Akkermansia biwaensis for preventing or treating metabolic disorders and usese thereof

Abstract

The present invention relates to a novel strain of Akkermansia biwaensis species having an effect of preventing or treating metabolic diseases, and to a pharmaceutical composition, a food composition, a veterinary composition, and a cosmetic composition containing the strain, a culture or dried product thereof, which are effective in preventing or treating metabolic diseases. The next-generation pharmabiotic strain of the present invention is excellent in the effect of preventing or treating metabolic diseases to the extent that it can be used as a new preventive and therapeutic tool.

Description

AKKERMANSIA BIWAENSIS FOR PREVENTING OR TREATING METABOLIC DISORDERS AND USESE THEREOF

[0001] The present invention relates to a novel microbial strain having an effect for preventing or treating metabolic diseases and a use thereof, and more specifically, toAkkermansiabiwaensis,atype of pharmabiotics, and a composition containing the same for preventing, improving, or treating metabolic diseases.

[0002] A metabolic disease refers to a disease in which several diseases such as obesity, diabetes, hypertension, dyslipidemia, coronary arteriosclerosis, 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.

[0003] Until now, in order to treat metabolic diseases with these complex symptoms, hypoglycemic agents, antihypertensive agents, and hyperlipidemia agents 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.

[0004] 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.

[0005] Korean Patent No. 10-0996577 disclosesLactobacillus curvatus, which lowers blood cholesterol and inhibits obesity, and Korean Patent Application Publication No. 2010-0010015 disclosesLactobacillus johnsonii, which lowers blood cholesterol and inhibits obesity at the same time.

[0006] 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.

[0007] 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 novelAkkermansia biwaensisstrain having an effect in preventing or treating metabolic diseases.

[0008] Another object of the present invention is to provide a pharmaceutical composition effective for preventing or treating metabolic diseases containingAkkermansia biwaensis.

[0009] Still another object of the present invention is to provide a food composition helpful in preventing or improving metabolic diseases, containingAkkermansia biwaensis.

[0010] Still another object of the present invention is to provide a veterinary composition or feed additive that helps prevent or improve metabolic diseases containingAkkermansia biwaensis.

[0011] Still another object of the present invention is to provide a cosmetic composition containingAkkermansia biwaensis.

[0012] An aspect of the present invention for achieving the above-described objects relates toAkkermansia biwaensisthat exhibits an effect in preventing or treating metabolic diseases.

[0013] TheAkkermansia biwaensismay have a nucleotide sequence of a 16S rRNA gene that is 98%, 99%, 99.5%, or 99.9% identical to SEQ ID NO: 1.

[0014] Another aspect of the present invention for achieving the above-described objects relates to a pharmaceutical composition for preventing or treating metabolic diseases, containingAkkermansia biwaensis, a culture of the strain, or a dried product of the strain as an active ingredient.

[0015] Still another aspect of the present invention relates to a food composition for preventing or improving metabolic diseases, containingAkkermansia biwaensis, a culture of the strain, or a dried product of the strain as an active ingredient.

[0016] Still another aspect of the present invention relates to a veterinary composition or a feed composition (feed additives) for preventing or improving metabolic diseases, containingAkkermansia biwaensis, a culture of the strain, or a dried product of the strain as an active ingredient.

[0017] Still another aspect of the present invention relates to a cosmetic composition containingAkkermansia biwaensis, a culture of the strain, or a dried product of the strain as an active ingredient.

[0018] The pharmaceutical composition containingAkkermansia biwaensisof the present invention as an active ingredient 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.

[0019] The pharmaceutical composition containingAkkermansia biwaensisof 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 level.

[0020] TheAkkermansia biwaensisof 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.

[0021] FIG. 1 shows the results of microscopic observation of theAkkermansia biwaensisstrain EB-ABDH76 of the present invention and the two type strains,Akkermansia biwaensisWON2089 andAkkermansia muciniphilaATCC BAA-835.

[0022] FIG. 2 shows the results of PCR analysis of theAkkermansia biwaensisstrain EB-ABDH76 of the present invention and the two type strains,Akkermansia biwaensisWON2089 andAkkermansia muciniphilaATCC BAA-835.

[0023] FIG. 3 shows the results of Random Amplified Polymorphic DNA (RAPD) analysis of the genomic DNA of theAkkermansia biwaensisstrain EB-ABDH76 of the present invention and theAkkermansia biwaensisstrain WON2089.

[0024] FIG. 4 is a comparative diagram showing the phylogenetic relationship betweenAkkermansia biwaensisstrain of the present invention and otherAkkermansiastrains.

[0025] FIG. 5 shows the results of comparing the presence or absence of genes based on the full-length genome of theAkkermansia biwaensisstrain EB-ABDH76 of the present invention and theAkkermansia biwaensisstrain WON2089.

[0026] FIG. 6 shows the results of comparing the expression levels of genes related to functional indicator substances of theAkkermansia biwaensisstrain EB-ABDH76 of the present invention and otherAkkermansiastrains.

[0027] FIG. 7 shows the results of confirming whether theAkkermansia biwaensisstrainEB-ABDH76 of the present inventionpossesses hemolytic activity.

[0028] FIGs. 8a and 8b show the cytotoxicity test results of theAkkermansia biwaensisstrain EB-ABDH76 of the present invention.

[0029] FIG. 9 shows the results of analyzing body weight changesAkkermansia biwaensisadministration group of the present invention(EB-ABDH76), the obesity-induced group (HFD), theAkkermansia muciniphilaadministration group (ATCC BAA-835), and the Orlistat (ORL) administration group.

[0030] FIG. 10 shows the results of analyzing the changes in the amount of subcutaneous fat, epididymal fat, and mesenteric fat, and the changes in the weight of liver tissue theAkkermansia biwaensisstrain EB-ABDH76 administration group of the present invention (EB-ABDH76), the obesity-induced group (HFD), theAkkermansia muciniphilaadministration group (ATCC BAA-835), and the Orlistat (ORL) administration group.

[0031] FIG. 11 shows the results of measuring changes in　the levels of serum total cholesterol and serum triglycerides theAkkermansia biwaensisstrain EB-ABDH76 administration group of the present invention (EB-ABDH76), the obesity-induced group (HFD), theAkkermansia muciniphilaadministration group (ATCC BAA-835), and the Orlistat (ORL) administration group using ELISA.

