Isolated akkermansia muciniphila, composition containing same, and use thereof

Abstract

The present invention provides an isolated Akkermansia muciniphila, a composition containing same, and a use thereof, which can be used for treating, preventing, or alleviating obesity, metabolic disorders caused by obesity, diabetes, inflammation, liver and kidney disease, liver disease, cardiovascular and cerebrovascular diseases, tumors, etc.

Description

Isolated akkermansia muciniphila, compositions comprising same and uses thereof TECHNICAL FIELD

[0001] The present disclosure relates to the field of microorganisms, more particularly to newly isolated strains of Akkermansia muciniphila species, compositions comprising same and uses thereof. BACKGROUND

[0002] Akkermansia muciniphila is a mucin-degrading bacterium that is normally colonized in the gut of humans and many animals.

[0003] Screening of new strains of Akkermansia muciniphila is very difficult because it mainly exists in the human or animal gut, is a strict anaerobic microorganism, has very high requirements for nutrition and culture environment, and has a long growth cycle. In addition, when verifying the efficacy, the bacteria of this genus are difficult to verify through in vitro cell experiments due to anaerobicity; in the process of animal in vivo experiments, the bacteria of this genus are difficult to maintain a stable number of viable bacteria due to the difficulty of culture, high anaerobicity, and lack of reproducibility. These technical problems related to in vitro and in vivo limit the discovery and application of new strains of Akkermansia muciniphila.

[0004] In addition, Akkermansia muciniphila grows slowly and is difficult to compete with other microbial strains, so there is a problem of acquisition difficulty in the screening process.

[0005] CN116925975B discloses that the hydrophobicity of AKK PROBIO strain reaches 31% at 60 min; the self-aggregation tends to be stable at 20 h, and remains at about 52%. Some documents consider that AKK PROBIO has weak ability to form a biofilm, and the in vitro test results show that the colonization ability of AKK PROBIO in the intestinal tract is low (https: / / doi.org / 10.3390 / foods13030442, section 3.1). If the colonization and adhesion ability of AKK strain is weak, it will limit the commercial product development thereof.

[0006] The prevalence of metabolic diseases such as obesity, diabetes, hypertension, hyperlipidemia, fatty liver and the like is increasing, and studies have also shown that these diseases are related to microbial flora imbalance. In many populations, Akkermansia muciniphila is negatively correlated with obesity, diabetes, cardiovascular disease and low-grade inflammation. Akkermansia muciniphila can protect the integrity of intestinal epithelial cells and mucus layer to exert metabolic protection.

[0007] In the prior art, a mixture of multiple microorganisms is usually used for treating metabolic and obesity-related diseases or disorders, such as probiotics or fecal microbiota transplantation (FMT). However, the mixture of multiple microorganisms makes the mechanism more complex, the mutual influence between the microorganisms is not maturely studied, and the use of the mixture of microorganisms usually breaks the intestinal flora homeostasis. In addition, the use of the mixture of microorganisms also needs to consider whether the microorganisms affect each other's activity, and it cannot be determined whether the synergistic effect of the mixture of microorganisms makes it play a role in the treatment or prevention of diseases or whether a single bacterium plays a role in the treatment or prevention of diseases.

[0008] The number of microbial resources is extremely large, and it is a great challenge to screen new strains or species from them that can be effectively used for treating or preventing diseases such as obesity, diabetes, hypertension, hyperlipidemia, liver and kidney function diseases, tumors, etc., but also represents a great unmet need.

[0009] SUMMARY

[0010] The present disclosure isolates a new strain of Akkermansia muciniphila. The new strain of the present disclosure has stronger epithelial cell adhesion ability than the existing Akkermansia muciniphila strain, can well colonize in the intestinal tract of humans or animals, thereby prolonging the residence time in the intestinal tract and better exerting its efficacy in the intestinal tract; at the same time, due to the excellent colonization and adhesion ability of the strain of the present disclosure, it can effectively prevent pathogenic bacteria from adhering, which is conducive to maintaining intestinal homeostasis and health. In addition, the strain of the present disclosure can synthesize a large amount of short-chain fatty acids, thereby effectively exerting the effects of treating or preventing metabolic diseases, reducing the body weight, body fat, blood glucose, blood lipids, cholesterol of the subject, improving or alleviating non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), repairing the intestinal tissue mucosa, and promoting the expression of type I interferon IFNβ of the subject, thereby improving immunity, and thus having the potential to prevent or treat viral infections and inhibit tumor growth.

[0011] In a first aspect, the present disclosure provides an isolated Akkermansia muciniphila having an average nucleotide identity (ANI) value of at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, or at least 97.5% to Akkermansia muciniphila (GCF_000020225.1) strain, and / or a 16S rRNA sequence having at least 98.65% identity to the sequence shown in SEQ ID NO. 1.

[0012] In some embodiments, the Akkermansia muciniphila has an alignment fraction (AF) of at least 90%, at least 90.5%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% to Akkermansia muciniphila (GCF_000020225.1) strain.

[0013] In some embodiments, the Akkermansia muciniphila has a 16S rRNA sequence that is 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, at least 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to the sequence set forth in SEQ ID NO. 1.

[0014] In some embodiments, the Akkermansia muciniphila is a new strain of Akkermansia muciniphila species having an average nucleotide identity (ANI) value of at least 95%, for example, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% to Akkermansia muciniphila (GCF_000020225.1).

[0015] In some embodiments, the metabolite of Akkermansia muciniphila comprises short chain fatty acids, including at least one of acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, hexanoic acid, and decanoic acid; preferably, the Akkermansia muciniphila is capable of high-yield production of acetic acid and / or propionic acid. In some embodiments, the Akkermansia muciniphila secretes acetic acid at a content of no less than 1000 pg / mL, 1100 pg / mL, 1200 pg / mL, 1300 pg / mL, 1400 pg / mL, or 500 pg / mL. In some embodiments, the Akkermansia muciniphila has a cell body with acetic acid content of no less than 50 pg / mg, 60 pg / mg, 70 pg / mg, 80 pg / mg, 90 pg / mg, or 100 pg / mg. In some embodiments, the Akkermansia muciniphila secretes propionic acid at a content of no less than 180 pg / mL, 190 pg / mL, 200 pg / mL, 210 pg / mL, 220 pg / mL, or 230 pg / mL. In some embodiments, the Akkermansia muciniphila has a cell body with propionic acid content of no less than 5 pg / mg, 6 pg / mg, 7 pg / mg, 8 pg / mg, 9 pg / mg, 10 pg / mg, 11 pg / mg, 12 pg / mg, 13 pg / mg, 14 pg / mg, or 15 pg / mg.

[0016] In some embodiments, the Akkermansia muciniphila MNH19250 is deposited with the Guangdong Microbial Culture Collection Center (GDMCC) on June 21, 2024, and has a deposit number of GDMCC No: 63782.

[0017] In a second aspect, the present disclosure provides a composition comprising the Akkermansia muciniphila or culture thereof or metabolite thereof or secreted protein thereof of the first aspect.

[0018] In some embodiments, the culture of Akkermansia muciniphila comprises a solid culture of Akkermansia muciniphila, a fermentation culture, or a supernatant of a fermentation culture.

[0019] In some embodiments, the fermentation culture or supernatant of a fermentation culture is obtained using a liquid medium under anaerobic culture conditions.

[0020] In some embodiments, the composition is provided in a liquid form or a solid form.

[0021] In some embodiments, the composition comprises 1 x 10 4 to 1 x 10 12 cfu / mL or 1 x 10 4 to 1 x 1012 The cfu / mg of the live Akermansia myxophilus bacteria.

[0022] In some embodiments, the composition contains 1×10 5 Up to 1×10 11 cfu / mL or 1×10 5 Up to 1×10 11 The cfu / mg of the live Akermansia myxophilus bacteria.

[0023] In some embodiments, the composition contains 1×10 6 Up to 1×10 10 cfu / mL or 1×10 6 Up to 1×10 10 The cfu / mg of the live Akermansia myxophilus bacteria.

[0024] In some embodiments, the composition contains 1×10 7 Up to 1×10 9 cfu / mL or 1×10 7 Up to 1×10 9 The cfu / mg of the live Akermansia myxophilus bacteria.

[0025] In some embodiments, each gram of the composition contains 1 × 10⁻⁶ 3 Up to 1×10 17 Bacteria with colony-forming units (CFU); for example, 1 × 10⁶. 4 Up to 1×10 12 1×10 5 Up to 1×10 11 One or 1×10 6 Up to 1×10 10 A colony-forming unit (CFU) of bacteria, specifically, for example, 1 × 10⁶. 3 2×10 3 3×10 3 4×10 3 5×10 3 6×10 3 7×10 3 8×10 3 9×10 3 1×10 4 2×10 4 3×10 4 4×10 4 5×10 4 6×10 4 7×10 4 8×10 4 9×10 4、1×10 5 、2×10 5 、3×10 5 、4×10 5 、5×10 5 、6×10 5 、7×10 5 、8×10 5 、9×10 5 、1×10 6 、2×10 6 、3×10 6 、4×10 6 、5×10 6 、6×10 6 、7×10 6 、8×10 6 、9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、4×10 10 、5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、1×10 11 、2×1011 3 x 10 11 4 x 10 11 5 x 10 11 6 x 10 11 7 x 10 11 8 x 10 11 9 x 10 11 1 x 10 12 2 x 10 12 3 x 10 12 4 x 10 12 5 x 10 12 6 x 10 12 7 x 10 12 8 x 10 12 9 x 10 12 1 x 10 13 2 x 10 13 3 x 10 13 4 x 10 13 5 x 10 13 6 x 10 13 7 x 10 13 8 x 10 13 9 x 10 13 or any value therebetween, of colony forming units (CFU) of bacteria.

[0026] In some embodiments, the Akkermansia muciniphila in the composition is an attenuated bacteria, a killed bacteria, a lyophilized bacteria or an irradiated bacteria, for example can be heat-inactivated bacteria, preferably pasteurized.

[0027] In some embodiments, the composition is in the form of a liquid, a foam, a cream, a spray, a powder (e.g. a lyophilized powder) or a gel.

[0028] In some embodiments, the composition is in the form of a powder, a microencapsulated powder, a capsule, a tablet, a lozenge, a granule, an oral liquid, a suspension, an emulsion, a liquid preparation, a sustained-release preparation, a nano-preparation or a micro-encapsulated capsule.

[0029] In some embodiments, the composition is in the form of an oral agent or an injection agent.

[0030] In some embodiments, the composition further comprises one or more pharmaceutically acceptable carriers or excipients or adjuvants.

[0031] The pharmaceutically acceptable adjuvants are well known to those skilled in the art.

[0032] In some embodiments, the adjuvant can be at least one selected from a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener and a flavoring agent.

[0033] In some embodiments, the composition comprises one or more of a buffer (e.g., sodium bicarbonate, infant formula, or sterile human milk or other agent that allows the bacteria to survive and grow (e.g., survive in the acidic environment of the stomach and grow in the intestinal environment)), a lyoprotectant, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegrant, an antioxidant, a flavoring agent, a sweetener, and a coloring agent.

[0034] In some embodiments, the composition further comprises one or more other active agents for preventing or treating metabolic diseases and / or tumors.

[0035] The other active agent has at least one of the following functions: (a) suppressing appetite, (b) preventing metabolic diseases, (c) treating metabolic diseases, (d) preventing tumors, (e) treating tumors.

[0036] In some embodiments, the suppressing appetite comprises reducing food intake and / or reducing appetite.

[0037] In some embodiments, the other active agent is selected from the group consisting of: a GLP-1 receptor agonist, a dual agonist of GLP-1 receptor and GCG receptor, a triple agonist of GLP-1 receptor, GIP receptor and GCG receptor, an AMPK agonist, or an active pharmaceutical that promotes GLP-1 secretion.

[0038] In some embodiments, the other active agent is selected from the group consisting of: metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin, pioglitazone, rosiglitazone, pentoxifylline, omega-3-fatty acids, statins, ezetimibe, ursodeoxycholic acid, semaglutide, liraglutide, exenatide, and bexarotene.

[0039] In some embodiments, the other active agent can be an agent for preventing or treating tumors.

[0040] In some embodiments, the other active agent can be one or more than one of probiotics, prebiotics, or a combination thereof.

[0041] Preferably, the probiotics are selected from the group consisting of lactic acid bacteria, lactobacilli, lactococci, butyrate-producing bacteria, bifidobacteria, Streptococcus thermophilus, Streptococcus faecalis, Streptococcus mesentericus.

[0042] Preferably, the prebiotic is selected from the group consisting of inulin, mulberry leaf extract, berberine, ganoderma, green coffee bean extract, oat, pectin, potato or extract thereof, citrus polyphenol, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidin, resistant dextrin, yeast beta-glucan, ginseng or extract thereof, nutritional compound, biotin, polydextrose, fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, mannan-oligosaccharide, mannan-oligosaccharide (MOS), inulin enriched with fructo-oligosaccharide, gluco-oligosaccharide, tagatose, trans-galacto-oligosaccharide, pectin, resistant starch, xylo-oligosaccharide (XOS), and any combination thereof.

