Isolated mucinophilic akkermansia, compositions comprising the same and uses

Abstract

The present application provides isolated mucinophilic Akkermansia, compositions comprising the same and uses thereof, which can be used for treating, preventing or relieving obesity, metabolic disorders caused by obesity, diabetes, inflammation, liver and kidney diseases, liver diseases, cardiovascular and cerebrovascular diseases, tumors and the like.

Description

Technical Field

[0001] This disclosure relates to the field of microbiology, and more specifically to novel strains of *Ackermania* strains isolated from the microorganisms, compositions comprising the microorganisms, and their uses. Background Technology

[0002] Akkermansia muciniphila is a mucin-degrading bacterium that typically colonizes the intestines of humans and many animals.

[0003] The screening of new strains of *Ackermania myxophilus* faces significant challenges due to its primary presence in the human and animal intestines. As a strictly anaerobic microorganism, it has extremely high requirements for nutrition and culture conditions, and exhibits a long growth cycle. Furthermore, its anaerobic nature makes in vitro cell experiments difficult to validate drug efficacy; in in vivo animal experiments, the high degree of anaerobicness and difficulty in maintaining stable viable cell counts lead to insufficient reproducibility. These in vitro and in vivo technical challenges limit the discovery and application of new *Ackermania myxophilus* strains.

[0004] In addition, Akkermansia myxophilus grows slowly and has difficulty competing with other microbial strains, thus posing a challenge to its availability during the screening process.

[0005] The hydrophobicity of the AKKPROBIO strain disclosed in CN116925975B reached 31% after 60 min; its self-aggregation stabilized at around 52% after 20 h. Some literature suggests that AKKPROBIO has a weak biofilm-forming ability, and in vitro tests indicate that its colonization ability in the intestine is low (https: / / doi.org / 10.3390 / foods13030442, Section 3.1). The weak colonization and adhesion ability of the AKK strain will limit its commercial product development.

[0006] The prevalence of metabolic diseases such as obesity, diabetes, hypertension, hyperlipidemia, and fatty liver is constantly increasing, and studies have also shown that these diseases are related to the ecological imbalance of the gut microbiota. In many populations, *Ackermania myxotropica* is negatively correlated with obesity, diabetes, cardiovascular disease, and low-grade inflammation. *Ackermania myxotropica* can play a metabolic protective role by protecting the integrity of intestinal epithelial cells and the mucus layer.

[0007] Existing technologies often utilize mixtures of multiple microorganisms to treat metabolic and obesity-related diseases or disorders, such as probiotics or fecal microbiota transplantation (FMT). However, the use of mixtures of multiple microorganisms complicates the mechanisms, and the interactions between the microorganisms are not well understood. Furthermore, the use of microbial mixtures often disrupts the homeostasis of the gut microbiota. In addition, the use of microbial mixtures requires further consideration of whether the individual microorganisms affect each other's activity, and it remains unclear whether the synergistic effect of the mixture or a single microorganism plays a role in the treatment or prevention of the disease.

[0008] The number of microbial resources is extremely large, and screening out new strains or microorganisms that can be effectively used to treat or prevent diseases such as obesity, diabetes, hypertension, hyperlipidemia, liver and kidney dysfunction, and tumors is a huge challenge, but it also represents a huge unmet need. Summary of the Invention

[0009] This disclosure discloses the isolation of a novel Akkermansia muciniphila strain. Compared to existing Akkermansia muciniphila strains, this novel strain exhibits stronger epithelial cell adhesion ability, enabling it to colonize well in the human or animal intestines, thereby prolonging its residence time in the intestines and better exerting its pharmacological effects. Simultaneously, due to the superior colonization and adhesion ability of this strain, it can effectively prevent pathogen adhesion, thus contributing to the maintenance of intestinal homeostasis and health. Furthermore, this strain can synthesize large amounts of short-chain fatty acids, thereby effectively treating or preventing metabolic diseases, reducing body weight, body fat, blood sugar, blood lipids, and cholesterol, improving or alleviating non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), repairing intestinal mucosal tissue, and promoting the expression of type I interferon IFNβ, enhancing immunity. Therefore, it has the potential to prevent or treat viral infections and inhibit tumor growth.