[0032] FIGs. 12a-b are images and a graph showing the size of fat cells formed in mesenteric fat tissue and the　degree of accumulation of subcutaneous fat theAkkermansia biwaensisstrain EB-ABDH76 administration group of the present invention (EB-ABDH76), the obesity-induced group (HFD), theAkkermansia muciniphilaadministration group (ATCC BAA-835), and the Orlistat (ORL) administration group.

[0033] Hereinafter, the present invention will be described in more detail with reference to the attached drawings.

[0034] In the present application, "Akkermansia biwaensis" is a new species belonging to the phylumVerrucomicrobiota, and is distinct fromAkkermansia muciniphilaorAkkermansiaglycaniphila.

[0035] In this specification, "metabolic disease" refers to the overall symptoms of diseases such as obesity, diabetes, hypertension, dyslipidemia, 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.

[0036] 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. In the case of Westerners, when the body mass index (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.

[0037] 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.

[0038] 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.

[0039] When "about" comes before a number, it means ±20%, preferably ±10% of the numeric value.

[0040] TheAkkermansia biwaensiscan also be substantially purified. The term "substantially purified" as used herein refers to a bacterial strain or a mixture of more than one bacterial strain (e.g.,Bacteroidetes, Firmicutes, Proteobacteria, orVerrucomicrobia) 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.

[0041] In the present invention, theAkkermansia biwaensismay be included in the form of viable (live) bacteria, pasteurized bacteria, or inactivated bacteria (heat-killed). As used herein, the "pasteurized bacteria" meansAkkermansia biwaensisthat has been heat-treated at a low temperature. In an embodiment, the pasteurizedAkkermansia biwaensismeansAkkermansia biwaensisthat has been heat-treated at a temperature of 50℃ to 110℃ for 5 minutes or more and less than 30 minutes.

[0042] In this application, the term "inactivated bacteria" means bacteria 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 wall, and bacteriocin; polysaccharides; and / or organic acids,etc.

[0043] As used herein, the term "culture" refers to a product obtained by culturingAkkermansia biwaensis, and may include a fermentation product. In an embodiment, the culture may be a fermentation product obtained by culturingAkkermansia biwaensisin 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.

[0044] The culture (or fermentation) may be a whole culture ofAkkermansia biwaensis, a dilution thereof, a concentrate thereof, a dried product thereof, a lyophilized product thereof, a lysate 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 lysate may be obtained by physically or ultrasonically treating the strain or culture. The fraction may be obtained by subjecting the culture, the lysate,etc. to a method such as centrifugation and chromatography.

[0045] 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.

[0046] As used herein, the terms such as "treat", "treating", and "to treat" mean to impede, slow down, halt, or reverse the progression or severity of an existing condition, disease, disorder, or symptom.

[0047] In this specification, the term 'functional health food' is the same as Food for Special Health Use (FoSHU), and refers to a food composition 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.

[0048] An aspect of the present invention relates toAkkermansia biwaensisthat exhibits an effect in preventing or treating metabolic diseases.

[0049] TheAkkermansia biwaensisof the present invention was isolated from the feces of a healthy Korean person, is an oval-celled, monococcus or diplococcus, which is an anaerobic, non-motile, gram-negative, non-endospore-forming, mucus-decomposing bacterium. TheAkkermansia biwaensisproduces several mucus-decomposing enzymes and is thus able to use mucus as a carbon and nitrogen source, metabolizes D-glucose, D-mannitol, lactose, and D-mannose, and produces short-chain fatty acids such as propionic acid and acetic acid as main metabolites.

[0050] As a result of sequencing the 16S rRNA gene, theAkkermansia biwaensisof the present invention can have a nucleotide sequence of the 16S rRNA gene that is 98%, 99%, 99.5%, or 99.9% identical to SEQ ID NO: 1. Through phylogenetic tree analysis, it was confirmed that theAkkermansia biwaensisbelongs to theAkkermansiasp., but as a result of analyzing the whole genome sequence, it was confirmed that theAkkermansia biwaensishas an average nucleotide identity (ANI) of 81.75% with its closely related type strainAkkermansia muciniphilaACTC BAA-835T.

[0051] The genome-wide average nucleotide identity (gANI) of theAkkermansia biwaensistoAkkermansia muciniphilaorAkkermansia glycaniphilais less than 95%, which confirms that theAkkermansia biwaensisof the present invention belongs to theAkkermansiasp. but is a new species distinct fromAkkermansiamuciniphilaandAkkermansia glycaniphila.

[0052] In an embodiment, theAkkermansia biwaensismay be anAkkermansia biwaensis　strain EB-ABDH76 having the Accession No. KCCM13454P.

[0053] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating metabolic diseases, containingAkkermansia biwaensis, a culture of the strain, or a dried product of the strain as an active ingredient. An example of theAkkermansia biwaensisis theAkkermansia biwaensisstrain EB-ABDH76 having the Accession No. KCCM13454P.

[0054] In the present invention,Akkermansia biwaensisis not anAkkermansiamuciniphilaorAkkermansiaglycaniphilastrain. It was clearly confirmed thatAkkermansia biwaensisof the present invention is not anAkkermansia muciniphilaorAkkermansia glycaniphilastrain by genome-based phylogenetic analysis and the average nucleotide identity (ANI) value analysis.

[0055] The genome-wide average nucleotide identity (gANI) betweenAkkermansia biwaensisof the present invention andAkkermansia muciniphilaorAkkermansia glycaniphilais less than about 95%. In the present invention,Akkermansia biwaensishas 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 ofAkkermansia muciniphila. In some embodiments, in the composition of the present invention,Akkermansia biwaensis(e.g.,Akkermansia biwaensisstrain EB-ABDH76 (KCCM13454P) may have a gANI of less than 95%, such as about any of 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 ofAkkermansia glycaniphila.

[0056] TheAkkermansia biwaensisof 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 syndrome in which various diseases occur simultaneously, by inhibiting weight gain and body fat gain, reducing insulin resistance, lowering blood total cholesterol level, and reducing the content of glutamic pyruvic transaminase (GPT), which is an indicator of blood hepatotoxicity.

[0057] In one embodiment of the invention,Akkermansia biwaensismay be in the form of viable cells. In another embodiment of the invention,Akkermansiabiwaensismay be in the form of non-viable cells. In one embodiment, metabolically activeAkkermansiabiwaensiscells are used in the present invention. In one embodiment, strains ofAkkermansiabiwaensisare not metabolically inactivated, wherein metabolic inactivation may result, for example, from autoclave treatment.