[0043] In some embodiments, the composition can be formulated as a frozen composition, e.g., a frozen composition prepared by flash freezing and drying, or lyophilization, for storage and / or transport.

[0044] In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained by electrostatic spray drying.

[0045] In some embodiments, the strains in the composition are freeze-dried or spray-dried. In some embodiments, the strains in the composition are electrostatically spray-dried. In some embodiments, the strains in the composition are freeze-dried or spray-dried and are viable. In some embodiments, the strains in the composition are freeze-dried or spray-dried and are capable of partial or complete colonization of the intestine. In some embodiments, the strains are reconstituted prior to administration. In some cases, the reconstitution is by use of a diluent described herein.

[0046] In some embodiments, the composition can be administered alone or in combination with a carrier, such as a pharmaceutically acceptable carrier or a biocompatible scaffold.

[0047] In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric formulation. In some embodiments, the enteric formulation is a dosage form with an enteric coating. For example, the enteric formulation can be an enteric granule, an enteric tablet, or an enteric capsule. In some embodiments, the composition is a capsule. In some embodiments, the capsule is a hard capsule or a soft capsule; or the capsule is a sustained release capsule, a controlled release capsule, or an enteric capsule, or the capsule can be a microencapsulated capsule, or a microcapsule.

[0048] In some embodiments, the composition is a medicament, or a nutraceutical, or a food product.

[0049] In some embodiments, the composition is an infant-appropriate dosage form, a child-appropriate dosage form, or an adult-appropriate dosage form.

[0050] In some embodiments, the composition is a gastrointestinal administration dosage form or a parenteral administration dosage form.

[0051] In a third aspect, there is provided use of the Akkermansia muciniphila of the first aspect or the composition of the second aspect in the preparation of a medicament, health product or food for treating, preventing or alleviating inflammatory diseases, liver and kidney diseases, cardiovascular and cerebrovascular diseases, metabolic diseases, intestinal barrier damage diseases, immune-related diseases, tumors.

[0052] In some embodiments, the metabolic disease is a metabolic disease, a metabolic disorder, or a disease caused by a metabolic disorder, including but not limited to at least one of liver diseases, obesity and obesity-related diseases, cardiovascular diseases, cardiovascular and cerebrovascular diseases, diabetes, dyslipidemia, glucose intolerance, type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, alcoholic fatty liver hepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic disease, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), atherosclerosis, and kidney diseases.

[0053] In some embodiments, the liver and kidney disease includes liver diseases, liver function damage related diseases, kidney damage, abnormal kidney function, and other kidney diseases.

[0054] The liver diseases include, but are not limited to, at least one of fatty liver, NAFLD / NASH, liver dysfunction, extrahepatic cholestasis, hepatitis, liver damage, intrahepatic cholestasis, liver fibrosis, cirrhosis, and liver cell damage.

[0055] The composition comprises a population of isolated and purified live microorganisms to reduce serum levels of at least 2 U / L, 5 U / L, or 10 U / L, 25 IU / L, 30 IU / L, 50 IU / L of one or more of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymes in a subject compared to the levels of ALT and / or AST in the subject prior to administration of the population of isolated and purified microorganism species; the composition comprises a population of isolated and purified live microorganisms to reduce serum levels of at least 2 mmol / L, 5 mmol / L, 10 mmol / L of urea nitrogen BUN, creatinine CRE in the serum of a subject compared to the serum levels of BUN, CRE in the serum of a subject prior to administration of the population of isolated and purified microorganism species.

[0056] As used herein, a microbiota generally refers to a population of microorganisms that consists essentially of a single strain, species, or genus, which can be the case when a population is cultured from a subpopulation of an isolated and purified strain, species, or genus. Thus, for a given population of microorganisms, if cultured from an isolated microorganism species or strain, such population will be referred to herein as purified or substantially pure. The resulting population can be at least 80% pure with respect to the microorganism species or strain, at least 90% pure with respect to other microorganism species or strains within that particular population, at least 95% pure, at least 98% pure, at least 99% pure, at least 99.5% pure, or at least 99.9% pure. Conversely, the level of non-desired strains in any particular desired population of microorganisms will be less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%. For example, the level of impurities in a purified population of microorganisms, such as other undesired microorganism strains or species, can be proportionally at or below the above levels for each desired population. Less than 2%, less than 1%, less than 0.5%, or less than 0.1%. In the case where a composition comprises a consortium of multiple populations of microorganisms, each population can have the above purity, either prior to its incorporation into the composition or when measured in aggregate with respect to the consortium. For example, the level of impurities in a purified population of microorganisms, such as other undesired microorganism strains or species, can be proportionally at or below the above levels for each desired population. Less than 2%, less than 1%, less than 0.5%, or less than 0.1%. In the case where a composition comprises a consortium of multiple populations of microorganisms, each population can have the above purity, either prior to its incorporation into the composition or when measured in aggregate with respect to the consortium. For example, the level of impurities in a purified population of microorganisms, such as other undesired microorganism strains or species, can be proportionally at or below the above levels for each desired population.

[0057] The pharmaceutical compositions of the present disclosure can also include cell components, metabolites, secreted molecules and compounds, and the like, that are metabolized by Akkermansia muciniphila. These can be recovered, for example, by recovering the supernatant of an Akkermansia muciniphila culture or by extracting cell components or cell fractions, metabolites or secreted compounds from an Akkermansia muciniphila culture; can correspond to isolated forms of components from Akkermansia muciniphila, or any mixture of one or more components from Akkermansia muciniphila.

[0058] In some embodiments, the Akkermansia muciniphila of the present disclosure can be used for treating or preventing a liver function impairment-related disease, including at least one of fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, liver cirrhosis, liver cancer, or liver cell damage.

[0059] In some embodiments, the predisposing factor of the liver disease includes, but is not limited to, at least one of high-fat diet, high-cholesterol diet, high-sugar diet, hyperlipidemia, hyperglycemia, or high cholesterol.

[0060] In some embodiments, the liver disease includes at least one of high-fat diet-induced, high-cholesterol diet-induced, high-sugar diet-induced, high-fat high-cholesterol-induced, high-fat high-sugar-induced, and / or high-fat high-cholesterol high-sugar-induced.

[0061] In some embodiments, the Akkermansia muciniphila of the present disclosure can be used for treating, preventing, or alleviating a liver function impairment-related disease, which includes at least one of fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, liver cirrhosis, or liver cell damage.

[0062] In particular, the Akkermansia muciniphila of the present disclosure or the composition comprising the same can be used for treating or preventing liver impairment. The liver impairment can be caused by long-time labor, long-term alcohol consumption, fatty liver, drug-induced liver disease, or genetic metabolic factors.

[0063] In some embodiments, the Akkermansia muciniphila of the present disclosure or the composition comprising the same can reduce the levels of ALT and / or AST.

[0064] In some embodiments, the Akkermansia muciniphila of the present disclosure or the composition comprising the same can reduce the weight of the liver.

[0065] In some embodiments, the kidney disease includes, but is not limited to, kidney impairment, abnormal kidney function, etc. The kidney disease includes, but is not limited to, primary glomerulonephritis, hypertensive renal arteriosclerosis, diabetic nephropathy, secondary glomerulonephritis, tubulointerstitial lesions (e.g., chronic pyelonephritis, chronic uric acid nephropathy, obstructive nephropathy, drug-induced nephropathy, etc.), ischemic nephropathy, etc. In some embodiments, the predisposing factor of the kidney disease includes at least one of high-fat diet, high-sugar diet, and high-cholesterol diet. In some embodiments, the kidney disease is manifested as elevated blood creatinine.

[0066] In some embodiments, the obesity and obesity-related diseases include, but are not limited to, overweight, obesity, metabolic syndrome, cardiovascular disease, cardio-cerebrovascular disease, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, and fatty liver hepatitis.

[0067] In some embodiments, the predisposing factor of the obesity and obesity-related diseases includes, but is not limited to, at least one of high-fat diet, high-sugar diet, high-cholesterol, hyperlipidemia, hyperglycemia, NAFLD, or NASH.

[0068] In some embodiments, the obesity and obesity-related diseases include, but are not limited to, obesity induced by high-fat diet, obesity induced by high-cholesterol diet, obesity induced by high-sugar diet, obesity induced by high-fat high-cholesterol diet, obesity induced by high-fat high-sugar diet, obesity induced by high-fat high-cholesterol high-sugar diet, or obesity in a patient with NAFLD or NASH.

[0069] In some embodiments, the obesity and obesity-related diseases include at least one of obesity, metabolic syndrome, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, obesity-related gastroesophageal reflux, and fatty liver hepatitis.

[0070] Compositions and methods for treating, reducing, controlling, decreasing, or preventing the symptoms, signs, or indicators of liver, kidney diseases, and the onset of the diseases themselves are provided. The compositions and methods include microbial compositions selected to improve the intestinal function of a subject to whom they are administered, thereby treating liver, kidney diseases, and / or the signs, symptoms, and indicators of these diseases.

[0071] In some embodiments, the obesity is peripheral obesity, and / or central obesity. In some embodiments, the obesity is diet-induced obesity, and / or metabolic obesity. In particular, the obesity is abdominal obesity or apple-shaped obesity, e.g., excess visceral fat.

[0072] In some embodiments, the Akkermansia muciniphila of the present application or a composition comprising the same is capable of reducing body fat and / or visceral fat and / or abdominal fat.

[0073] In some embodiments, the diabetes includes, but is not limited to, type I diabetes, type II diabetes, gestational diabetes, diabetes mediated by HDAC activity, diabetic nephropathy, diabetic neuropathy, diabetic eye disease, diabetic retinopathy, diabetic foot, diabetes caused by damage to pancreatic B cells, diabetes caused by insulin resistance, diabetes caused by obesity.

[0074] In some embodiments, the diabetes is induced by at least one of pancreatic cell dysfunction, decreased insulin secretion, increased insulin resistance, high-fat diet, high-sugar diet, high-cholesterol, high blood lipids, high blood glucose, NAFLD, or NASH.

[0075] In some embodiments, the diabetes is induced by at least one of high-fat diet, high-sugar diet, high-cholesterol diet.

[0076] In some embodiments, the diabetes is manifested as hyperglycemia caused by low levels of insulin and / or peripheral insulin resistance.

[0077] In some embodiments, the metabolic disorder includes, but is not limited to, (1) diabetes caused by sugar metabolism disorder, or (2) diabetes caused by abnormal glucose tolerance or reduced glucose tolerance, or (3) diabetes caused by impaired insulin B cells, or (4) diabetes caused by insulin resistance.

[0078] In some embodiments, the Akkermansia muciniphila of the present disclosure has the effect of increasing the secretion level of glucagon-like peptide-1 (GLP-1), thereby regulating the blood glucose balance of the body, improving the glucose tolerance of the body, further improving the insulin sensitivity and leptin sensitivity of the body, and thereby achieving the effect of preventing and / or treating diabetes and / or hyperlipidemia.

[0079] In some embodiments, the cardiovascular disease or cardio-cerebrovascular disease includes, but is not limited to, atherosclerosis, coronary heart disease, hypertension, cardiovascular disease in patients with NAFLD or NASH, cardio-cerebrovascular disease in patients with NAFLD or NASH, and high cholesterol disease.

[0080] In some embodiments, the cause of the cardiovascular disease or cardio-cerebrovascular disease includes, but is not limited to, at least one of atherosclerosis, NAFLD, NASH, hyperlipidemia, hyperglycemia, or high cholesterol.

[0081] In some embodiments, the inflammatory disease is selected from diseases related to inflammation of the bronchus, such as bronchitis; diseases related to inflammation of the cervix, such as cervicitis; diseases related to inflammation of the conjunctiva, such as conjunctivitis; diseases related to inflammation of the esophagus, such as esophagitis; diseases related to inflammation of the myocardium, such as myocarditis; diseases related to inflammation of the rectum, such as proctitis; diseases related to inflammation of the sclera, such as scleritis; diseases related to inflammation of the gums; diseases related to inflammation of the bone, lung inflammation (alveolitis), respiratory tract inflammation (e.g., asthma, such as bronchial asthma), acute respiratory distress syndrome (ARDS); inflammatory skin diseases, such as contact dermatitis, atopic dermatitis; fibrotic diseases (e.g., pulmonary fibrosis); encephalitis.

[0082] In some embodiments, the Akkermansia muciniphila of the present disclosure has the effect of treating / preventing damage to the intestinal barrier. In some embodiments, the cause of the damage to the intestinal barrier includes at least one of a high-fat diet, a high-sugar diet, and a high-cholesterol diet.

[0083] In some embodiments, examples of the tumor or cancer include, but are not limited to, solid tumors and non-solid tumors.

