingredients in Health Functional Foods." The cell lines used in the test were colon epithelial cell lines (HT-29 and Caco-2), and Akkermansia massiliensis EB-AMDK39 was subjected to 10 3 ~10 7 The samples were treated with CFU / mL and tested. Pseudomonas aeruginosa strain ATCC 17831 was used as a positive control group and treated with the same concentration as EB-AMDK39. The results, including cytotoxicity calculated using the formula below and converted to cell viability, are shown in Figure 7.

[0148]

number

[0149] As can be seen in Figure 7, the Ackermansia massiliensis EB-AMDK39 strain of the present invention was confirmed to be non-cytotoxic at all concentrations tested.

[0150] The strain Akkermansia massiliensis EB-AMDK39, isolated from human feces, was identified using biochemical (API) and molecular biological methods (16s rRNA sequence analysis, RAPD, and full-length gene analysis) with Akkermansia muciniphila ATCC BAA-835 as a control group. Furthermore, the isolated strain was confirmed to be a safe strain capable of probiotic function through antibiotic resistance testing, hemolytic activity, pathogenic factors, and cytotoxicity assessment. Based on these results, the isolated Akkermansia massiliensis EB-AMDK39 strain was deposited with the Korea Center for Biotechnology's Korea Reference Culture Collection (KCTC) and assigned accession number KCTC 13765BP.

[0151] Example 3: Confirmation of anti-obesity efficacy by administration of live Ackermansia massiliensis EB-AMDK39 in an obesity-inducing mouse model. 3.1. Strain samples The Akkermansia muciniphylla ATCC BAA-835 (control group) and Akkermansia massiliensis EB-AMDK39 strains used in this example were prepared for use in animal studies, with 1 × 10⁶ live cells. 8 Prepared at a CFU / 100μL PBS (25% glycerol, 0.05% cysteine / PBS) concentration.

[0152] 3.2. Animal Testing Animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) Animal Use and Care Protocol. Seven-week-old male C57BL / 6 mice were purchased and, after a one-week adaptation period, were housed for eight weeks. The housing environment was maintained at a constant temperature (22°C) and relative humidity (40-60%), with light and darkness adjusted in 12-hour cycles. A high-fat diet (60 kcal% fat; Research Diets Inc., NJ, USA) was given to induce obesity, while the normal control group was fed a 10 kcal% fat diet. Drinking water was provided free access.

[0153] The experimental groups were divided into five groups as shown in Table 11. Each group was: Normal (normal control group with a normal diet), HFD (obesity induction group with a high-fat diet), ORL (obesity induction group with a high-fat diet + orlistat administration group), ATCC BAA-835 (obesity induction group with a high-fat diet + administration of Akkermansia muciniphila ATCC BAA-835 strain), and Akkermansia massiliensis EB-AMDK39 (obesity induction group with a high-fat diet + administration of Akkermansia massiliensis EB-AMDK39 strain). The ATCC BAA-835 strain (live bacteria) and orlistat (ORL; Xenical, 10 mg / kg), an oral treatment for obesity, were used as positive control groups.

[0154] [Table 11]

[0155] 3.3. Confirmation of weight and weight gain During the 8-week experiment, the body weight of each experimental group was measured weekly, and the weight gain was calculated. The results are shown in Figure 8. Referring to Figure 8, compared to the obesity-inducing group that consumed only high-fat feed, the groups treated with Akkermansia muciniphila ATCC BAA-835 strain and Akkermansia massiliensis EB-AMDK39 strain showed a significant decrease in both body weight and weight gain. These results confirm that Akkermansia massiliensis EB-AMDK39 strain induces weight loss.

[0156] 3.4. Confirmation of weight changes in adipose tissue and liver tissue At the end of the experiment, the mice were anesthetized with CO2, and the weights of subcutaneous fat, epididymal fat, mesenteric fat, and liver tissue, which were removed by sacrificing the mice, were measured. The results are shown in Figure 9.

[0157] Referring to Figure 9, the weight of subcutaneous fat, epididymal fat, and mesenteric fat tissue was significantly increased in the obesity-induced group compared to the normal diet group. Conversely, in the group that consumed the same high-fat diet as the obesity-induced group but was administered the Akkermansia massiliensis EB-AMDK39 strain, the weight of adipose tissue decreased compared to the obesity-induced group, and statistical significance was confirmed for subcutaneous fat and epididymal fat.