[0058] In the present invention, pasteurization ofAkkermansiabiwaensismeans heating at a temperature of 50℃ or higher and 110℃ or lower for 5 minutes or longer and 30 minutes or less. For example, the pasteurization may be performed at 70℃ for 30 minutes. The pasteurizedAkkermansia biwaensis　strain EB-ABDH76 can reduce body fat accumulation more compared to the probiotic. The reason why the pasteurizedAkkermansia biwaensisis 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 ofAkkermansia biwaensisenhance metabolic benefits in the host.

[0059] TheAkkermansia biwaensisof 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.

[0060] SinceAkkermansia biwaensisof the present invention is sensitive to oxygen, it is preferable to cultureAkkermansia biwaensisunder anaerobic conditions (80-90% nitrogen, 0-5% hydrogen, and 5-20% carbon dioxide).

[0061] 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 ofAkkermansia biwaensisof the present invention.

[0062] 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 plant peptones such as tryptone, peptone, soy peptone, L-glutamic acid, and ammonium as a nitrogen source.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] The composition of the present invention may further contain, in addition to the above-mentioned active ingredients, a pharmaceutically acceptable carrier, excipient, diluent, cryoprotectant, or antioxidant, and may be formulated and prepared together with various additives commonly used pharmaceutically, such as binders, disintegrants, coating agents, and lubricants.

[0067] The pharmaceutical composition of the present invention may be formulated in the form of a powder, granule, tablet, capsule, or liquid by mixing theAkkermansia biwaensisof 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. The enteric-coated capsule may contain fatty acids, wax, shellac, resin, plant fibers, or any combination thereof.

[0068] 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 pregelantinized 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, hydroxypropylmethylcellulose, ethylcellulose, hydroxypropylethylcellulose, and sodium carboxymethylcellulose; and polymers such as polyvinylpyrrolidone, polyvinyl alcohol, polyvinylacetate, polyethylene glycol, polyacrylic acid, polymethacrylic acid, and vinyl acetate resin.

[0069] The pharmaceutical composition of the present invention may include one or more cryoprotectants. Such cryoprotectants may be used, for example, so as to maintain the viability ofAkkermansia biwaensiscells in the pharmaceuticalcomposition when frozen or lyophilized during transport and / or storage prior to use. In some cases, the one or more cryoprotectants may be glycerol, dimethylsulfoxide (DMSO), ethylene glycol, propylene glycol, 2-methyl-2,4-pentanediol, trehalose, sucrose, diethyl glycol, triethylene glycol, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), saccharose, formamide, glycerol 3-phosphate, proline, methyl alcohol, glucose, bovine serum albumin, polyvinyl alcohol, hydroxyethyl starch, sorbitol, or combinations thereof. In a preferred embodiment of the present invention, the cryoprotectant is glycerol.

[0070] The pharmaceutical composition of the present invention may further contain an antioxidant to provide anaerobic conditions during storage or transportation and to protect the strain, as an active ingredient, from reactive oxygen species. Examples of the antioxidant to be used in the present invention may include ascorbic acid, dithiothreitol, glutathione, phenolic acid, phenolic diterpenes, flavonoids, volatile oils, tocopherol, trolox, vitamin A, vitamin C, coenzyme Q10, manganese, iodide, melatonin, alpha-carotene, astaxanthin, beta-carotene, canthaxanthin, cryptoxanthin, lutein, lycopene, zeaxanthin, luteolin, tangeretin, flavonol, isorhamnetin, kaempferol, myricetin, proanthocyanidin, quercetin, eriodictyol, hesperetin, naringenin, catechin, gallocatechin, epicatechin, epigallocatechin, theaflavins, thearubigins, isoflavone phytoestrogens, daidzein, genistein, glycitein, stilbenoids such as resveratrol, pterostilbene, anthocyanins, cyanidins, delphinidins, malvidin, pelargonidin, peonidin, petunidin, chicoric acid, chlorogenic acid, cinnamic acid, ellagic acid, ellagitannins, gallic acid, gallotannins, rosmarinic acid, curcumin, xanthone, capsaicin, bilirubin, citric acid, oxalic acid, phytic acid, N-acetylcysteine, L-cysteine, L-glutamate, L-proline, anthocyanins, copper, cryptoxanthin, indole, isoflavonoids, lignans, selenium, zinc, or combinations thereof.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] The administration dose of theAkkermansia biwaensisstrain, which is an active ingredient 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.

[0075] The pharmaceutical composition of the present invention containsAkkermansia biwaensisas an active ingredient in an amount of 102CFU to 1015CFU based on the total weight of the composition, or contains a culture having an equivalent number of viable cells.

[0076] Generally, for adult patients, 1Х102or more live, pasteurized, or inactivated bacteria, preferably 1Х102to 1Х1015of live, pasteurized, 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 condition.

[0077] In an embodiment of the present invention, the composition containsAkkermansia biwaensisin the range of about 1Х102to about 1Х1015cells / g composition, about 1Х103to about 1Х1014cells / g composition, preferably about 1Х104to about 1Х1013cells / g composition, more preferably about 1Х105to about 1Х1012cells / g composition, even more preferably about 1Х106to about 1Х1011cells / g composition, about 1Х107to about 1Х1010cells / g composition, about 1Х108to about 1Х1012cells / g composition.

[0078] In an embodiment of the present invention, the composition containsAkkermansia biwaensisin the range of about 1Х102to about 1Х1015cells / mL composition, about 1Х103to about 1Х1014cells / mL composition, preferably about 1Х104to about 1Х1013cells / mL composition, more preferably about 1Х105to about 1Х1012cells / mL composition, even more preferably about 1Х106to about 1Х1011cells / mL composition, about 1Х107to about 1Х1010cells / mL composition, about 1Х108to about 1Х1012cells / mL composition.

[0079] In another embodiment of the present invention, the composition containsAkkermansia biwaensisin the range of about 1Х106to about 1Х1010cells / g or cells / mL composition, preferably about 1Х108to about 1Х1010cells / g or cells / mL composition, more preferably about 1Х109to about 1Х1010cells / g or cells / mL composition.

[0080] 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 toAkkermansia biwaensis.

[0081] Other probiotic strains may include probiotics from the phylaBacteroidetes,Firmicutes,Actinobacteria,orProteobacteria. In an embodiment, the other probiotics may be one or more strains of theFirmicutessp.,Ruminococcussp.,Clostridiumsp.,Akkermansia sp., Faecalibacterium sp.,Bacteroidessp.,Lactobacillussp., orBifidobacteriumsp. In addition, it may be one or more strains ofNeglectasp.,Egerthellasp.,Clostridiaceaesp.,Parabacteroidessp.,Bilophilasp.,Doreasp., andCollinsellasp.