[0084] In some embodiments, the drug or health product or food has at least one effect selected from the group consisting of: reducing liver weight; treating initial fatty liver inflammation lesions; slowing down liver cell fat accumulation; reducing serum AST, ALT; reducing abdominal white fat inflammatory lesions; reducing the weight of the mammal; reducing the food intake of the mammal; reducing the body fat of the mammal; reducing at least one of the following in the serum of the mammal: total cholesterol level, low-density lipoprotein and triglyceride level; increasing the level of high-density lipoprotein in the serum of the mammal; improving impaired oral glucose tolerance of the mammal; reducing the fasting blood glucose of the mammal; reducing the HOMA-IR index of the mammal; reducing at least one of the following in the mammal: epididymal fat weight, perirenal fat weight, visceral fat weight and inguinal fat weight; repairing digestive tract mucosa damage; increasing colon mucus layer thickness; regulating the immune system of the body; increasing the expression of IFNβ; promoting the transcriptional activity of IFNβ; treating, preventing or alleviating coronary heart disease; treating, preventing or alleviating atherosclerosis; treating, preventing or alleviating hyperglycemia; treating, preventing or alleviating hyperlipidemia; treating, preventing or alleviating high cholesterol; treating, preventing or alleviating liver function damage; treating, preventing or alleviating fatty liver; treating, preventing or alleviating NAFLD or NASH; treating, preventing or alleviating hypertension; treating, preventing or alleviating diabetes, preferably gestational diabetes or type II diabetes or diabetes mediated by HDAC activity; treating, preventing or alleviating obesity; treating, preventing or alleviating metabolic syndrome; treating, preventing or alleviating local seborrhea, excessive inguinal fat, excessive epididymal fat and / or excessive brown fat; treating, preventing or alleviating tumors.

[0085] The Akkermansia muciniphila MNH19250 isolated by the present disclosure has strong autoaggregation ability and hydrophobicity, is easy to adhere to epithelial cells, and has good pH, NaCl and bile salt tolerance, and thus can quickly and stably colonize in the intestinal tract of humans or animals.

[0086] The Akkermansia muciniphila MNH19250 isolated by the present disclosure can also synthesize a large amount of short-chain fatty acids, and thus can effectively treat or prevent metabolic diseases, reduce the weight, body fat, blood glucose, blood lipids and cholesterol of the subject, improve or alleviate non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), repair intestinal tissue mucosa, promote the expression of type I interferon IFNβ of the subject, improve immunity, and has the potential to prevent or treat viral infections and inhibit tumor growth.

[0087] Therefore, in some embodiments, the drug or health product or food can regulate metabolism while also regulating immunity.

[0088] In some embodiments, the drug or health product or food product can reduce abdominal fat while also repairing intestinal barrier damage.

[0089] In some embodiments, the drug or health product or food product can improve liver damage while also reducing weight.

[0090] In some embodiments, the drug or health product or food product can reduce weight while also reducing inflammation.

[0091] In some embodiments, the drug or health product or food product can reduce lipid while also reducing inflammation. The lipid reduction can be abdominal obesity, or visceral fat reduction.

[0092] In some embodiments, the drug or health product or food product can reduce lipid while also repairing intestinal barrier damage.

[0093] In some embodiments, the drug or health product or food product can reduce lipid while also reducing serum AST and / or ALT. The lipid reduction can be abdominal obesity, or visceral fat reduction.

[0094] In some embodiments, the drug or health product or food product can improve liver damage while also improving kidney damage.

[0095] In some embodiments, the drug or health product or food product can improve diabetes while also improving liver damage.

[0096] In some embodiments, the drug or health product or food product can improve diabetes while also improving kidney damage.

[0097] In some embodiments, the drug or health product or food product can improve atherosclerosis while also improving liver damage.

[0098] In some embodiments, the drug or health product or food product can improve atherosclerosis while also reducing lipid. The lipid reduction can be abdominal obesity, or visceral fat reduction.

[0099] In some embodiments, the Akkermansia muciniphila or the composition of the present application has an effect of affecting or modulating immune signaling, affecting intestinal barrier function, affecting or modulating fasting glucose homeostasis, affecting or modulating cholesterol homeostasis, triglyceride homeostasis, repairing liver damage, kidney damage, promoting local adipose tissue metabolism in a subject in need thereof.

[0100] In some embodiments, the Akkermansia muciniphila or the composition described in the present application has an effect of reducing liver and / or kidney injury, the reduction of liver injury is reducing one or more elevated liver injury or liver disease indicators of the subject, for example, reducing the serum level of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and / or liver weight indicators of the subject; the reduction of kidney injury is reducing urea nitrogen (BUN) in the serum of the subject and / or reducing the serum CRE (creatinine) indicator. BRIEF DESCRIPTION OF DRAWINGS

[0101] The above and other aspects and advantages of the present application will become more apparent by describing in detail the embodiments thereof with reference to the attached drawings in which:

[0102] Figure 1 shows the colony morphology photo of strain MNH19250.

[0103] Figure 2 shows the gram staining photo of strain MNH19250.

[0104] Figure 3 shows the electron microscope photo of strain MNH19250.

[0105] Figure 4 shows the tolerance results of strain MNH19250 to different pH. The abscissa is pH value, and the ordinate is survival percentage.

[0106] Figure 5 shows the tolerance results of strain MNH19250 to different concentrations of NaCl.

[0107] Figure 6 shows the tolerance results of strain MNH19250 to different concentrations of bile salts.

[0108] Figure 7 shows the 6h auto-aggregation ability of strain MNH19250.

[0109] Figure 8 shows the phylogenetic tree of strain MNH19250.

[0110] Figure 9 shows the columnar distribution diagram of relative fluorescence value of IFNβ expression amount in the presence of strain MNH19250.

[0111] Figure 10 shows that strain MNH19250 can significantly reduce liver function indicators (a: ALT; b: AST).

[0112] Figure 11: shows that the strain MNH19250 can significantly improve nonalcoholic fatty liver and nonalcoholic fatty liver disease (NAFLD / NASH) (a: liver weight; b: liver weight percentage of body weight; c: liver steatosis; d: liver lobular inflammation; e: liver cell ballooning; f: nonalcoholic fatty liver disease activity score (NAS); g: liver HE tissue section).

[0113] Figure 12: shows that the strain MNH19250 can improve kidney function indicators (a: urea nitrogen BUN; b: creatinine CRE).

[0114] Figure 13: shows that the strain MNH19250 can significantly reduce the oral glucose tolerance of high-fat diet-induced type 2 diabetic mice (a. Oral glucose tolerance results; b. Oral glucose tolerance area under the curve results; data is shown as mean ± standard deviation (Mean ± SD). Statistical analysis uses Student’s t test analysis method; *, p < 0.05 compared with the HFD-Control group; **, p < 0.01 compared with the HFD-Control group).

[0115] Figure 14: shows that the strain MNH19250 can significantly reduce the fasting blood glucose of high-fat diet-induced type 2 diabetic mice (data is shown as mean ± standard deviation (Mean ± SD). Statistical analysis uses Student’s t test analysis method; *, p < 0.05 compared with the HFD-Control group).

[0116] Figure 15: shows that the strain MNH19250 can significantly reduce the body weight of high-fat diet-induced obese mice (a. Body weight results; b. Body weight change ratio results; data is shown as mean ± standard deviation (Mean ± SD). Statistical analysis uses Student’s t test analysis method; *, p < 0.05 compared with the HFD-Control group; **, p < 0.01 compared with the HFD-Control group).

[0117] Figure 16: shows that the strain MNH19250 can significantly reduce the blood lipids of high-fat diet-induced obese mice (a. Total cholesterol in serum (TCHO) results; b. Serum triglyceride (TG); c. Serum low-density lipoprotein cholesterol (LDL-C); d. Low-density lipoprotein cholesterol / high-density lipoprotein cholesterol (LDL-C / HDL-C); data is shown as mean ± standard deviation (Mean ± SD). Statistical analysis uses Student’s t test analysis method; *, p < 0.05 compared with the HFD-Control group).

[0118] Figure 17: shows that strain MNH19250 can significantly reduce the fat weight and the ratio of visceral fat of high-fat diet-induced obese mice (a. epididymal fat; b. perirenal fat; c. mesenteric fat; d: visceral fat; e: the ratio of visceral fat; f: inguinal fat; data is shown as Mean ± SD. Statistical analysis was performed using Student’s t-test; *, p < 0.05 compared with the HFD-Control group).

[0119] Figure 18: shows that strain MNH19250 can significantly increase the mucus layer thickness of high-fat diet-induced obese mice (a. colon Alcian blue staining map; b. colon mucus layer thickness; data is shown as Mean ± SD. Statistical analysis was performed using Student’s t-test; *, p < 0.05 compared with the HFD-Control group).

[0120] Preservation of the strain

[0121] The strain Akkermansia muciniphila MNH19250 is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) with the preservation number GDMCC No: 63782, the preservation time is June 21, 2024, the address is No. 59, Building 5, Guangzhou Xianlie Middle Road 100, Guangdong Microbial Institute, and the preservation name is Akkermansia muciniphila MNH19250. DETAILED DESCRIPTION

[0122] The present disclosure isolates a new strain of Akkermansia muciniphila species with the preservation number GDMCC No: 63782, and identifies it using traditional classification methods and molecular biology methods. The identification results show that the strain belongs to a new strain of Akkermansia muciniphila species. Further, the present disclosure studies the biochemical properties and therapeutic uses of the strain.

[0123] It is known in the art that the classification and identification of bacterial species can be performed by traditional classification methods and molecular biology methods. Traditional classification methods include, but are not limited to, for example, cell morphology observation, Gram staining, flagella staining, various metabolic experiments, etc. Molecular biology methods include, but are not limited to, ribosomal RNA sequence determination method, whole genome sequencing-based determination method, etc.

[0124] The term “prebiotic” as used herein can be a general term referring to a chemical substance and / or component that can affect the growth and / or activity of microorganisms in the host (e.g., can allow specific changes in the composition and / or activity of the microbiome).

[0125] The terms "subject," "object," "individual," "host," and "patient" are used interchangeably herein to refer to any animal subject, including: humans, mammals, experimental animals, livestock, and domestic pets.

[0126] The compositions or formulations of the present disclosure can be administered as a pharmaceutical formulation, a therapeutic composition, a dietary supplement, a nutritional supplement, a medical probiotic, or a medical food. In certain instances, the composition is administered in the form of a pharmaceutical formulation. In certain instances, the composition is administered in the form of a nutritional supplement. In certain instances, the composition is administered in the form of a dietary supplement. In certain instances, the composition is administered in the form of a medical food. In certain instances, the composition is administered in the form of a medical probiotic. In certain instances, the composition (e.g., a dietary supplement, a nutritional supplement, a medical probiotic, or a medical food) can be administered orally, for example, as a capsule, pill, or tablet.

[0127] In the context of the present invention, the term "polypeptide" is equivalent to "protein." A polypeptide has a specific amino acid sequence. A "variant" of a polypeptide of the present invention preferably has an amino acid sequence that has at least 50% sequence identity to a polypeptide of the present invention.

[0128] The term "homeostasis" is a mechanism that facilitates processes that maintain internal balance of the organism, for example, glucose regulation homeostasis, triglyceride homeostasis, cholesterol homeostasis.

[0129] 16S rRNA is a ribosomal RNA of prokaryotes, and the 16S rRNA gene is composed of variable regions and conserved regions. The conserved regions are shared by all bacteria, while the variable regions differ to varying degrees among different bacteria. By comparing the 16S rRNA gene sequences of bacteria, an evolutionary tree can be drawn according to the evolutionary distance based on the number of sequence differences. When the sequence identity between two strains of 16S rRNA genes is less than 98.65%, they can be judged to belong to different species (see Kim, M., Oh, H.-S., Park, S.-C., & Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary Microbiology, 64(Pt 2), 346-351, and Liu, C., Du, M.-X., Abuduaini, R., Yu, H.-Y., Li, D.-H., Wang, Y.-J., Liu, S.-J. (2021). Enlightening the taxonomy darkness of human gut microbiomes with a cultured biobank. Microbiome, 9(1), P23).

[0130] The "identity" between the sequences of two nucleic acid molecules can be determined using known computer algorithms, such as the "FASTA" program, the GCG package, BLASTN or FASTA. Commercially or publicly available programs can also be, for example, the DNAStar "MegAlign" program.

[0131] The second-generation sequencing technology can also be used for bacterial strain identification based on whole genome sequencing, which makes the bacterial strain identification result more accurate. Average nucleotide identity (ANI) of bacterial genome refers to the similarity of homologous genes between two bacterial genomes. ANI value can be calculated by BLAST and other methods. In the field of bacterial taxonomy, it is generally believed that ANI value needs to reach more than 95% to be identified as belonging to the same bacterial species (Jain C, Rodriguez-R L M, Phillippy A M, et al. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries [J]. Nature Communications, 2018, 9(1): 5114.).

[0132] The existing mature ANI value calculation tools can be used, such as local operation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), online calculation tools ANI caculator (http: / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani) and ANItools.

[0133] Using the above method, a person skilled in the art can determine whether a separated strain belongs to the Akkermansia muciniphila strain identified by the present inventors. For example, when the average nucleotide identity ANI value with Akkermansia muciniphila (GCF_000020225.1) is at least 95%, such as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, it can be determined that it belongs to the same bacterial species.

[0134] For example, when its 16S rRNA sequence has at least 98.65% identity, such as at least 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or is 100% to the sequence set forth in SEQ ID NO. 1, it can be determined to belong to the same species.