[0010] In a first aspect, this disclosure provides isolated Akkermansia muciniphila strains having an average nucleotide identity (ANI) 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% with respect to the Akkermansia muciniphila strain (GCF_000020225.1), and / or having a 16S rRNA sequence having at least 98.65% identity with the sequence shown in SEQ ID NO.1.

[0011] In some embodiments, the comparison score (AF) of the Akkermansia muciniphila strain to the Akkermansia muciniphila (GCF_000020225.1) strain is 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%.

[0012] In some embodiments, the Akkermansia muciniphila has a 16S rRNA sequence having 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% identity with the sequence shown in SEQ ID NO.1.

[0013] In some embodiments, the Akkermansia muciniphila is a novel strain of Akkermansia muciniphila that has an average nucleotide identity (ANI) value of at least 95% with Akkermansia muciniphila (GCF_000020225.1), 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%... 0.3%, 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%.

[0014] In some embodiments, the metabolites of *Ackermania myxophilus* include short-chain fatty acids, including at least one selected from acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, hexanoic acid, and decanoic acid; preferably, *Ackermania myxophilus* is capable of producing high levels of acetic acid and / or propionic acid. In some embodiments, the acetic acid content secreted by *Ackermania myxophilus* is not less than 1000 μg / mL, 1100 μg / mL, 1200 μg / mL, 1300 μg / mL, 1400 μg / mL, or 500 μg / mL. In some embodiments, the acetic acid content of the *Ackermania myxophilus* cells is not less than 50 μg / mg, 60 μg / mg, 70 μg / mg, 80 μg / mg, 90 μg / mg, or 100 μg / mg. In some embodiments, the propionic acid content secreted by the *Ackermania myxophilus* is not less than 180 μg / mL, 190 μg / mL, 200 μg / mL, 210 μg / mL, 220 μg / mL, or 230 μg / mL. In some embodiments, the propionic acid content of the *Ackermania myxophilus* cells is not less than 5 μg / mg, 6 μg / mg, 7 μg / mg, 8 μg / mg, 9 μg / mg, 10 μg / mg, 11 μg / mg, 12 μg / mg, 13 μg / mg, 14 μg / mg, or 15 μg / mg.

[0015] In some embodiments, the Akkermansia muciniphila MNH19250 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC No: 63782 and deposit date of June 21, 2024.

[0016] In a second aspect, this disclosure provides compositions comprising Akkermansia myxophilus or its cultures or its metabolites or its secreted proteins as described in the first aspect.

[0017] In some embodiments, the culture of Akkermansia myxophilus includes a solid culture of Akkermansia myxophilus, a fermentation culture, or the supernatant of a fermentation culture.

[0018] In some embodiments, the fermentation culture or fermentation culture supernatant is a fermentation culture or fermentation culture supernatant obtained using a liquid culture medium under anaerobic culture conditions.

[0019] In some embodiments, the composition is provided in liquid or solid form.

[0020] In some embodiments, the composition contains 1×10 4 Up to 1×10 12 cfu / mL or 1×10 4 Up to 1×1012 The cfu / mg of the live Akermansia myxophilus bacteria.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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 3×10 11 4×10 11 5×10 11 6×10 11 7×10 11 8×10 11 9×10 11 1×10 12 2×10 12 3×10 12 4×10 12 5×10 12 6×10 12 7×10 12 8×10 12 9×10 12 1×10 13 2×10 13 3×10 13 4×10 13 5×10 13 6×10 13 7×10 13 8×10 13 9×10 13 A number of colony-forming units (CFU) or any number of colonies forming units (CFU) of bacteria.

[0025] In some embodiments, the Akermansia muciniphila in the composition is an attenuated bacterium, a killed bacterium, a freeze-dried bacterium, or an irradiated bacterium, for example, it may be a heat-inactivated bacterium, preferably pasteurized.

[0026] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (e.g., lyophilized powder), or gel.

[0027] In some embodiments, the composition is in the form of a powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral liquid, suspension, emulsion, liquid formulation, sustained-release formulation, nanoformulation, or microencapsulated capsule.

[0028] In some embodiments, the composition is in the form of an oral or injectable dosage form.

[0029] In some embodiments, the composition further comprises one or more pharmaceutically acceptable carriers, excipients, or excipients.