[0082] 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.

[0083] Another aspect of the present invention relates to a food composition containingAkkermansia biwaensis, a culture or dried product thereof.

[0084] 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.

[0085] 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.

[0086] Sweeteners may be natural or synthetic. Natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] Another aspect of the present invention may be a cosmetic composition, in which the cosmetic composition containsAkkermansia biwaensis, preferablyAkkermansia biwaensisstrain EB-ABDH76 (Accession No. KCCM13454P), a culture of the strain or a dried product of the strain, as an active ingredient. The cosmetic composition of the present invention may be a cosmetic composition for promoting weight loss. The cosmetic composition of the present invention may be used as an inner beauty product.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] Still another aspect of the present invention provides a veterinary composition for preventing or treating metabolic diseases, or a feed additive for preventing or improving metabolic diseases, containingAkkermansia biwaensis, a culture of the strain, or a dried product of the strain.

[0096] The veterinary composition or feed additive for preventing or treating metabolic diseases may be prepared by adding theAkkermansia biwaensisstrain EB-ABDH76 (Accession No. KCCM13454P) at an appropriate effective concentration range according to various feed preparation methods known in the art. TheAkkermansia biwaensis(Accession No. KCCM13454P) is the same as described above.

[0097] 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.

[0098] Examples

[0099] Example 1: Isolation and identification of Akkermansia biwaensis strain EB-ABDH76

[0100] 1.1. Isolation and identification of the strain

[0101] In order to isolate a strain ofAkkermansia sp. from the feces of a Korean (female, 35 years old, BMI 17.9), the strain was isolated after selective cultivation using a mucin medium (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.

[0102] In order to confirm whether the isolate was indeed a strain of theAkkermansiasp., the isolate was observed under a microscope, and the results are shown in FIG. 1. In addition, PCR analysis was performed using the AM-specific primers shown in Table 1 below, and the results are shown in FIG. 2.

[0103] In FIG. 1, A shows a microscopic image of anAkkermansia muciniphilastrain ATCC BAA-835, B and C show microscopic images ofAkkermansia biwaensisstrain WON2089 (DSM 114407) andAkkermansia biwaensisstrain EB-ABDH76 observed at 1000X magnification, respectively. In FIG. 2, lane M indicates a DNA size marker, lanes 1 and 2 indicateAkkermansia biwaensisstrainsEB-ABDH76 and WON2089, respectively, lane 3isAkkermansia muciniphilastrain ATCC BAA-835, and lane 4 shows the result of the negative control (distilled water).

[0104] DesignationDirectionSequence (5'→3')Amplicon sizeSEQ ID NO:AM1ForwardCAG CAC GTG AAG GTG GGG AC327 bpSEQ ID NO :2AM2ReverseCCT TGC GGT TGG CTT CAG ATSEQ ID NO :3

[0105] 1.2. Random Amplified Polymorphic DNA (RAPD) analysis

[0106] In order to confirm whether the isolate as above is a sttrain of theAkkermansiasp., RAPD, a type of molecular typing, was performed. To this end, genomic DNA extracted from the bacterial body was amplified using the universal primers shown in Table 2 below, and then electrophoresed on a 1% agarose gel for 1 hour and 30 minutes, and the DNA fragmentation pattern was compared on a UV perforator. The results are shown in FIG. 3.

[0107] DesignationDirectionSequence (5'→3')SEQ ID NO:ERIC-1ForwardATG TAA GCT CCT GGG GAT TCA CSEQ ID NO: 4ERIC-2ReverseAAG TAA GTG ACT GGG GTG AGC GSEQ ID NO: 5(GTG)5Forward / ReverseGTG GTG GTG GTG GTGSEQ ID NO: 6

[0108] As can be seen in FIG. 3, it was confirmed that theAkkermansia biwaensisstrain EB-ABDH76 of the present invention showed a band pattern almost similar to that ofAkkermansia biwaensisstrain WON2089, but one band position was different (arrow) at (GTG)5.It was confirmed that theAkkermansia biwaensisstrain EB-ABDH76 of the present invention is a strain different fromAkkermansia biwaensisstrain WON2089.

[0109] 1.3. Phylogenetic tree analysis using full-length 16S rRNA gene sequences

[0110] For the 16S rRNA gene sequence analysis of the EB-ABDH76 strain isolated as above, the 16S rRNA gene was amplified using the 27F and 1492R primers shown in Table 3 below, and the resulting nucleotide sequence was determined using a 3730xl DNA analyzer. Using the 16S rRNA gene nucleotide sequences of EB-ABDH76 obtained in this way and other species of the same genus that have already been published, a phylogenetic tree was created and the results are shown in FIG. 4.

[0111] DesignationDirectionSequence (5'→3')Amplicon sizeSEQ ID NO:27FForwardAGA GTT TGA TCM TGG CTC AG1,473 bp71492RReverseGGT TAC CTT GTT ACG ACT TC8

[0112] As shown in FIG. 4, the EB-ABDH76 strain was genetically confirmed to beAkkermansia biwaensisas a result of analyzing the evolutionary relationship through a phylogenetic tree by 16S rRNA gene sequence analysis.

[0113] 1.4. Whole genome sequencing

[0114] In order to analyze the similarity between theAkkermansia biwaensisstrainEB-ABDH76 and theAkkermansia muciniphilastrain ATCC BAA-835 at the genome level, whole genome sequencing based on the Illumina system (Novaseq6000) was performed, and the assembly to secure the full genome was performed using the SPAdes (v3.15.4) program. The genome was analyzed and compared with those ofAkkermansia muciniphilastrain ATCC BAA-835 andAkkermansia biwaensisstrain WON2089 (DSM 114407), and the results are shown in Table 4 below.