[0135] A "strain" refers to a member of a bacterial species that has genetic characteristics that distinguish it from closely related members of the same bacterial species. The genetic characteristics can be the absence of all or part of at least one gene, the absence of all or part of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the absence of at least one native plasmid ("cure"), the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one exogenous gene (a gene from another species), the presence of at least one mutated regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. The genetic characteristics between different strains can be identified by PCR amplification, optionally followed by DNA sequencing of the genomic region of interest or the entire genome. In the case where a strain gains or loses antibiotic resistance or gains or loses biosynthetic capabilities (e.g., an auxotrophic strain) compared to another strain of the same species, the strains or nutrients / metabolites can be distinguished by selection or counter-selection using the antibiotic.

[0136] "Supernatant" or "supernate" within the meaning herein refers to a culture supernatant of a bacterial strain according to the present disclosure, optionally comprising compounds and / or cell debris of said strain, and / or metabolites and / or molecules secreted by said strain.

[0137] Compositions can be prepared using the mucinophilic Akkermansia described herein, for example by using pharmaceutically acceptable excipients. The pharmaceutical compositions comprise a pharmaceutically effective amount of the mucinophilic Akkermansia, for example the mucinophilic Akkermansia having the accession number GDMCC NO: 63782. Likewise, the mucinophilic Akkermansia having the accession number GDMCC NO: 63782 can also be prepared into a pharmaceutical composition, for example by using pharmaceutically acceptable excipients, comprising a pharmaceutically effective amount of the mucinophilic Akkermansia.

[0138] Suitable pharmaceutically acceptable excipients that can be used are, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings.

[0139] The compositions herein can be formulated in any form suitable for enhancing the abundance of Akkermansia muciniphila in a subject. The compositions can be administered by oral administration (e.g., by oral gavage), intramuscular injection, inhalation, intracranial, intralymphatic, intraocular, intraperitoneal, intrapleural, intrathecal, intratracheal, intrauterine, intravascular, intravenous, intravesical, intranasal, gastrointestinal, biliary perfusion, cardiac perfusion, pre-anal, rectal, spinal subcutaneous, sublingual, topical, intravaginal, transdermal, ureteral, or urethral routes, among others.

[0140] Examples of suitable dosage forms for the compositions herein include, but are not limited to, tablets, aerosols, chewable bars, capsules, capsules containing coated granules, capsules containing sustained-release granules, capsules containing sustained-release granules, concentrates.

[0141] In some embodiments, the compositions are sugar-coated tablets, gel capsules, gels, emulsions, tablets, tablet-like capsules, hydrogels, nanofiber gels, electrospun fibers, food bars, candies, fermented milk, fermented cheese, chewing gum, powders, or toothpaste, among others.

[0142] In some embodiments, administration can also be by inclusion in the subject’s diet, for example, in a functional food for a human or companion animal.

[0143] The compositions provided herein can include a pharmaceutically acceptable excipient, diluent, or carrier. Pharmaceutically acceptable excipients, diluents, or carriers are well known in the art.

[0144] In some embodiments, the Akkermansia muciniphila in the compositions of the present disclosure are lyophilized. In some embodiments, the Akkermansia muciniphila in the compositions of the present disclosure are spray-dried. In some embodiments, the Akkermansia muciniphila in the compositions of the present disclosure are lyophilized or spray-dried and are viable. In some embodiments, the Akkermansia muciniphila in the compositions of the present disclosure are lyophilized or spray-dried and are partially or completely colonized in the intestine. In some embodiments, the lyophilized Akkermansia muciniphila are reconstituted prior to administration. In some embodiments, the reconstitution is performed using a diluent described herein.

[0145] In some embodiments, the compositions of the present disclosure are administered orally. Oral administration can involve swallowing, so that the composition enters the gastrointestinal tract, and / or administration through the mouth, tongue, or sublingually.

[0146] In some embodiments, the compositions are prepared by freeze-drying, or spray-drying.

[0147] The compositions of the present disclosure include pharmaceutical compositions, nutraceuticals, or food products.

[0148] The subject of the present disclosure can be a human or an animal, including but not limited to a cow, a sheep, a cat, a dog, a horse, a rabbit, a monkey, a mouse, a rat, an alpaca, a camel, etc.

[0149] The pharmaceutical composition of the present disclosure can be used for treating, preventing, or alleviating a metabolic disease or a disease caused by a metabolic disorder.

[0150] The pharmaceutical composition of the present disclosure can be used for treating, preventing, or alleviating a tumor.

[0151] In some embodiments, the metabolic disease, metabolic disorder, or disease caused by a metabolic disorder includes, but is not limited to, at least one of a liver disease, obesity and obesity-related diseases, cardiovascular disease, diabetes, dyslipidemia, cardio-cerebral vascular disease, glucose intolerance, atherosclerosis, coronary heart disease or hypertension, type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, alcoholic fatty liver hepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic disease, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), atherosclerosis, and renal disease.

[0152] Diabetes

[0153] Diabetes includes type I diabetes (T1D), type II diabetes (T2D), and gestational diabetes (GDM). Type I diabetes is a kind of diabetes caused by autoimmune damage or idiopathic reasons, characterized by absolute destruction of islet function, often occurs in children and adolescents, and must be treated with insulin to achieve satisfactory effect, otherwise it will be life-threatening. Type II diabetes is a multifactorial syndrome characterized by abnormal carbohydrate / fat metabolism, usually including hyperglycemia, hypertension, and cholesterol abnormalities. Type II diabetes is caused by ineffective action of insulin (low receptor binding content), so not only fasting blood glucose but also 2-hour postprandial blood glucose should be checked, and especially islet function should be checked. There are two cases of diabetes during pregnancy, one is that diabetes is diagnosed before pregnancy, which is called "diabetes combined with pregnancy"; the other is that the pregnant woman has normal glucose metabolism or potential impaired glucose tolerance before pregnancy, and the diabetes appears or is diagnosed during pregnancy, which is also called "gestational diabetes mellitus (GDM)", and more than 80% of pregnant women with diabetes are GDM.

[0154] 4 kinds of metabolic disease related models, high fat diet (HFD) induced mouse obesity model, high fat high sugar high cholesterol induced mouse NASH model, high fat diet combined with streptozotocin (Streptozotocin) (HFD-STZ) induced mouse type II diabetes model, leptin receptor gene deficiency mouse model (db / db), are commonly used metabolic disease mouse models, and model mice are usually accompanied by obesity, insulin resistance, hyperglycemia, hyperlipidemia, high cholesterol, NAFLD / NASH and other metabolic diseases.

[0155] Insulin resistance refers to the decrease in the efficiency of insulin in promoting glucose uptake and utilization due to various reasons, and the body compensates by secreting too much insulin to produce hyperinsulinemia to maintain blood glucose stability. Insulin resistance can easily lead to metabolic syndrome and type II diabetes.

[0156] Oral glucose tolerance is used to determine the function of pancreatic beta cells and the body's ability to regulate blood glucose, and is currently recognized as a diagnostic indicator for diagnosing diabetes. When sugar metabolism is disturbed, the blood glucose rises sharply after a certain amount of glucose is taken orally, or the rise is not obvious, but it cannot be reduced to the fasting level or the original level within a short period of time, which is abnormal glucose tolerance or decreased glucose tolerance; abnormal glucose tolerance indicates that the body's ability to metabolize glucose is decreased, which is commonly seen in type II diabetes and obesity.

[0157] HOMA-IR is an index for evaluating the level of insulin resistance in individuals, and is currently widely used in clinical evaluation of insulin sensitivity in diabetic patients. The calculation method is: fasting blood glucose level (FPG, mmol / L) x fasting insulin level (FINS, μU / mL) / 22.5. The HOMA-IR index of normal individuals is 1. As the level of insulin resistance increases, the HOMA-IR index will be higher than 1.

[0158] L cells in the intestinal tract can secrete glucagon-like peptide-1 (GLP-1), which can promote the production of insulin by pancreatic beta cells and inhibit the production of glucagon by pancreatic alpha cells, thereby regulating the body's blood glucose balance and improving the body's glucose tolerance.

[0159] Liver function disease

[0160] Hepatic disease is a liver function abnormality or liver function impairment. Alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) are sensitive markers of liver function disease. When the liver function of the body is abnormal (e.g., liver damage, nonalcoholic fatty liver disease (NAFLD), or nonalcoholic steatohepatitis (NASH)), the ALT and / or AST in the blood is significantly elevated. Generally, the sensitivity of ALT to acute liver damage is higher than that of AST. Persistent elevation of ALT indicates chronic liver damage. In the case of chronic hepatitis, liver cirrhosis, liver cancer, etc., AST is significantly elevated and can exceed ALT. The AST level is a marker of the chronicity, extent, and severity of liver lesions, and even indicates the prognosis of chronic liver disease.

[0161] Common liver diseases with elevated ALT and / or AST include, but are not limited to, acute viral hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E); EB virus, cytomegalovirus infection; chronic hepatitis B or chronic hepatitis C; autoimmune liver disease; alcoholic liver disease (ALD); nonalcoholic fatty liver disease (NAFLD or NASH); drug-induced / toxic liver damage; cirrhosis; liver cancer; hepatolenticular degeneration; alpha 1-antitrypsin deficiency; hemochromatosis, etc.

[0162] In addition, intrahepatic fat accumulation is an important factor in the development of nonalcoholic fatty liver disease (NAFLD or NASH), so a decrease in ALT and / or AST levels and a decrease in liver weight after drug intervention can show that the drug has a certain therapeutic improvement effect.

[0163] Nonalcoholic fatty liver disease (NAFLD) refers to the accumulation of excess fat in the liver in the form of triglycerides (TG) (steatosis). Some patients with NAFLD also have liver cell damage and inflammation (steatohepatitis) in addition to excess fat, i.e., nonalcoholic steatohepatitis (NASH). NASH is widely considered to be a liver manifestation of metabolic syndrome, such as type II diabetes, insulin resistance, central obesity, hyperlipidemia (low high-density lipoprotein cholesterol, hypertriglyceridemia), and hypertension.

[0164] Hepatorenal disease

[0165] Hepatorenal disease refers to functional acute renal failure that occurs in severe liver disease, decompensated cirrhosis, and hepatorenal syndrome due to insufficient effective circulating blood volume, decreased prostaglandins, etc.

[0166] Cardiovascular disease

[0167] TG (triglycerides) are primarily involved in energy metabolism in the human body, producing heat energy. High levels of TG in the blood can cause blood to be thick and sticky, allowing lipids to deposit on the walls of blood vessels, gradually forming small plaques, i.e., atherosclerosis. Elevated LDL-C is a major, independent risk factor for the development and progression of atherosclerosis; elevated levels of LDL-C are also an indicator of coronary heart disease. HDL-C can transport cholesterol in the vessel wall to the liver for catabolism (i.e., reverse cholesterol transport), reducing the deposition of cholesterol in the vessel wall, and plays an anti-atherosclerotic role.

[0168] Inflammation

[0169] Lipopolysaccharide (LPS), also known as endotoxin, is a phospholipid that makes up the outer cell wall of gram-negative bacteria. In addition to ensuring the integrity of the bacterial structure, lipopolysaccharide also protects these bacteria from bile salt decomposition secreted by the gallbladder. Under normal circumstances, lipopolysaccharide is blocked by the tight junctions of the intestinal wall cells from entering the bloodstream. If lipopolysaccharide enters the blood, it can induce a strong inflammatory response in the animal body. Therefore, the level of lipopolysaccharide in the blood can reflect the level of inflammation.

[0170] Obesity

[0171] Obesity is a state of excessive accumulation of fat in the body, especially triglycerides, which is a risk of abnormal or excessive accumulation of fat that poses a risk to health. It is caused by excessive food intake or changes in body metabolism, resulting in excessive weight gain and causing pathological and physiological changes or latency in the human body. A body mass index of more than 25 is considered overweight, and more than 30 is considered obese. Obesity increases the risk of many physical and mental illnesses. It is mainly associated with metabolic syndrome, including type 2 diabetes, high blood pressure, high cholesterol, and high triglyceride levels. In general, the impact of obesity on health falls into two categories: diseases attributable to increased body fat (such as osteoarthritis, obstructive sleep apnea, etc.) and diseases attributable to increased number of fat cells (diabetes, dyslipidemia, cancer, cardiovascular disease, nonalcoholic fatty liver or nonalcoholic steatohepatitis, etc.). The "obesity-related disease" can be selected from the following diseases: overeating, binge eating, hyperphagia, hypertension, diabetes, elevated plasma insulin concentration, insulin resistance, hyperlipidemia, metabolic syndrome, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, arteriosclerosis, hypercholesterolemia, hyperuricemia, lower back pain, cardiac hypertrophy and left ventricular hypertrophy, adipose metabolism disorder, nonalcoholic steatohepatitis, cardiovascular disease, and polycystic ovary syndrome, as well as those subjects with these obesity-related diseases, including those who wish to lose weight.

[0172] The obesity-related diseases in the present disclosure include at least one of the following diseases: obesity, metabolic syndrome, cardiovascular disease, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, and fatty liver disease.