[0030] The pharmaceutically acceptable excipients are well known to those skilled in the art.

[0031] In some embodiments, the excipient may be at least one selected from carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings.

[0032] In some embodiments, the composition comprises one or more of the following: a buffer (e.g., sodium bicarbonate, infant formula or sterile human milk or other agents that allow bacteria to survive and grow (e.g., to survive in the acidic environment of the stomach and to grow in the intestinal environment)), a lyophilization protectant, a preservative, a stabilizer, a binder, a compactor, a lubricant, a dispersion enhancer, a disintegrant, an antioxidant, a flavoring agent, a sweetener, and a coloring agent.

[0033] In some embodiments, the composition further comprises one or more other active agents for the prevention or treatment of metabolic diseases and / or tumors.

[0034] The other active agents have at least one of the following functions: (a) suppressing appetite, (b) preventing metabolic diseases, (c) treating metabolic diseases, (d) preventing tumors, and (e) treating tumors.

[0035] In some implementations, the appetite suppression includes reducing food intake and / or reducing appetite.

[0036] In some embodiments, the other active agent is selected from: GLP-1 receptor agonists, dual agonists of GLP-1 and GCG receptors, triple agonists of GLP-1, GIP, and GCG receptors, AMPK agonists, or active pharmaceutical ingredients that promote GLP-1 secretion.

[0037] In some embodiments, the other active agents are selected from: metformin, sulfonylureas, chloroanisole, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin, pioglitazone, rosiglitazone, pentoxifylline, omega-3 fatty acids, statins, ezetimibe, ursodeoxycholic acid, smegglutide, liraglutide, exenatide, and benaglutide.

[0038] In some embodiments, the other active agent may be a drug for the prevention or treatment of tumors.

[0039] In some embodiments, the other active agents may be one or more of probiotics and prebiotics, or a combination thereof.

[0040] Preferably, the probiotics are selected from one or more of lactic acid bacteria, lactobacillus, lactococcus, butyric acid bacteria, bifidobacteria, thermophilic streptococcus, fecal streptococcus, and mesenteric streptococcus.

[0041] Preferably, the prebiotic is selected from inulin, mulberry leaf extract, berberine, Ganoderma lucidum, green coffee bean extract, oats, pectin, potato or its extract, citrus polyphenols, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidins, resistant dextrin, yeast β-glucan, ginseng or its extract, nutrient compounds, biotin, polydextrose, fructooligosaccharides (FOS), galactooligosaccharides (GOS), starch, cellulose, β-glucan, hemicellulose, lactulose, mannooligosaccharides, mannooligosaccharides (MOS), inulin rich in fructooligosaccharides, oligodextrose, tagatose, trans-galactooligosaccharides, pectin, resistant starch, xylooligosaccharides (XOS), and any combination thereof.

[0042] In some embodiments, the composition may be formulated as a frozen composition, such as a frozen composition prepared by quick-freezing and drying, or freeze-drying, for storage and / or transport.

[0043] In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained by electrostatic spray drying.

[0044] In some embodiments, the strain in the composition is freeze-dried or spray-dried. In some embodiments, the strain in the composition is electrostatically spray-dried. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is viable. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is capable of partially or completely colonizing the intestine. In some embodiments, the strain is reconstituted prior to administration. In some cases, the reconstitution is performed using the diluent described herein.

[0045] In some embodiments, the composition may be administered alone or in combination with a carrier such as a pharmaceutically acceptable carrier or a biocompatible scaffold.

[0046] In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric-coated formulation. In some embodiments, the enteric-coated formulation is a dosage form with an enteric coating. For example, the enteric-coated formulation may be enteric-coated granules, enteric-coated tablets, or enteric-coated capsules. 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-coated capsule, or the capsule may be a microencapsulated capsule or microcapsule.

[0047] In some embodiments, the composition is a drug.

[0048] In some embodiments, the composition is an infant-specific dosage form, a pediatric-specific dosage form, or an adult-specific dosage form.

[0049] In some embodiments, the composition is a gastrointestinal or non-gastrointestinal dosage form.