[0115] Genome StatisticsStrainATCC BAA-835(GCF_000020225.1)WON2089(GCF_026072915.1)EB-ABDH76(Enterobiome)Genome Size (bp)2,664,1023,175,5253,150,821GC Content (%)55.76256.75656.803Assembly_LevelComplete genomeComplete genomeDraft genomeNo. of Sequences11137Sequence CategoryChromosomeChromosomeContigsGenes2,2022,6152,598CDS2,1382,5512,545CDS_Coding2,1222,5402,521Pseudo_Genes161124RNA646453rRNA993rRNA_5S3 (complete)3 (complete)1 (complete)rRNA_16S3 (complete)3 (complete)1 (complete)rRNA_23S3 (complete)3 (complete)1 (complete)tRNA525247Repeat_Region233CRISPR266ANI Similarity (%)*--81.75 / 99.93*ANI similarity is a similarity to ATCC BAA-835 or WON2089 (DSM 114407).

[0116] As can be seen in Table 4 above, there were differences when comparing the whole genome statistics of theAkkermansia biwaensisstrain EB-ABDH76 of the present invention and theAkkermansia muciniphilastrain ATCC BAA-835, and the average nucleotide identity (ANI) similarity value was confirmed to be 81.75%, which is lower than the cut-off line (95-96%) that determines the species boundary. In addition, theAkkermansia biwaensisstrain EB-ABDH76 of the present invention was confirmed to have a homology of 99.93% withAkkermansia biwaensisstrain WON2089 (DSM 114407) of the same species.

[0117] The genome information of theAkkermansia biwaensisstrain EB-ABDH76 obtained in the examples above was functionally classified. The protein coding genes were functionally classified according to the cluster of orthologous groups (COG) definition, and are shown in Table 5 below. As described in Table 5, the function of each of these genes in the genome of theAkkermansia biwaensisstrain EB-ABDH76 was confirmed based on COG, a search-oriented database for comparison to find orthologous genes, and compared with theAkkermansia biwaensisstrain WON2089 (DSM 114407) of the same species, and the results are shown in Table 5 below.

[0118] CODECOG Functional DescriptionWON2089EB-ABDH76JTranslation, ribosomal structure and biogenesis193194ARNA processing and modification00KTranscription7675LReplication, recombination and repair107101BChromatin structure and dynamics00DCell cycle control, cell division, chromosome partitioning2625YNuclear structure00VDefense mechanisms6262TSignal transduction mechanisms9292MCell wall / membrane / envelope biogenesis243245NCell motility1516ZCytoskeleton33WExtracellular structures1010UIntracellular trafficking, secretion, and vesicular transport3637OPosttranslational modification, protein turnover, chaperones113112XMobilome: prophages, transposons1816CEnergy production and conversion99101GCarbohydrate transport and metabolism162161EAmino acid transport and metabolism150151FNucleotide transport and metabolism5858HCoenzyme transport and metabolism9494ILipid transport and metabolism6061PInorganic ion transport and metabolism9395QSecondary metabolites biosynthesis, transport and catabolism1817RGeneral function prediction only193187SFunction unknown115115-Not assigned504493

[0119] As can be seen in Table 5, it was confirmed thatAkkermansia biwaensisstrain WON2089 (DSM 114407) and theAkkermansia biwaensisstrain EB-ABDH76 of the present invention 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 terms of detailed COG functional classification, it was confirmed that theAkkermansia biwaensisstrain WON2089 (DSM 114407) has a classification characteristic different from that of theAkkermansia biwaensisstrain EB-ABDH76. Therefore, the two strains show clear differences in the whole genome.

[0120] As a result of excluding the cases where there were errors in the genome sequence or incomplete annotation, 16 genes specific to the EB-ABDH76 strain were derived and the results are shown in Table 6 and FIG. 5.

[0121] NoGeneAnnotationSize (bp)BSR toWON20891EB-ABDH76_000689autotransporter-associated beta strand repeat-containing protein87930.99912EB-ABDH76_000740autotransporter domain-containing protein47110.99443EB-ABDH76_000884pseudouridine synthase17280.99754EB-ABDH76_000945hypothetical protein50220.99545EB-ABDH76_001037hypothetical protein8910.96456EB-ABDH76_001516hypothetical protein5580.98017EB-ABDH76_001585four helix bundle protein6480.97688EB-ABDH76_001889hypothetical protein63609EB-ABDH76_001890iron-containing alcohol dehydrogenase11580.533210EB-ABDH76_001891flavodoxin family protein5370.136511EB-ABDH76_001892DUF3737 family protein8730.773812EB-ABDH76_001893pyridoxal phosphate-dependent aminotransferase11910.380713EB-ABDH76_001894DUF362 domain-containing protein1005014EB-ABDH76_002301hypothetical protein3090.834115EB-ABDH76_002543autotransporter-associated beta strand repeat-containing protein87120.995216EB-ABDH76_002544autotransporter-associated beta strand repeat-containing protein57780.9981

[0122] Referring to Table 6 and FIG. 5, the blast score ratio (BSR) was calculated through Large-Score Blast Score Ratio (LS-BSR) for 16 genes specific to theAkkermansia biwaensisstrain EB-ABDH76 of the present invention. As a result, it was confirmed that the EB-ABDH76_001889 and EB-ABDH76_001894 genes were not present in the genome of theAkkermansia biwaensisstrain WON2089, and were specifically present in the genome of theAkkermansia biwaensisstrain EB-ABDH79.

[0123] 1.5. Confirmation of ability of Akkermansia biwaensis strain to produce short-chain fatty acids (SCFAs)

[0124] Short-chain fatty acids (SCFAs), such as butyric acid, acetic acid, and propionic acid, are metabolites produced by intestinal bacteria and play an important role in the host's energy metabolism, and are involved in energy balance as signaling mediators acting on G protein-coupled receptors (GPR41 and GPR43).

[0125] Short-chain fatty acids (SCFAs) decrease intestinal motility and increase intestinal transit speed through GPR41 in enteroendocrine cells. This induces the secretion of peptide YY (PYY), thereby reducing energy intake and preventing obesity. In addition, GPR43 induced 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 adipose tissue, thereby preventing fat accumulation. Short-chain fatty acids (SCFAs) can enhance glucose metabolism and activate intestinal gluconeogenesis (IGN), which can reduce food intake through the gut-brain neural circuit.

[0126] TheAkkermansia biwaensisstrain EB-ABDH76 was cultured in a test tube, and the content of short-chain fatty acids (SCFAs) contained in the cultured solution was analyzed by gas chromatography (GC). To this end, the cultured solution 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 7 below, and the analysis results are shown in Table 8 below.