[0173] Interferon (IFN) receptor proteins are a class of cytokines secreted by host cells, which can regulate immune responses. Viruses, bacterial endotoxins, artificially synthesized double-stranded RNA, etc. can stimulate the production of interferons. Macrophages, lymphocytes, somatic cells, etc. in the human body can produce interferons. Among them, IFNβ belongs to type I interferon, which can promote the activity of NK cells, macrophages and T lymphocytes, thereby playing the roles of anti-virus, anti-tumor, immune regulation, etc.

[0174] The Akkermansia muciniphila or product thereof provided by the present application can significantly promote the transcriptional activity of IFNβ. Type I interferon IFNβ has been confirmed to be able to regulate immunity and reconstruct the synergistic effect of innate immunity and acquired immunity in the tumor microenvironment, and is used for treating refractory drug-resistant cancer (see "Targeting the tumor Microenvironment with interferon-β Bridges innate and adaptive immune responses, Yang X1, cancer cell, 2014, doi:10.1016 / j.ccr.2013.12.004."). Therefore, these results show that the strain MNH19250 or the Akkermansia muciniphila or product thereof provided by the present application can regulate immunity and achieve anti-tumor through immune regulation, and has a potential functional effect on anti-viral tumors.

[0175] Short-chain fatty acids are one of the important metabolites of intestinal microorganisms, which have an impact on a series of activities of the host as signal molecules, mainly in the form of acetate, propionate and butyrate. Short-chain fatty acids can reduce the pH value of the intestinal tract and inhibit the growth of pathogens. Short-chain fatty acids can activate target pathways such as GPR41, GPR43, GPR109A and GPCR81, improve the integrity and function of colon epithelial cells, enhance the intestinal barrier function, promote the secretion of hormones such as GLP-1 and PYY in intestinal endocrine cells, increase insulin sensitivity, increase energy consumption, promote fat decomposition, inhibit the production of pro-inflammatory cytokines, and maintain intestinal immune homeostasis. Short-chain fatty acids have certain beneficial effects on metabolic diseases such as obesity, diabetes, non-alcoholic fatty liver, non-alcoholic steatohepatitis, ulcerative colitis, radiation proctitis and Crohn's disease.

[0176] Propionic acid, butyric acid can be used as HDAC inhibitors, change the expression of various functional genes by inhibiting HDAC, regulate cell proliferation, apoptosis and differentiation, thereby preventing the occurrence and development of inflammation while improving immunity.

[0177] Amuc-1100, Amuc_1631 (P9) protein is an outer membrane protein derived from Akkermansia muciniphila. Amuc-1100 protein can interact with host cell Toll-like receptor 2 (TLR2), affect intestinal health and immune regulation (Wang J, Xu W, Wang R, et al., The outer membrane protein Amuc_1100 of Akkermansia muciniphila promotes intestinal 5-HT biosynthesis and extracellular availability through TLR2 signalling. [J]. Food & function, 2021, 12 (8): 3597-3610. DOI: 10.1039 / d1fo00115a.). Amuc-1100 has the effect of promoting GLP-1 secretion, which helps to improve glycemic control and metabolic disorders; "YOON H S, CHO C H, YUN M S, et al., Akkermansia muciniphila secretes a glucagon-like peptide-1-inducing protein that improves glucose homeostasis and ameliorates metabolic disease in mice [J]. Nature Microbiology, 2021, 6: 563-573"; Amuc-1100 remains stable in pasteurization and can inhibit the expression of CB1 receptor in the intestine and enhance the expression of tight junction protein; "Ding, G., Yang, X., Li, Y. et al., Gut microbiota regulates gut homeostasis, mucosal immunity and influences immune-related diseases. Mol Cell Biochem (2024). https: / / doi.org / 10.1007 / s11010-024-05077-y".

[0178] Amuc_1631 (P9) protein can promote GLP-1 secretion of intestinal L cells, and has the potential to treat diabetes, obesity and other metabolic diseases (Wenxuan D, Yuchen Z, Xinyuan Z, et al., Heterologous expression of P9 from Akkermansia muciniphila increases the GLP-1 secretion of intestinal L cells [J]. World Journal of Microbiology & Biotechnology, 2024 (7): 40. DOI: 10.1007 / s11274-024-04012-z.). P9 protein also shows strong anti-inflammatory effect, and can regulate intestinal barrier function, thereby playing a role in inflammatory bowel disease. P9 protein enhances the function of intestinal barrier by increasing the number of goblet cells, mucus production and differentiation of stem cells into secretory cells. "Patrice D. Cani, Clara Depommier, Muriel Derrien, et al., Akkermansia muciniphila: paradigm for next-generation beneficial microorganisms. Nature Reviews Gastroenterology & Hepatology volume 19, pages 625-637 (2022)".

[0179] Therefore, the Akkermansia muciniphila involved in the present application can affect or regulate immune signaling and / or affect intestinal barrier function and / or affect glucose homeostasis and / or cholesterol homeostasis and / or triglyceride homeostasis.

[0180] The scheme of the present application will be explained below in combination with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the application. If the specific technology or condition is not specified in the examples, the technology or condition described in the literature of the art or according to the product or instrument instruction is used. If the manufacturer of all reagents or instruments is specified, they can be purchased in the market.

[0181] Examples

[0182] The liquid MM01 medium involved in the embodiments has the following composition: 5 g / L of proteose peptone, 5 g / L of trypticase, 10 g / L of yeast extract, 5 g / L of beef extract, 5 g / L of glucose, 2 g / L of K2HPO4, 2 g / L of sodium acetate, 1 mL / L of Tween 80, 5 mg / L of hemin, 0.5 g / L of L-cysteine hydrochloride, 1 μL / L of vitamin K1, and 8 mL / L of an inorganic salt solution (containing 0.25 g of calcium chloride, 1 g of K2HPO4, 1 g of KH2PO4, 0.5 g of magnesium sulfate, 10 g of sodium bicarbonate, and 2 g of sodium chloride per 1 L).

[0183] The solid MM01 medium involved in the embodiments has the following composition: 5 g / L of proteose peptone, 5 g / L of trypticase, 10 g / L of yeast extract, 5 g / L of beef extract, 5 g / L of glucose, 2 g / L of K2HPO4, 2 g / L of sodium acetate, 1 mL / L of Tween 80, 5 mg / L of hemin, 0.5 g / L of L-cysteine hydrochloride, 1 μL / L of vitamin K1, 8 mL / L of an inorganic salt solution (containing 0.25 g of calcium chloride, 1 g of K2HPO4, 1 g of KH2PO4, 0.5 g of magnesium sulfate, 10 g of sodium bicarbonate, and 2 g of sodium chloride per 1 L), and 15 g / L of agar.

[0184] Anaerobic blood agar plates, purchased from Hangzhou Microorganism, have the following formula: 10 g / L of trypticase, 3 g / L of heart infusion tryptose, 1 g / L of corn starch, 5 g / L of pepsin, 5 g / L of yeast extract, 5 g / L of sodium chloride, 15 g / L of agar, and 50-100 mL / L of sterile defibrinated sheep blood, with a pH of 7.3±0.2.

[0185] The above-mentioned culture media can be prepared by using conventional preparation methods and sterilization methods.

[0186] Example 1. Isolation and identification of strains

[0187] 1.1 Isolation and purification of strain MNH19250

[0188] The enteric bacterial strain numbered MNH19250 was isolated from a sample taken from a healthy male volunteer in Guangzhou, Guangdong Province, China. The isolation method used a conventional bacterial strain isolation method, gradient dilution was used, and then single colonies were picked by isolation culture and purified, and cultured anaerobically at 37°C. The purified bacterial strain was prepared into a 20% glycerol / water-bacterial liquid, and stored at -80°C.

[0189] Specifically, the bacterial strain isolation method is as follows:

[0190] 5 g of the sample taken by the donor was placed into a sample collection and storage tube, and after homogenization, the treated sample was placed in an ice box and delivered to the laboratory within 24 hours for bacterial strain isolation.

[0191] Dispense physiological saline in a biological safety cabinet, 9 mL / tube; prepare anaerobic blood agar plates for strain isolation, and transfer them into the anaerobic workstation 24 h in advance, label sample information, medium type, isolation date, etc.

[0192] Take a new sample and place it in the anaerobic workstation, mix it well using a vortex shaker for 1 min, take 1 mL of the sample and mix it with 9 mL of physiological saline, mix well to 10 -1 Dilute the solution, then dilute it to 10 -6 Dilute the solution, then dilute it to 10

[0193] Take 10 -6 Dilute the solution to 10 μL / dish, spread evenly, and after the surface of the plate dries, invert the plate and incubate at 37°C for 3-5 days.

[0194] Observe the growth of the strain on the isolation medium (anaerobic blood agar plate) and pick single colonies with a sterile toothpick for strain purification. Purify the strain and incubate it at 37°C under anaerobic conditions. Prepare 20% glycerol / water-bacterial liquid from the pure culture strain and store it at -80°C.

[0195] 1.2 Morphological characteristics of strain MNH19250

[0196] Culture and morphological characteristics

[0197] Inoculate strain MNH19250 into MM01 medium and incubate it at 37°C under anaerobic conditions for 72 h. Visible colonies form on the MM01 plate medium, which are round, regular and smooth in edge, about 0.5 mm in diameter, light yellow and translucent. The strain is gram-negative. Microscopic observation shows that it is non-flagellated, non-motile and rod-shaped, about 0.5-1 μm x 1.5-3 μm in size. See Figure 1 for the colony morphology of strain MNH19250 after 72 h of incubation on MM01 plate medium. See Figure 2 for the gram staining of strain MNH19250 and Figure 3 for the electron microscope image.

[0198] 1.3 Physiological characteristics of strain MNH19250

[0199] Strain MNH19250 does not grow under aerobic conditions, but only under anaerobic conditions. It can grow at a pH of 6.0-9.0, with an optimal growth pH of about 8.0 (see Figure 4 for the results of the strain's tolerance to different pH values). Growth is significantly inhibited on medium with a NaCl content of more than 2% (w / v) (see Figure 5 for the results of the strain's tolerance to different concentrations of NaCl). Strain MNH19250 can survive and grow at a bile salt concentration of 0%-0.25% (w / v), and growth is significantly inhibited at a bile salt concentration of 0.3% or more (see Figure 6 for the results of the strain's tolerance to different concentrations of bile salt).

[0200] 1.4 Biochemical identification of strain MNH19250 by API 20A

[0201] Strain MNH19250 was subjected to biochemical identification by API 20A (purchased from BioMerieux, CN2030025), and the specific experimental operation can be found in the conventional API reagent operation guide. The culture conditions of strain MNH19250: 37°C, anaerobic. The experimental results are shown in Table 1.

[0202] Table 1. API 20A test results of strain MNH19250

[0203] MNH19250 can ferment glucose, lactose, and mannose to produce acid. Therefore, glucose, lactose, mannose, and their derivatives can be used as carbon sources during fermentation or culture of strain MNH19250.

[0204] MNH19250 can hydrolyze escin (ESC), i.e., can synthesize β-glucosidase. β-glucosidase belongs to the cellulase class and can hydrolyze cellobiose and short-chain cellotriose to produce glucose.

[0205] MNH19250 can hydrolyze gelatin (GEL), i.e., can synthesize protease. The function of protease is to convert non-absorbable proteins into polypeptides or amino acids that can penetrate the bacterial cell. This enzyme is mainly an extracellular enzyme that can first hydrolyze gelatin into polypeptides and further hydrolyze them into amino acids, losing the gelatin property and liquefying, allowing the black substance in the reaction well to diffuse and show a positive reaction.

[0206] 1.5 Antibiotic minimum inhibitory concentration test of strain MNH19250

[0207] The antibiotic minimum inhibitory concentration of strain MNH19250 was determined using E-test (purchased from Liofilchem), and the test results are shown in Table 2.

[0208] Table 2. Antibiotic minimum inhibitory concentration test results of strain MNH19250

[0209] The results show that MNH19250 is sensitive to ampicillin, chloramphenicol, clindamycin, amoxicillin, rifampicin, imipenem, penicillin, and cefquinome. It can be seen that MNH19250 is sensitive to most types of antibiotics, and the risk of developing antibiotic resistance in the subject due to long-term use of MNH19250 is low.

[0210] 1.6 Determination of the autoaggregation ability of strain MNH19250

[0211] Strain MNH19250 was inoculated in MM01 liquid medium and cultured anaerobically at 37°C for 48 hours. 20 mL of the fermentation broth was collected and centrifuged (4500 rpm, 4°C, 10 min) to collect the bacterial cells, which were then washed twice with sterilized PBS (pH 7.2), resuspended in PBS buffer, and adjusted to an OD600 value of 0.5, denoted as A0.

[0212] The bacterial solution was incubated at 37°C (directly in a colorimetric cup) and the OD600 value was measured every 30 min, denoted as At, for 6 hours, with three replicates.

[0213] The auto-aggregation ability of the strain was calculated according to the following formula: Strain auto-aggregation ability (%) = [1-(At / A0)] x 100%.