[0158] Furthermore, while the liver tissue weight was significantly increased in the obesity-induced group (HFD) compared to the normal diet group (Normal), it was significantly reduced by administration of the Akkermansia massiliensis EB-AMDK39 strain. These results suggest that administration of the Akkermansia massiliensis EB-AMDK39 strain of the present invention reduces body fat and suppresses obesity.

[0159] 3.5. Analysis of Biochemical Indicators of Blood Lipids After fasting the experimental animals for 18 hours, blood was collected and centrifuged to obtain serum. Using this serum, the levels of total cholesterol (TC) and triglycerides (TG), which are lipid content indicators, were measured and shown graphically in Figure 10. The concentrations of total cholesterol and triglycerides were measured using Wako's LabAssay. TM Cholesterol and LabAssay TM Measurements were taken using a triglyceride product.

[0160] Referring to Figure 10, analysis of total blood cholesterol levels showed that the relevant values ​​were significantly increased in the obesity-induced group (HFD) compared to the normal diet group (Normal), and decreased again after oral administration of the Ackermansia massiliensis EB-AMDK39 strain.

[0161] In terms of serum triglyceride (TG) levels, the obesity-induced group (HFD) showed a significant increase compared to the normal diet group. Furthermore, serum triglyceride levels were significantly lower in the group administered with the Ackermansia massiliensis EB-AMDK39 strain compared to the obesity-induced group (HFD).

[0162] 3.6. Comparison of the size of fat cells To measure and compare the diameter of adipocytes, subcutaneous fat was extracted from sacrificial mice and fixed in formalin. After embedding in paraffin, tissue sections were prepared to a thickness of 4 μm. To observe the adipocytes under a microscope, the tissue section slides were stained with hematoxylin and eosin, and the diameter of the adipocytes was measured using the ImageJ program and is shown in Figure 11.

[0163] Referring to Figure 11, inducing obesity through a high-fat diet significantly increased the diameter of adipocytes in the HFD group. Conversely, the groups orally administered Ackermansia muciniphila ATCC BAA-835, Ackermansia massiliensis EB-AMDK39, and orlistat (ORL) showed a significant decrease compared to the HFD group.

[0164] Oral administration of Ackermansia massiliensis EB-AMDK39 can be confirmed to reduce the size of adipocytes and fat accumulation.

[0165] The present invention can be implemented in various forms of modification and variation without departing from its spirit and scope, and this fact should be obvious to the ordinary person in the art. The specific embodiments described herein are merely for illustrating preferred embodiments of the invention and should not be construed as limiting the invention. The scope of protection of the present invention should be determined by the appended claims, and the aforementioned various modifications and variations are intended to be included within the scope of protection of the present invention. [Accession Number]

[0166] Contracting organization name: Korean standard strain culture collection Accession number: KCTC 13765BP Date of acceptance: 20181205 TIFF2026526152000014.tif148170TIFF2026526152000015.tif137170

Claims

1. A composition comprising the strain of Akkermansia massiliensis, or a culture or dried product of the said strain.

2. The composition according to claim 1, characterized in that the Akkermansia massiliensis strain is not an Akkermansia muciniphila or Akkermansia glycaniphila strain.

3. The composition according to claim 2, characterized in that the Akkermansia massiliensis strain has a genome-wide mean nucleotide identity (gANI) of less than 95% compared to Akkermansia muciniphila or Akkermansia glycaniphila.

4. The composition according to claim 1, characterized in that the Akkermansia massiliensis strain is the Akkermansia massiliensis strain with deposit number KCTC 13765BP.

5. The composition according to claim 1, characterized in that it comprises live Akkermansia massiliensis strains, pasteurized strains, or dead Akkermansia massiliensis strains.

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

7. The composition contains Akkermansia muciniphila strains at 1×10 2 to 1×10 15 CFU, 1×10 3 to 1×10 14 CFU, 1×10 4 to 1×10 13 CFU, or 1×10 5 to 1×10 12 CFU, or 1×10 6 to 1×10 11 CFU, 1×10 7 to 1×10 10 CFU, or 1×10 8 to 1×10 12 CFU, or contains a culture having an equivalent number of viable bacteria agents, low-temperature sterilizing agents or dead bacteria agents, and the composition according to claim 1 is characterized by this.

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

9. The composition according to claim 1, characterized in that the composition further comprises a pharmaceutically acceptable carrier or excipient.

10. The composition according to claim 1, characterized in that it further comprises other probiotic strains or one or more prebiotics.

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

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