[0127] FlowH2: 40 mL / min, Air: 350 mL / minInjector 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 Ratio20:1

[0128] StrainProduction of Short-Chain Fatty Acid (㎍ / mL)AcetatePropionateSuccinateATCC BAA-835653.4 ± 78.841415.97 ± 145.017.23 ± 6.98EB-ABDH76606.09 ± 75.301429.03 ± 165.4210.20 ± 28.50

[0129] As can be seen in Table 8, theAkkermansia biwaensisstrain EB-ABDH76 of the present invention exhibited a short-chain fatty acid-producing ability similar to that ofAkkermansia muciniphilastrain ATCC BAA-835.

[0130] 1.6. Analysis of gene expression related to Akkermansia functional indicator substances

[0131] An extracellular polypeptide derived fromAkkermansiamuciniphilawas identified that it can modulate or promote the intestinal mucosal immune system function, maintain or restore the metabolic state, and increase the physical integrity of the intestinal mucosal barrier in mammals.

[0132] FIG. 6 is a graph comparing the expression levels of genes related to functional indicator substances of theAkkermansia biwaensisstrain EB-ABDH76 (AmIV) of the present invention withAkkermansiastrains of other clades (BAA-835; AmIa, EB-AMDK19; AmIb, EB-AMDK39; AmII). Referring to FIG. 6, it was confirmed that　theAkkermansia biwaensis　strainEB-ABDH76 of the present invention has a significantly higher expression level of Amuc_1100 and Amuc_1984 genes compared to otherAkkermansiastrains.

[0133] Example 2: Mycological characteristics and safety analysis of Akkermansia biwaensis strain EB-ABDH76

[0134] 2.1. Confirmation of sugar utilization of Akkermansia biwaensis strains

[0135] In order to determine the sugar utilization of theAkkermansia biwaensisstrain EB-ABDH76 of the present inventionisolated above, the strain was cultured using the API50CH kit (Biomerieux, France) and the growth utilizing each sugar was compared with theAkkermansia biwaensisstrain WON2089 andAkkermansia muciniphila　strain ATCC BAA-835. The results are shown in Table 9 below.

[0136] NoCarbohydratesBAA-835WON2089EB-ABDH76NoCarbohydratesBAA-835WON2089EB-ABDH760Negative control---25Esculine---1Glycerol---26Salicine---2Erythritol---27D-Cellobiose---3D-Arabinosew++28D-Maltose-++4L-Arabinosew--29D-Lactose(bovine origin)+++5Ribose+w-30D-Melibiose---6D-Xylosew--31D-Saccharose(sucrose)---7L-Xylosew--32D-Trehalose---8Adonitol---33Inuline---9β-Methyl-xyloside---34D-Melezitose---10D-Galactosew++35D-Raffinose---11D-Glucose+++36Amidon (starch)---12D-Fructosew++37Glycogene---13D-Mannose+++38Xylitol---14L-Sorbose---39Gentiobiose---15L-Rhamnose---40D-Turanose---16Dulcitol---41D-Lyxosew--17Inositol---42D-Tagatose---18D-Mannitol-++43D-Fucose---19D-Sorbitol---44L-Fucose+++20Methyl-α D-mannopyranoside---45D-Arabitol---21Methyl-α D-glucopyranoside---46L-Arabitol---22N-Acetylglucosamine+++47Potassium Gluconate---23Amygdaline---48Potassium 2-Ketogluconate---24Arbutine---49Potassium 5-Ketogluconatew--+ : growth, w : weak growth, - : no growth,

[0137] As can be seen in Table 9 above, it was confirmed that theAkkermansia biwaensisstrain EB-ABDH76 of the present invention has differences in the ability to utilize D,L-arabinose, ribose, D,L-xylose, D-galactose, D-fructose, D-mannitol, D-maltose, D-lyxose, and potassium 5-ketogluconate, compared toAkkermansia muciniphilastrain ATCC BAA-835, while being similar to theAkkermansia biwaensisstrain WON2089.

[0138] 2.2. Confirmation of antimicrobial susceptibility of Akkermansia biwaensis strains

[0139] In order to determine the susceptibility ofAkkermansia biwaensisstrain EB-ABDH76 to antimicrobial agents, the minimum inhibitory concentration (MIC) for a total of 15 antimicrobial agents, including piperacillin-tazobactam (PTZ), ceftizoxime (CTZ), chloramphenicol (CHL), clindamycin (CLI), 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) guideline and the EFSA guideline. The results are shown in Table 10 below.

[0140] Cut-off Values(mg / L)AntibioticsPTZCTZCHLCLIMXFMTZTETCLSI(Anaerobes)≥128 / 6≥128≥32≥8≥8≥32≥16EFSA(E.coli, G-)N / AN / A>16N / AN / AN / A>8ATCC WON208916 / 4(S)256(R)2(S)≤0.125(S)32(R)2(S)2(S)EB-ABDH7616 / 4(S)128(R)2(S)≤0.125(S)>32(R)2(S)2(S)Cut-off values(mg / L)AntibioticsAMPGENKANSTRNALSMZTMPAPRCLSI(Anaerobes)≥2N / AN / AN / AN / AN / AN / AN / AEFSA(E.coli, G-)>8>2>8>16>16>256>2>8ATCC WON208964(R)>256(R)>256(R)>512(R)64(R)8(S)0.5(S)>128(R)EB-ABDH7664(R)>256(R)>256(R)>512(R)64(R)8(S)0.5(S)>128(R)PTZ: Piperacillin-tazobactam, CTZ: ceftizoxime, CHL: chloramphenicol, CLI: clindamycin, MXF: moxifloxacin, MTZ: metronidazole, TET: Tetracycline, AMP: Ampicillin, GEN: Gentamicin, KAN: Kanamycin, STR: Streptomycin, NAL: Nalidixic acid, SMZ: Sulfamethoxazole, TMP: Trimethoprim, APR: Apramycin

[0141] As can be seen in Table 10 above, theAkkermansia biwaensisstrain EB-ABDH76 of the present invention was confirmed to be resistant to ampicillin, which is a beta-lactam antibiotic, ceftizoxime, which is a cephalosporin antibiotic, gentamicin, kanamycin, streptomycin, and apramycin, which are aminoglycoside antibiotics, and nalidixic acid and moxifloxacin, which are quinoline based antibiotics and to be susceptible to antibacterias except these. In addition, when compared to the WON2089 strain, the antibacterial resistance pattern was found to be the same.