[0214] The 6h auto-aggregation ability of MNH19250 was 54.21% (see Figure 7), which was 20% stronger than that of other strains in the literature (Xin Ma 1, Meng Tian 2, Xueping Yu, et al., Foods. 2024, Jan 30; 13(3): 442. Characterization and Preliminary Safety Evaluation of Akkermansia muciniphila PROBIO (https: / / doi.org / 10.3390 / foods13030442); Figure 1d, A. muciniphila PROBIO auto-aggregation ability was about 30% (8h)). Strains with strong auto-aggregation ability have strong epithelial cell adhesion ability and can better colonize the human intestinal tract to exert probiotic effects.

[0215] 1.7 Determination of the surface hydrophobicity of strain MNH19250

[0216] Strain MNH19250 was inoculated in MM01 liquid medium and cultured anaerobically at 37°C for 48 hours. 20 mL of the fermentation broth was collected and centrifuged (4500 rpm, 4°C, 10 min) to collect the bacterial cells, which were then washed twice with sterilized PBS (pH 7.2), resuspended in PBS buffer, and adjusted to an OD600 value of 0.5, denoted as A0.

[0217] 4 mL of xylene was added to 4 mL of the resuspended solution, and the two-phase system was vortexed for 5 min. After incubation at room temperature for 1 h, the xylene phase was carefully removed, and the absorbance A of the water phase was measured at 600 nm, with three replicates. The surface hydrophobicity of the test strain was calculated according to the following formula:

[0218] H% = [(A0-A) / A0] x 100, wherein A0 and A represent the absorbance values before and after organic solvent extraction, respectively.

[0219] The results of the determination of the surface hydrophobicity of strain MNH19250 were 57.06%, which was stronger than the surface hydrophobicity of other strains in the literature by more than 25% (Xin Ma 1, Meng Tian 2, Xueping Yu, et al., Foods. 2024, Jan 30; 13(3): 442. Characterization and Preliminary Safety Evaluation of Akkermansia muciniphila PROBIO (https: / / doi.org / 10.3390 / foods13030442); Figure 1c, A. muciniphila PROBIO surface hydrophobicity was 31% (1h)), and the strain with strong surface hydrophobicity had strong epithelial cell adhesion ability, could better colonize in the human intestinal tract to play a probiotic effect, and at the same time could inhibit the adhesion of other pathogenic bacteria, playing a role in intestinal probiotics.

[0220] 1.8 Amplification of 16S rRNA gene of strain MNH19250

[0221] Fresh culture of strain MNH19250 was taken to extract the strain genomic DNA. The extracted strain genomic DNA was used as a template for 16S rRNA gene amplification.

[0222] The primer pair used for 16S rRNA gene PCR was:

[0223] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID No. 2)

[0224] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID No. 3).

[0225] The PCR reaction program was as follows:

[0226] Pre-denaturation: 94°C, 4 min; denaturation: 94°C, 50 sec; annealing: 52°C, 40 sec; extension: 72°C, 70 sec; final extension: 72°C, 10 min (cycling 36 times).

[0227] 1.9 16S rRNA gene sequencing

[0228] The PCR product was purified and subjected to 16S rRNA gene sequencing by Shenguo Company, and the 16S rRNA gene sequence (1355 bp) was obtained, as shown in SEQ ID No. 1.

[0229] 1.10 Identification of strain MNH19250

[0230] The measured 16S rRNA gene sequence as shown in SEQ ID No. 1 was used to analyze the 16S rRNA gene of the strain using the NCBI Basic Local Alignment Search Tool, and the strain classification information was confirmed.

[0231] The measured sequence was analyzed by BLAST with the data in GenBank, and the alignment results showed that the strain with the highest similarity to MNH19250 was Akkermansia muciniphila, with a similarity of 100%, so it was judged that strain MNH19250 was a strain under the Akkermansia muciniphila species.

[0232] The 16S rRNA gene sequence of strain MNH19250 was compared with the 16S rRNA gene sequences of Akkermansia sp. related strains retrieved from databases such as GenBank, and a phylogenetic tree was constructed.

[0233] The 16S rRNA gene sequence of strain MNH19250 was subjected to multiple sequence alignment with the sequences of strains with high similarity of 16S rRNA gene sequences in the NCBI database, and then a phylogenetic tree was constructed using software MEGA 5 (the phylogenetic tree was constructed using the maximum likelihood method) (see Figure 8), and in Figure 8, only Bootstrap values greater than 50% are shown in the phylogenetic tree nodes.

[0234] From the phylogenetic tree, it can be seen that strain MNH19250 is clustered with Akkermansia sp. and Akkermansia muciniphila Muc AY271254, so it is judged that strain MNH19250 is a new strain under the Akkermansia muciniphila species.

[0235] 1.11 Genome analysis of strain MNH19250

[0236] Genome analysis of strain MNH19250

[0237] The genome of the MNH19250 strain was sequenced by ultrasonic fragmentation, and then an Illumina sequencing library was constructed using a standard DNA library kit (NEB UltraTM). The constructed sequencing library was sequenced by NovaSeq (Illumina) with a double-end 150bp sequencing. The sequencing obtained 3.01Gbp data, of which the Q20 accounted for 97.42%.

[0238] The raw sequencing data of the genome was filtered using fastp (version: 0.20.0). The filtered raw data was used for genome assembly using SPAdes (version: v3.14.0). The genome assembly obtained a total length of 2.82Mbp, a N50 length of 378.4kbp, and a GC content of 55.19%.

[0239] The genome genes were analyzed for genome gene prediction using the prokka (version: 1.14.5) of the prokaryotic analysis software. A total of 2376 CDS sequences were predicted, with an average CDS sequence length of 1039bp.

[0240] The potential antibiotic resistance genes in the genome were analyzed using RGI (version: 4.2.2), and the antibiotic resistance gene database was CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). For detailed comparison information, refer to Table 3.

[0241] Table 3. Drug resistance gene information list

[0242] The analysis of potential virulence factors and related genes in the genome used NCBI blastp (version: 2.7.1+) to align the virulence factor database VFDB (virulence factor database, http: / / www.mgc.ac.cn / cgi bin / VFs / v5 / main.cgi, updated on September 19, 2019) for analysis. The detailed comparison results are shown in Table 4.

[0243] Table 4. MNH-19250 potential virulence gene list

[0244] The analysis of potential secondary metabolic gene clusters in the genome used antiSMASH6 (version: 6.0.1). The detailed comparison results are shown in Table 5.

[0245] Table 5. MNH-19250 potential secondary metabolic gene cluster list

[0246] The analysis of potential primary metabolism gene clusters in the genome was performed using gutSMASH5 (version: 1.0.0). The detailed alignment results are shown in Table 6.

[0247] Table 6. MNH-19250 potential primary metabolism gene cluster list

[0248] Analysis of Amuc_1100 protein and P9 protein encoding genes of strain MNH19250. BLAST was used to align the Amuc_1100 protein sequence (WP_197738471) and the Amuc_1631 (P9) protein sequence (ACD05451).

[0249] Table 7. MNH19250 Amuc_1100 protein encoding gene analysis

[0250] Sequence:

[0251] >MNH19250_01469 hypothetical protein

[0252] Table 8. MNH19250 P9 protein encoding gene analysis

[0253] Sequence:

[0254] >MNH19250_00667 Tail-specific protease

[0255] Example 2. Analysis of fatty acid composition of strain MNH19250

[0256] 2.1 Analysis of fatty acid composition of strain MNH19250

[0257] Strain MNH19250 was inoculated on MM01 plates and incubated anaerobically at 37°C for 72 hours. After that, the bacterial cells were collected and subjected to fatty acid extraction and methylation treatment. The fatty acid composition of strain MNH19250 was analyzed using the full-automatic bacterial identification system of MIDI (Microbial ID, Inc., Newark, Del).

[0258] The main fatty acid (>10%) of the experimental strain MNH19250 was C15:0 anteiso 48.73%.

[0259] Bacterial cell preparation

[0260] Strain MNH19250 was inoculated in MM01 liquid medium and cultured anaerobically at 37°C for 48 hours. The bacterial cells and supernatant were collected by centrifugation and stored at -80°C.

[0261] Pre-treatment of supernatant sample

[0262] (1) After thawing the sample, vortex for 3 min to mix evenly;

[0263] (2) Transfer 50 μL of the sample to a 1.5 mL centrifuge tube, add 100 μL of phosphoric acid solution (0.5%, v / v), and vortex at 2500 r / min for 3 min;

[0264] (3) Add 750 μL of MTBE extractant containing an internal standard, vortex at 2500 r / min for 3 min, ultrasonic at 4 m for 5 min, and centrifuge at 4 m and 12000 r / min for 10 min;

[0265] (4) Take 200 μL of the supernatant to the inner tube of the sample injection bottle, and store it in the -20°C refrigerator for GC-MS / MS analysis.

[0266] Pre-treatment of bacterial cell sample

[0267] (1) After thawing the sample, add 100 μL of frozen ultrapure water extract solution to resuspend evenly;

[0268] (2) Transfer 50 μL of the bacterial cell suspension sample to a 1.5 mL centrifuge tube, add 100 μL of phosphoric acid solution (0.5%, v / v), and vortex for 3 min to mix evenly;

[0269] (3) Soak in liquid nitrogen for 2 min, take out and completely thaw on ice, vortex at 2500 r / min for 3 min, and repeat 3 times;

[0270] (4) Add 150 μL of MTBE extractant containing an internal standard, vortex at 2500 r / min for 3 min, ultrasonic at 4 m for 5 min, and centrifuge at 4 m and 12000 r / min for 10 min;

[0271] (5) Take 90 μL of the supernatant to the inner tube of the sample injection bottle for GC-MS / MS analysis.

[0272] (6) The remaining 50 μL of bacterial cell suspension was repeatedly frozen and thawed in liquid nitrogen for 3 times, and the supernatant was collected after centrifugation at 12000 r / min for 10 min, and the protein concentration was determined by BCA method.

[0273] Standard preparation: Prepare 0.005 μg / mL, 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 8 μg / mL, 10 μg / mL, 20 μg / mL of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid as standard solution respectively. Chromatography mass spectrometry detection is carried out using the conditions shown in Table 9.

[0274] Obtain the chromatographic peak intensity data of the corresponding quantitative signal of each concentration standard. Draw the standard curve of different substances with the external standard and internal standard concentration ratio (Concentration Ratio) as the abscissa and the external standard and internal standard peak area ratio (Area Ratio) as the ordinate.

[0275] Table 9. Main conditions of chromatography mass spectrometry

[0276] The integral peak area ratio of all detected samples is substituted into the standard curve linear equation for calculation. After substituting into the sample calculation formula, the content data of the substance in the actual sample is finally obtained. The results show that the supernatant and bacterial cells of the bacterial solution contain short-chain fatty acids acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid, among which the contents of acetic acid and propionic acid are shown in Table 10.

[0277] Table 10. Content detection of short-chain fatty acids and medium-chain fatty acids in MNH19250 bacterial solution supernatant and bacterial cells

[0278] As can be seen from the detection results, the strain MNH19250 can synthesize short-chain fatty acids during growth, including a large amount of acetic acid and propionic acid.

[0279] Example 3. Effect of strain MNH19250 on IFNβ expression

[0280] To verify whether MNH19250 can promote the expression of IFNβ, the present study used THP-1 cells carrying the IFNβ gene promoter reporter gene (THP-1-IFNβ-promoter reporter cells, a cell line constructed by Muen Company) to evaluate the effect of MNH19250 on the transcriptional activity of IFNβ.

[0281] The construction of THP-1-IFNβ-promoter reporter cells includes the following steps: inserting the reporter gene into a vector, infecting cells with the vector, and screening to obtain a cell line expressing the reporter gene (see the following references for details: Huashan Du, Tianmin Xu, Manhua Cui, “cGAS-STING signaling in cancer immunity and immunotherapy” Biomedicine & Pharmacotherapy 133 (2021) 110972; Jiang et al., “cGAS-STING, an important pathway in cancer immunotherapy” Journal of Hematology & Oncology (2020) 13:81; Khiem C. Lam et al., “Microbiota triggers STING-type I IFN-dependent monocyte reprogramming of the tumor microenvironment” Cell 184, 5338-5356).

[0282] Preparation of culture supernatant of strain MNH19250: inoculate strain MNH19250 in MM01 liquid medium, cultivate anaerobically at 37°C for 48 hours, centrifuge to remove bacterial bodies, filter the culture supernatant with a 0.22 μm filter, aliquot, and store the collection at -80°C.

[0283] Control group (Control): DMEM complete medium (Gibco, containing 10% FBS) containing 10% volume of MM01 liquid medium;

[0284] MSA-2 group (positive control group): DMEM complete medium containing 10 μM MSA-2 (purchased from Taotao Biological);

[0285] Strain MNH19250 group: DMEM complete medium containing 10% volume of culture supernatant of strain MNH19250.

[0286] THP-1-IFNβ-promoter reporter cells were inoculated in a 96-well plate at 1×10 5One cell. The cells were treated according to the set group. After 24 hours of continuous culture, the cells were centrifuged at 300g for 5 minutes, the culture supernatant was removed, 50 μL of 1x Luminescence (Promega) was added to the control group (Control) for normalization, and the effect of strain MNH19250 on the transcriptional activity of IFNβ was evaluated.