[0142] The bioinformatics-based PlasmidFinder (https: / cge.cbs.dtu.dk / services / PlasmidFinder / ), PHASTER (https: / phaster.ca / ), and Mobile Element Finder (cge.cbs.dtu.dk / services / MobileElementFinder) programs were applied to the complete genome of theAkkermansia biwaensisstrain EB-ABDH76 of the present invention so as to confirm whether it was obtainable or endogenous. As a result, chromosomal Mmobile Genetic Elements (MEGs)factors such as plasmids, transposons, and prophages, and antibiotic-resistant genes, were not detected in the Akkermansia biwaensis strain EB-ABDH76.

[0143] 2.3. Identification of hemolytic activity and virulence factors of Akkermansia biwaensis strains

[0144] In order to verify the safety of theAkkermansia biwaensisstrain EB-ABDH76 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. 7. As can be seen from FIG. 7, theAkkermansia biwaensis　strain EB-ABDH76 of the present invention did not exhibit beta-hemolysis (a completely transparent area around the colony) associated with pathogenicity.

[0145] In order to confirm the presence or absence of possible toxigenic genes in the EB-ABDH76 strain, 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.

[0146] Using the Virulence Finder analysis tool, the presence of virulence factor genes was confirmed by comparative analysis ofAkkermansia biwaensisstrains WON2089 and EB-ABDH76 with well-known virulence bacteria (E. coli, Enterococcus, Listeria,andS. aureus), and as a result, no virulence factors were detected in both strains.

[0147] As a result of performing virulence factor analysis based on VFDB, six and five possible virulence factors were identified in the whole genomes ofAkkermansia biwaensisstrains WON2089 and EB-ABDH76, 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 11). 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.

[0148] MatchFunctionAccession(VFDB)Locus_tag, Identity (%)WON2089EB-ABDH76Glucose-1-Phosphate Thymidylyltransferase[LPS (VF0542)]CapsuleVFG047039OQH67_RS08715,(60.69)EB-ABDH76_000838(60.69)GDP-mannose 4,6-dehydratase[LPS (VF0367)]CapsuleVFG002225OQH67_RS06570,(66.185)EB-ABDH76_001304,(66.185)Chaperonin GroEL[GroEL (VF0594)]AdherenceVFG012095OQH67_RS01900,(61.059)EB-ABDH76_000592,(61.248)Elongation factor Tu[EF-Tu (VF0460)]AdherenceVFG046465OQH67_RS06160,(72.589)EB-ABDH76_001388,(72.589)Catalase[KatA (VF0454)]StressVFG037028OQH67_RS06715,(72.671)EB-ABDH76_001276,(72.671)Trifunctional Thioredoxin / Methionine Sulfoxide Reductase A / B protein[MsrAB (VF0456)]StressVFG037100OQH67_RS04495,(62.59)-

[0149] 2.4. Confirmation of cytotoxicity of EB-ABDH76 strain

[0150] In order to confirm the cytotoxicity of theAkkermansia biwaensisstrain EB-ABDH76 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 described 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, Caco-2), and the test was performed by treating EB-ABDH76 at a concentration of 103CFU / mL to 107CFU / mL.Pseudomonas aeruginosaATCC 17831 strain was used as a positive control group and treated with the same concentration as EB-ABDH76. As a result, the cytotoxicity was calculated according to the equation below, converted into cell viability, and the results are shown in FIGs. 8a-b.

[0151] [Equation 1]

[0152]

[0153] As can be seen from FIGs. 8a and 8b,　theAkkermansia biwaensisstrain EB-ABDH76 of the present invention was confirmed to be non-cytotoxic at all tested concentrations.

[0154] TheAkkermansia biwaensisstrain EB-ABDH76 isolated from human feces was identified by biochemical (API) and molecular biological methods (16S rRNA sequence analysis, RAPD, and full-length genome analysis,etc.), and was confirmed to be a safe strain by showing no antibiotic resistance genes, hemolytic activity, virulence factors, and cytotoxicity.

[0155] In conclusion, although the 16S rRNA sequencing phylogeny, genomic features and phenotypic features indicated that the EB-ABDH76 strain was closely related toAkkermansia muciniphilastrain ATCC BAA-835, the 16S rRNA and ANI similarities indicated that the EB-ABDH76 strain represents a new species in the genusAkkermansia. Therefore, the strain isolated from a human as described in the present invention with excellent therapeutic activity for metabolic disease was namedAkkermansia biwaensisstrain EB-ABDH76 and deposited with the Korea Culture Center for Microorganism (KCCM) on January 12, 2024, under the Accession No. KCCM13454P.

[0156] Example 3: Confirmation of anti-obesity efficacy by administering live Akkermansia biwaensis strain EB-ABDH76 in obesity-induced mouse model

[0157] 3.1. Strain samples

[0158] TheAkkermansiamuciniphilastrain ATCC BAA-835 (control) andAkkermansiabiwaensisstrain EB-ABDH76 used in this experiment were prepared at a concentration of 1Х108CFU / 100 ㎕ PBS (25% glycerol, 0.05% cysteine / PBS) for use in animal testing.

[0159] 3.2. Animal Testing

[0160] 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, and 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. To induce obesity, the animals were fed a high-fat diet (60 kcal% fat; Research Diets Inc., NJ, USA), while the normal control group (Normal) was fed a 10 kcal% fat feed. Drinking water was providedad libitum. The experimental groups were divided into five groups as shown in Table 12 below. Each of the groups was Normal (normal control group), HFD (obesity-induced group), Orlistat (ORL) (high-fat diet-fed obesity-induced group + Orlistat administration group), BAA-835 (high-fat diet-fed obesity-induced group +Akkermansia muciniphila　strain ATCC BAA-835 administration group), and EB-ABDH76 (high-fat diet-fed obesity-induced group +Akkermansia biwaensisstrain EB-ABDH76 administration group). Orlistat (ORL; Xenical, 10 mg / kg), which is an oral obesity treatment drug, were used as positive controls. Oral administration was started simultaneously with the feeding of a high-fat feed and was performed 5 times a week for 8 weeks.

[0161] Group name　Feed for FeedingOral Administration Substances　1NormalNormal diet (10 Kcal% fat)PBS (25% glycerol)2HFDHigh-fat diet (60 Kcal% fat)PBS (25% glycerol)3BAA-835High-fat diet (60 Kcal% fat)BAA-8354EB-ABDH76High-fat diet (60 Kcal% fat)EB-ABDH765ORLHigh-fat diet (60 Kcal% fat)Orlistat 10 mg / kg

[0162] 3.3. Confirmation of body weight and weight gain

[0163] 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. 9. Referring to FIG. 9, compared to the obesity-induced group (HFD) that only consumed a high-fat diet, the body weight and body weight gain were statistically significantly reduced in the BAA-835 strain administration group and the EB-ABDH76 strain administration group. Through these results, it can be confirmed that theAkkermansia biwaensisstrain EB-ABDH76 of the present invention induces weight loss.