[0287] The experimental results are shown in Figure 9. Strain MNH19250 can significantly promote the transcriptional activity of IFNβ. Type I interferon IFNβ has been proven to be able to regulate immunity and achieve anti-tumor effect through immune regulation, and has potential functional effects on anti-viral tumors. Therefore, these results show that MNH19250 and its metabolites can regulate the body's immunity and achieve anti-tumor effect through immune regulation, and have potential preventive and inhibitory effects on tumors.

[0288] Example 4: Strain MNH19250 improves liver and kidney function and related diseases in a mouse model of high-fat diet-induced liver and kidney injury

[0289] The strain MNH19250 was used to study the improvement of liver and kidney function and related diseases in a mouse model of high-fat diet-induced liver and kidney injury. The experimental protocol has been reviewed by the Muen Biological Animal Care and Use Committee.

[0290] 4.1 Experimental method

[0291] 1) Experimental animals: The experimental mice were C57BL / 6J mice, 5-6 weeks old, purchased from Jiangsu Jucu Yakang Biotechnology Co., Ltd.

[0292] 2) Preparation of test substance of strain MNH19250: After thawing the glycerol stock tube of strain MNH19250 at 37°C, inoculate it on MM01 plates in an anaerobic workstation for activation. Inoculate the activated strain into MM01 liquid medium and anaerobically culture to obtain sufficient amount of culture. Centrifuge the cultured bacterial liquid, resuspend the bacterial body with PBS containing 0.05% L-Cys HCl, and obtain the test substance with purity and viable bacterial count (2x10 9 CFU / mL) meeting the requirements of animal experiments.

[0293] 3) Negative control: PBS containing 0.05% L-Cys HCl was used as the negative control.

[0294] 4) Experimental process: 5-6 weeks old C57BL / 6J male mice, after quarantine period, high-fat diet for 10 weeks, select 12 mice with body weight range of 35.50g-44.49g, according to the body weight, random stratified grouping, 6 mice / group, divided into 2 groups (experimental group and control group). After grouping (D1), start dosing, the experimental group is given the above test substance, the control group is given the negative control, 2 times a day, a total of 28 days, during the experiment, the mice are free to drink water and eat, using 12h / 12h day-night cycle. During the experiment, general clinical observation was performed once after each dosing. The end of the experiment was the day after the end of dosing (D29), the end of the experiment was dissection, data analysis of each dissection data and serum test data. All data are expressed in the form of Mean ± SD, and are plotted and statistically analyzed using GraphPad Prism software. For pairwise comparison, the t-test (Student's t-test) analysis method was used. Significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0295] Referring to FIG. 10, it is shown that the strain MNH19250 can significantly reduce liver function indicators. Specifically, FIG. 10 shows that the strain MNH19250 can significantly reduce alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, thus the strain MNH19250 can improve liver dysfunction caused by high-fat diet, and has activity of treating or preventing liver function damage.

[0296] Referring to FIG. 11, according to the NAS scoring system proposed by the American Liver Disease Association (2005), it is shown that the strain MNH19250 has activity of treating or preventing liver function damage, fatty liver NAFLD / NASH. Specifically, FIG. 11 shows that the strain MNH19250 can significantly improve non-alcoholic fatty liver and non-alcoholic fatty hepatitis (NAFLD / NASH): the strain MNH19250 can significantly reduce liver weight and significantly reduce the ratio of liver weight to body weight, significantly reduce liver steatosis, liver lobular inflammation, significantly reduce liver cell ballooning, and significantly reduce non-alcoholic fatty liver disease activity score (NAS). Therefore, the strain MNH19250 can significantly improve non-alcoholic fatty liver and non-alcoholic fatty hepatitis, significantly improve liver cell damage, and has activity of treating or preventing liver cell damage, NAFLD / NASH.

[0297] Referring to FIG. 12, it is shown that the strain MNH19250 can significantly reduce BUN (urea nitrogen) in serum and reduce creatinine (CRE) in serum. Therefore, the strain MNH19250 can significantly improve kidney dysfunction, and has activity of treating or preventing kidney damage and kidney dysfunction.

[0298] Example 5: Use of strain MNH19250 for preventing or treating diabetes

[0299] 5.1 Experimental method

[0300] 1) Experimental animals: The experimental mice were C57BL / 6J mice, 5-6 weeks old, purchased from Jiangsu Jicui Yekang Biotechnology Co., Ltd.

[0301] 2) Preparation of the test substance of strain MNH19250: After the glycerol frozen tube of the strain MNH19250 was thawed at 37°C, it was inoculated on MM01 plates in an anaerobic workstation for activation. The activated strain was inoculated in MM01 liquid medium and anaerobically cultured to obtain a sufficient amount of culture. The cultured bacterial liquid was centrifuged and concentrated, and then the bacterial bodies were resuspended with PBS containing 0.05% L-Cys HCl to obtain a test substance with a purity and viable bacterial count (2x10 9 CFU / mL) meeting the requirements of animal experiments.

[0302] 3) Negative control: PBS containing 0.05% L-Cys HCl was used as the negative control.

[0303] 4) Experimental process: 5-6 week old C57BL / 6J male mice were fed with high-fat diet for 10 weeks after the quarantine period. Twelve mice with body weight ranging from 35.50g to 44.49g were selected and randomly stratified according to body weight, with 6 mice per group, divided into 2 groups (experimental group and control group). After grouping (D1), the experimental group was given the test substance of strain MNH19250, and the control group was given the negative control. The mice were given the drug 2 times a day, for a total of 28 days. The experimental endpoint was the day after the end of drug administration (D29). The mice were allowed to drink and eat freely during the experiment, and a 12h / 12h day-night cycle was used. During the experiment, general clinical observation was performed once after each administration. OGTT was measured once in the last week of administration, and fasting blood glucose was measured at the end of the experiment (D29). All data were expressed as Mean ± SD, and were plotted and statistically analyzed using GraphPad Prism software. For pairwise comparisons, the t-test (Student's t-test) analysis method was used. Significant differences were indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0304] 5.2 Oral glucose tolerance test of high-fat diet-induced type 2 diabetic mice treated with strain MNH19250:

[0305] Oral glucose tolerance test (OGTT): OGTT was measured in the last week of administration as described above, with 12h fasting (overnight 20:30:00 fasting to the next day 08:30:00). The mice were weighed and fasted, and then administered glucose by gavage at a dose of 2g / kg (glucose g / mouse fasted body weight kg). Fasting blood glucose, 15min, 30min, 60min, 90min, 120min post-glucose values were measured. Each mouse was strictly timed and blood glucose values were measured at the 6 time points exactly.

[0306] 5.3 Experimental results of the effect of strain MNH19250 on oral glucose tolerance of high-fat diet-induced type 2 diabetic mice:

[0307] As shown in Figure 13, it can be seen that strain MNH19250 can significantly reduce the oral glucose tolerance of high-fat diet-induced type 2 diabetic mice. Specifically, the results shown in Figure 13 show that strain MNH19250 can significantly inhibit the increase in blood glucose after glucose administration in type 2 diabetic mice, significantly improve oral glucose tolerance, and significantly reduce the area under the curve of oral glucose tolerance in type 2 diabetic mice, indicating that strain MNH19250 can significantly control blood glucose and significantly improve glucose homeostasis in type 2 diabetic mice.

[0308] 5.4 Experimental results of the effect of strain MNH19250 on fasting blood glucose of high-fat diet-induced type 2 diabetic mice:

[0309] As shown in Figure 14, it can be seen that strain MNH19250 can significantly reduce the fasting blood glucose of high-fat diet-induced type 2 diabetic mice, and therefore strain MNH19250 has activity in preventing or treating diabetes.

[0310] Example 6: Use of strain MNH19250 for the treatment and prevention of obesity and related diseases

[0311] 6.1 Experimental methods:

[0312] 1) Experimental animals: The experimental mice were C57BL / 6J mice, 5-6 weeks old, purchased from Jiangsu Jizhu Pharmaceutical Biotechnology Co., Ltd.

[0313] 2) Preparation of test substance of strain MNH19250: After thawing the glycerol stock tube of strain MNH19250 at 37°C, inoculate it on MM01 plates in an anaerobic workstation for activation, inoculate the activated strain into MM01 liquid medium, and anaerobically culture to obtain sufficient amount of culture. Centrifuge the cultured bacterial liquid to concentrate it, then resuspend the bacterial bodies in PBS containing 0.05% L-Cys HCl to obtain a test substance with a purity and viable bacterial count (2x10 9 CFU / mL) that meets the requirements of animal experiments.

[0314] 3) Negative control: PBS containing 0.05% L-Cys HCl (cysteine hydrochloride) was used as the negative control

[0315] 4) Experimental procedure: 5-6 week old C57BL / 6J male mice were fed with high-fat diet for 10 weeks after quarantine. Twelve mice with body weight ranging from 35.50 g to 44.49 g were selected and randomly stratified according to body weight, with 6 mice per group, divided into 2 groups (experimental group and control group). The day of administration after grouping was D1. The experimental group was given strain MNH19250, and the control group was given the negative control. Administration was performed twice a day, for a total of 28 days. During the experiment, mice were allowed to drink water and eat freely, and a 12h / 12h circadian cycle was used. During the experiment, general clinical observation was performed once after administration. Animal body weight was measured twice a week during the administration period; animal body weight was measured before dissection at the end of the experiment.

[0316] Mouse food intake determination: The amount of feed given was weighed on Day n, and the amount of remaining feed was weighed on Day n+1 to obtain the food intake 24 hours before administration. The amount of feed given and the amount of remaining feed after 24 hours of mouse feed intake were measured once a week during the administration period. The end of the experiment was the day after administration ended (D29), and the end of the experiment was dissection for data collection, analysis of body weight and body weight change percentage, and results of serum detection data. All data were expressed as Mean ± SD, and were plotted and statistically analyzed using GraphPad Prism software. For pairwise comparisons, the t-test (Student's t test) analysis method was used. Significant differences were indicated by *, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0317] 6.2 Effect of strain MNH19250 on body weight of obese model mice:

[0318] As shown in Figure 15, strain MNH19250 significantly reduced the body weight and body weight gain of high-fat diet-induced obese mice, and after four weeks of gavage intervention, the body weight was reduced by about 10% compared with before intervention, indicating that strain MNH19250 had a significant weight loss effect.

[0319] 6.3 Effect of strain MNH19250 on blood lipids of high-fat diet-induced obese model mice:

[0320] Referring to FIG. 16, the results show that the strain MNH19250 can significantly reduce the concentration of total cholesterol (TCHO), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in the serum of high-fat diet-induced obese mice, and significantly reduce the ratio of low-density lipoprotein cholesterol to high-density lipoprotein cholesterol. The above data show that the strain MNH19250 has the effect of treating / preventing hypercholesterolemia, hyperlipidemia and cardiovascular and cerebrovascular diseases.

[0321] 6.4 Effect of strain MNH19250 on body fat of high-fat diet-induced obese model mice:

[0322] Referring to FIG. 17, the results show that the strain MNH19250 can significantly reduce the weight of epididymal fat, perirenal fat, visceral fat and inguinal fat of high-fat diet-induced obese mice, and significantly reduce the ratio of visceral fat to body weight, indicating that the strain MNH19250 has the effect of treating / preventing high visceral fat ratio and treating / preventing obesity.

[0323] Example 7: Effect of strain MNH19250 on colonic mucosal repair of high-fat diet-induced intestinal barrier dysfunction model mice

[0324] 7.1 Experimental method:

[0325] 1) Experimental animals: The experimental mice were C57BL / 6J mice, 5-6 weeks old, purchased from Jiangsu Jicui Yekang Biotechnology Co., Ltd.

[0326] 2) Preparation of test substance of strain MNH19250: After thawing the glycerol stock tube of the strain MNH19250 at 37°C, inoculate it on MM01 plates in an anaerobic workstation for activation. Inoculate the activated strain into MM01 liquid medium and anaerobically culture to obtain sufficient amount of culture. Centrifuge the cultured bacterial liquid, concentrate it, and then resuspend the bacterial body with PBS containing 0.05% L-Cys HCl to obtain the test substance with a purity and viable bacterial count (2x10 9 CFU / mL) meeting the requirements of animal experiments.

[0327] 3) Negative control: PBS containing 0.05% L-Cys HCl was used as the negative control.