[0164] 3.4. Confirmation of weight changes in adipose tissue and liver tissue

[0165] 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. 10.

[0166] Referring to FIG. 10, the weights of subcutaneous fat, epididymal fat, and mesenteric fat tissues in the obesity-induced group (HFD) were significantly increased compared to the normal diet group. In contrast, in the EB-ABDH76 strain administration group, the weights of subcutaneous fat and epididymal fat were statistically significantly reduced compared to the obesity-induced group (HFD).

[0167] 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 theAkkermansia biwaensisstrain EB-ABDH76. Through these results, it can be confirmed that the administration of theAkkermansia biwaensisEB-ABDH76 strain inhibits obesity by reducing body fat and inhibits fat accumulation in the liver.

[0168] 3.5. Analysis of blood lipid biochemical indicators

[0169] 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 using LabAssay™ Cholesterol and LabAssay™ Triglyceride products (Wako), and the results are shown in FIG. 11.

[0170] Referring to FIG. 11, as a result of analyzing the total cholesterol (TC) and triglyceride (TG) levels, both lipid indices were statistically significantly increased in the obesity-induced group (HFD) compared to the normal diet group. However, they were significantly decreased following oral administration of　theAkkermansia biwaensisstrain EB-ABDH76. TheAkkermansia muciniphilatype strain (BAA-835) did not have a significant effect on TC and TG levels. However, it could be confirmed that the blood lipid indices were decreased as a result of the obesity inhibition effect ofAkkermansiabiwaensisstrain EB-ABDH76 of the present invention.

[0171] 3.6. Comparison of size of fat cells

[0172] In order to 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 FIGs. 12a and 12b.

[0173] Referring to FIGs. 12a and 12b, the diameter of adipocytes in the HFD group was statistically significantly increased as obesity was induced by feeding a high-fat diet. In contrast, the group orally administered withAkkermansia muciniphilastrain ATCC BAA-835,Akkermansiabiwaensisstrain EB-ABDH76, or Orlistat (ORL) showed a statistically significant reduction in adipocyte size compared to the HFD group.

[0174] It can be confirmed that the oral administration ofAkkermansia biwaensisstrain EB-ABDH76 decreased the adipocyte size and fat accumulation.

[0175] While the present invention has been described with reference to preferred examples thereof as described above, it will be understood by those skilled in the art that various modifications, changes, or alterations may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

[0176] [Accession No.]

[0177] Depository Institution: Korean Culture Center of Microorganisms

[0178] Accession No.: KCCM13454P

[0179] Deposition Date: January 12, 2024

[0180]

Claims

1.AnAkkermansia biwaensisstrain exhibiting an effect for preventing or treating metabolic diseases.2.TheAkkermansia biwaensisstrain of claim 1, wherein theAkkermansia biwaensishas a nucleotide sequence of a 16S rRNA gene that is 98%, 99%, 99.5%, or 99.9% identical to SEQ ID NO: 1.3.TheAkkermansia biwaensisstrain of claim 1, wherein theAkkermansia biwaensishas a genome-wide average nucleotide identity (gANI) of less than 95% compared toAkkermansia muciniphilaorAkkermansia glycaniphila.4.TheAkkermansia biwaensisstrain of claim 1, wherein theAkkermansia biwaensisis anAkkermansia biwaensisEB-ABDH76 strain with Accession No. KCCM13454P.5.A pharmaceutical composition for preventing or treating metabolic diseases, containingAkkermansia biwaensis, a culture of the strain, or a dried product of the strain as an active ingredient.6.The pharmaceutical composition of claim 5, wherein theAkkermansia biwaensishas a genome-wide average nucleotide identity (gANI) of less than 95% compared toAkkermansia muciniphilaorAkkermansia glycaniphila.7.The pharmaceutical composition of claim 5, wherein theAkkermansia biwaensisisAkkermansia biwaensisstrain EB-ABDH76 with Accession No. KCCM13454P.8.The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is for oral administration.9.The pharmaceutical composition of claim 5, wherein theAkkermansia biwaensisis a substantially purified and freeze-dried strain.10.The pharmaceutical composition of claim 5, wherein the metabolic diseases are obesity, diabetes, insulin resistance, hypertension, hyperlipidemia, dyslipidemia, coronary arteriosclerosis, arteriosclerosis, or non-alcoholic fatty liver disease.11.The pharmaceutical composition of claim 5, wherein the pharmaceutical composition comprises live, pasteurized, or inactivatedAkkermansia biwaensis.12.The pharmaceutical composition of claim 5, wherein the composition comprisesAkkermansia biwaensisas an active ingredient in an amount of 1Х102CFU to 1Х1015CFU, 1Х103CFU to 1Х1014CFU, 1Х104CFU to 1Х1013CFU, or 1Х105CFU to 1Х1012CFU, or 1Х106CFU to 1Х1011CFU, 1Х107CFU to 1Х1010CFU, or 108CFU to 1012CFU based on the total weight of the composition, or comprises a culture comprising an equivalent number of live bacteria, pasteurized strains, or inactivated bacteria.13.The pharmaceutical composition of claim 5, wherein the composition further comprises a different probiotic strain or one or more prebiotics.14.The pharmaceutical composition of claim 13, wherein the different probiotic strain is one or more strains among theFirmicutessp.,Ruminococcussp.,Clostridiumsp.,Akkermansiasp.,Faecalibacteriumsp.,Bacteroidessp.,Lactobacillussp.,or Bifidobacteriumsp.15.The pharmaceutical composition of claim 5, wherein the composition further comprises a carrier, an excipient, a diluent, a cryoprotectant, or an antioxidant.16.A composition containingAkkermansia biwaensis, a culture of the strain, or a dried product of the strain as an active ingredient.17.The composition of claim 16, wherein theAkkermansia biwaensisstrain isAkkermansia biwaensisEB-ABDH76 strain with Accession No. KCCM13454P.18.The composition of claim 16, wherein the composition is a veterinary composition, a feed additive, or a cosmetic composition for preventing or improving metabolic diseases.19.The composition of claim 18, wherein the cosmetic composition is a cosmetic composition for promoting weight loss.

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