[0328] 4) Experimental process: 5-6 weeks old C57BL / 6J male mice, after quarantine period, high-fat diet for 10 weeks, select 12 mice with body weight range of 35.50g-44.49g, according to the body weight of random stratified grouping, 6 / group, divided into 2 groups (experimental group and control group). After grouping (D1), start dosing, the experimental group is given MNH19250, the control group is given negative control, 2 times a day, a total of 28 days, during the experiment, the mice are free to drink water and eat, using 12h / 12h day-night cycle. During the test period, general clinical observation was carried out once after the end of dosing. The end of the experiment is the day after the end of dosing (D29), the colon is dissected at the end of the experiment, fixed with polyformaldehyde solution, the mucous layer is stained with AB-PAS staining solution alizarin blue, and the thickness of the mucous layer is measured. All data are expressed in the form of Mean ± SD, and are plotted and statistically analyzed using GraphPad Prism software. For pairwise comparison, t-test (Student's t-test) analysis method is used. Significant difference is indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0329] 7.2 Effect of strain MNH19250 on the repair of colonic tissue mucosa of mice with impaired intestinal barrier function induced by high-fat diet:

[0330] Referring to FIG. 18, the results show that strain MNH19250 can significantly increase the colonic mucous layer thickness of mice with impaired intestinal barrier function induced by high-fat diet, indicating that strain MNH19250 has the effect of treating / preventing impaired intestinal barrier.

[0331] Although the present application has been disclosed with reference to certain embodiments, it will be apparent that modifications and changes can be made without departing from the spirit and scope of the application as disclosed in this document and as provided in the claims that follow. Furthermore, it should be understood that although all examples in the disclosure illustrate embodiments of the present application, they are provided only as non-limiting examples, and therefore should not be considered as limiting the various aspects of the application thus illustrated. The present application is intended to have the full scope defined by the language of the disclosure, the language of the following claims, and any equivalents thereof. Therefore, the drawings and detailed description should be considered as illustrative rather than limiting.

Claims

1. An isolated Akkermansia muciniphila having an average nucleotide identity (ANI) value of at least 95% to Akkermansia muciniphila MNH19250 strain; and / or, having a 16S rRNA sequence which is at least 98.65% identical to the sequence as set forth in SEQ ID NO.

1.

2. The Akkermansia muciniphila of claim 1, comprising an Amuc-1100 protein which is at least 95% identical to the sequence as set forth in SEQ ID NO. 4; and / or comprising an Amuc_1631 protein which is at least 95% identical to the sequence as set forth in SEQ ID NO.

5.

3. The Akkermansia muciniphila of claim 1 or 2, having an average nucleotide identity (ANI) value of 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% to Akkermansia muciniphila MNH19250 strain; and / or having a 16S rRNA sequence which is at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to the sequence as set forth in SEQ ID NO.

1. the Akkermansia muciniphila comprises an Amuc-1100 protein which is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO. 4; and / or the Akkermansia muciniphila comprises an Amuc_1631 protein which is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO.

5.

4. The Akkermansia muciniphila of claim 1, wherein the Akkermansia muciniphila is Akkermansia muciniphila MNH19250 strain, deposited with Guangdong Microbial Culture Collection Center (GDMCC) on June 21, 2024, and has a deposit number of GDMCC NO: 63782.

5. A composition comprising the Akkermansia muciniphila of any one of claims 1-4, a culture thereof, a metabolite thereof, and / or a protein isolated from the Akkermansia muciniphila. The Akkermansia muciniphila has a 16S rRNA sequence with an average nucleotide identity (ANI) value of at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% to Akkermansia muciniphila MNH19250 strain, and / or has a 16S rRNA sequence with at least 98.65% identity, for example, at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to the sequence set forth in SEQ ID NO. 1; and / or the Akkermansia muciniphila comprises an Amuc-1100 protein with at least 96%, at least 97%, at least 98%, at least 99% identity to the sequence set forth in SEQ ID NO. 4; and / or The Akkermansia muciniphila comprises an Amuc_1631 protein with at least 96%, at least 97%, at least 98%, at least 99% identity to the sequence set forth in SEQ ID NO. 5; And / or the Akkermansia muciniphila is Akkermansia muciniphila MNH19250 strain, deposited with Guangdong Microbial Culture Collection Center (GDMCC) on June 21, 2024, and has a deposit number of GDMCC NO: 63782.

6. The composition of claim 5, wherein the Akkermansia muciniphila is selected from any one of live bacteria, attenuated bacteria, inactivated bacteria, and freeze-dried bacteria. Preferably, the inactivated bacteria are heat-inactivated bacteria, for example bacteria obtained by pasteurization. Preferably, the composition comprises as active ingredient Akkermansia muciniphila at a concentration of 10 7 to 10 12 CFU / g; Preferably, the culture of Akkermansia muciniphila is selected from any one of the following: a fermentation culture, a supernatant of a fermentation culture, or a dried product thereof. Preferably, the protein secreted by Akkermansia muciniphila is Amuc-1100 protein; and / or Amuc_1631 protein.

7. The composition of claim 5, further comprising one or more pharmaceutically acceptable carriers, food carriers, excipients and / or adjuvants.

8. The composition of claim 5, further comprising one or more other active agents for preventing or treating metabolic diseases, cardiovascular and cerebrovascular diseases, inflammatory diseases and / or tumors. Preferably, the other active agent is one or more of probiotics, prebiotics, anti-tumor agents, GLP-1 receptor agonists. Preferably, the prebiotics are selected from inulin, mulberry leaf extract, berberine, ganoderma, green coffee bean extract, oat, pectin, potato or extract thereof, citrus polyphenol, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidin, resistant dextrin, yeast beta-glucan, ginseng or extract thereof, nutritional compounds, biotin, polydextrose, fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, mannan oligosaccharide, mannan oligosaccharide (MOS), inulin rich in fructo-oligosaccharide, gluco-oligosaccharide, tagatose, trans-galacto-oligosaccharide, pectin, resistant starch, xylo-oligosaccharide (XOS), and any combination thereof. Preferably, the probiotics are selected from at least one of lactic acid bacteria, lactobacillus, butyric acid producing bacteria.

9. The composition of any one of claims 5-8, which is any one of a drug, a health product or a food product. Preferably, the health product or food product is any one of a nutritional composition, a food product, a candy, a food bar, a food additive, a drink additive, a dietary supplement.

10. Use of the Akkermansia muciniphila of any one of claims 1-4, or the composition of any one of claims 5-9 in the preparation of a medicament, health product or food for preventing, treating or alleviating inflammatory diseases, liver and kidney diseases, cardiovascular and cerebrovascular diseases, metabolic diseases, lipid-lowering, intestinal barrier damage diseases, immune diseases, tumors in a subject in need thereof, the Akkermansia muciniphila having an average nucleotide identity (ANI) value of at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% to the Akkermansia muciniphila MNH19250 strain, and / or having a 16S rRNA sequence that is at least 98.65% identical to the sequence set forth in SEQ ID NO. 1, for example at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical; and / or the Akkermansia muciniphila comprising an Amuc-1100 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO. 4; and / or the Akkermansia muciniphila comprising an Amuc_1631 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO.

5.

10. and / or the Akkermansia muciniphila is Akkermansia muciniphila MNH19250 strain, deposited in Guangdong Microbial Culture Collection Center (GDMCC), with the accession number of GDMCC NO: 63782, on June 21, 2024.

11. A method for preventing, treating or alleviating inflammatory diseases, hepatorenal diseases, cardiovascular and cerebrovascular diseases, metabolic diseases, intestinal barrier injury diseases, immune diseases, tumors, the method comprising administering to a subject in need thereof an effective amount of the Akkermansia muciniphila of any one of claims 1-4 or the composition of any one of claims 5-9, the Akkermansia muciniphila has an average nucleotide identity (ANI) value of at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% to the Akkermansia muciniphila MNH19250 strain, and / or has a 16S rRNA sequence that is at least 98.65% identical to the sequence set forth in SEQ ID NO. 1, for example, at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical; and / or the Akkermansia muciniphila comprises an Amuc-1100 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO. 4; and / or the Akkermansia muciniphila comprises an Amuc-1100 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO. 4; and / or the Akkermansia muciniphila comprises an Amuc_1631 protein having at least 96%, at least 97%, at least 98%, at least 99% identity to the sequence set forth in SEQ ID NO. 5; and / or the Akkermansia muciniphila is Akkermansia muciniphila MNH19250 strain, deposited with Guangdong Microbial Culture Collection Center (GDMCC) on June 21, 2024, and has a deposit number of GDMCC NO: 63782.

12. The use of claim 10 or the method of claim 11, wherein the inflammatory disease is selected from the group consisting of bronchial inflammation, cervical inflammation, conjunctival inflammation, esophageal inflammation, myocardial inflammation, rectal inflammation, scleral inflammation, gum inflammation, bone inflammation, lung inflammation, respiratory tract inflammation, inflammatory skin disease, fibrotic disease, brain inflammation; the liver and kidney disease is selected from any one of the following: non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFLD), liver fibrosis, liver cirrhosis, alcoholic liver disease and drug-induced liver injury, kidney injury, abnormal kidney function; preferably, the Akkermansia muciniphila or the composition reduces serum aspartate aminotransferase AST and / or alanine aminotransferase ALT and / or liver weight of the subject; and / or reduces the level of urea nitrogen BUN and / or creatinine CRE in the serum of the subject; the metabolic disease is selected from obesity, metabolic syndrome, insulin deficiency or insulin resistance related disorders, diabetes, glucose intolerance, abnormal lipid metabolism, hyperglycemia, dyslipidemia, irritable bowel syndrome, intestinal contraction disorder, hypercholesterolemia and / or hyperlipidemia; the cardiovascular disease is selected from hypertension, atherosclerosis, coronary heart disease and / or stroke; the intestinal barrier damage disease is selected from inflammatory bowel disease, Crohn's disease, ulcerative colitis; preferably, the Akkermansia muciniphila or the composition increases the thickness of the colon mucus layer; the lipid-lowering is selected from reducing visceral fat and / or local fat.

13. The use of claim 10 or the method of claim 11, wherein the Akkermansia muciniphila or the composition is capable of treating, preventing, alleviating or improving at least one of the following: (1) weight loss or weight control; (2) prevention, treatment, improvement or alleviation of liver damage, NAFLD or NASH; (3) prevention, treatment, improvement or alleviation of kidney damage, abnormal kidney function; (4) promoting the expression of type I interferon IFNβ in the subject, improving immunity; (5) prevention, treatment, improvement or alleviation of cancer; (6) prevention, treatment, improvement or alleviation of diabetes; (7) reducing the blood lipid and / or cholesterol level of the subject; (8) reducing the local fat, body fat rate and / or visceral fat of the subject; (9) improving the intestinal barrier function of the subject. (9) repairing the intestinal tissue mucosa of the subject; (10) preventing, treating, ameliorating hypertension, atherosclerosis.

14. The use of claim 10 or the method of claim 11, wherein the Akkermansia muciniphila or the composition has at least one property selected from the group consisting of: modulating immunity while modulating metabolism; reducing abdominal fat while repairing intestinal barrier damage; improving liver damage while reducing weight; reducing weight while anti-inflammation; reducing lipid while anti-inflammation; reducing lipid while repairing intestinal barrier damage; reducing lipid while reducing serum aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT); improving liver damage while improving kidney damage; improving diabetes while improving liver damage; improving diabetes while improving kidney damage; improving atherosclerosis while improving liver damage; improving atherosclerosis while reducing lipid.

15. The use of Akkermansia muciniphila of any one of claims 1-4, or the composition of any one of claims 5-9 comprising an Akkermansia muciniphila strain, in affecting or modulating at least one of immune signaling, intestinal barrier function, fasting glucose homeostasis, cholesterol homeostasis, triglyceride homeostasis, repairing liver damage, kidney damage, promoting local adipose tissue metabolism in a subject in need thereof. The Akkermansia muciniphila has a 16S rRNA sequence with an average nucleotide identity (ANI) value of at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% to the Akkermansia muciniphila MNH19250 strain, and / or has a 16S rRNA sequence that is at least 98.65% identical to the sequence set forth in SEQ ID NO. 1, for example, at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical; and / or The Akkermansia muciniphila comprises an Amuc-1100 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO. 4; and / or The Akkermansia muciniphila comprises an Amuc_1631 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO.

5. The Akkermansia muciniphila is Akkermansia muciniphila MNH19250 strain, deposited with the Guangdong Microbial Culture Collection Center (GDMCC) on June 21, 2024, and has a deposit number of GDMCC NO: 63782.

16. The use of Akkermansia muciniphila of any one of claims 1-4, or the composition of any one of claims 5-9, for reducing liver and / or kidney injury in a subject in need thereof, said reduction of liver injury is reducing one or more elevated liver injury or liver disease indicators in the subject, such as reducing serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and / or liver weight indicators in the subject; said reduction of kidney injury is reducing urea nitrogen (BUN) in serum and / or reducing creatinine (CRE) indicators in serum in the subject; said Akkermansia muciniphila has an average nucleotide identity (ANI) value of at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% to Akkermansia muciniphila MNH19250 strain, and / or, has a 16S rRNA sequence that is at least 98.65% identical to the sequence set forth in SEQ ID NO. 1, such as at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical; and / or said Akkermansia muciniphila comprises an Amuc-1100 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO. 4; and / or said Akkermansia muciniphila comprises an Amuc_1631 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence set forth in SEQ ID NO. 5; and / or the Akkermansia muciniphila is Akkermansia muciniphila MNH19250 strain, deposited with Guangdong Microbial Culture Collection Center (GDMCC) on June 21, 2024, and has the accession number of GDMCC NO: 63782.

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