[0050] Thirdly, the use of the *Akermansia myxophilus* described in the first aspect or the composition described in the second aspect in the preparation of medicaments for treating, preventing or alleviating inflammatory diseases, liver and kidney diseases, cardiovascular and cerebrovascular diseases, metabolic diseases, intestinal barrier damage diseases, immune-related diseases, and tumors is provided.

[0051] In some implementations, the metabolic disease is a metabolic disorder, metabolic disturbance, or a disease caused by a metabolic disturbance, including but not limited to: liver disease, obesity and obesity-related diseases, cardiovascular disease, cerebrovascular disease, diabetes, dyslipidemia, glucose intolerance, type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic diseases, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), atherosclerosis, and at least one of the following: kidney disease.

[0052] In some implementations, the liver and kidney diseases include liver diseases, liver function impairment-related diseases, kidney damage, kidney dysfunction, and other kidney diseases.

[0053] Liver diseases include, but are not limited to, at least one of the following: fatty liver, NAFLD / NASH, abnormal liver function, extrahepatic cholestasis, hepatitis, liver injury, intrahepatic cholestasis, liver fibrosis, cirrhosis, and hepatocellular damage.

[0054] The composition comprises an aggregate of isolated and purified live microbial communities to reduce serum levels of ALT and / or AST in subjects at least by 2 U / L, 5 U / L, 10 U / L, 25 IU / L, 30 IU / L, or 50 IU / L compared to pre-administration levels of ALT and / or AST in subjects; the composition comprises an aggregate of isolated and purified live microbial communities to reduce serum levels of blood urea nitrogen (BUN) and creatinine (CRE) in the subject's serum at least by 2 mmol / L, 5 mmol / L, or 10 mmol / L compared to pre-administration levels of ALT and / or AST in subjects.

[0055] As used herein, a microbiome generally refers to a group of microorganisms that consists essentially of a single strain, species, or genus, as may be the case when a subgroup of such strain, species, or genus is isolated and purified. Therefore, for a given microbial community, if cultured from an isolated microbial species or strain, such a community will be referred to herein as purified or substantially pure. The resulting community will typically be at least 80% pure with respect to the said microbial species or strain, and at least 90%, 95%, 98%, 99%, 99.5%, or 99.9% pure relative to other microbial species or strains within that particular community. Conversely, the level of undesired strains in any particular desired microbial community 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 the purified community aggregates, such as other undesirable microbial strains or species, may be proportional to or below the aforementioned levels for each desired community. Less than 2%, less than 1%, less than 0.5%, or less than 0.1%. In the case where the composition comprises an aggregate of multiple microbial populations, each population may have the above-mentioned purity, either before its incorporation into the composition or when polymerization measurements are performed on the aggregate. For example, the level of impurities in the purified aggregate, such as other undesirable microbial strains or species, may be proportionally at or below the above-mentioned level for each desired population. Less than 2%, less than 1%, less than 0.5%, or less than 0.1%. In the case where the composition comprises an aggregate of multiple microbial populations, each population may have the above-mentioned purity, either before its incorporation into the composition or when polymerization measurements are performed on the aggregate. For example, the level of impurities in the purified aggregate, such as other undesirable microbial strains or species, may be proportionally at or below the above-mentioned level for each desired population.

[0056] The pharmaceutical compositions disclosed herein may also include cellular components, metabolites, secreted molecules and compounds of *Ackermania myxophilus*, and the like. This can be achieved, for example, by recovering the supernatant of a *Ackermania myxophilus* culture or by extracting cellular components or cell fractions, metabolites, or secreted compounds from a *Ackermania myxophilus* culture; these may correspond to components in isolated forms from *Ackermania myxophilus*, or any mixture of one or more components from *Ackermania myxophilus*.

[0057] In some embodiments, the Akkermansia myxophilus of this disclosure can be used to treat or prevent liver function impairment-related diseases, including at least one of the following diseases: fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, liver cancer, or hepatocellular damage.

[0058] In some implementations, the causes of the liver disease include, but are not limited to, at least one of a high-fat diet, a high-cholesterol diet, a high-sugar diet, high blood lipids, high blood sugar, or high cholesterol.

[0059] In some implementations, the liver diseases include those caused by a high-fat diet, a high-cholesterol diet, a high-sugar diet, a high-fat and high-cholesterol diet, a high-fat and high-sugar diet, and / or a high-fat, high-cholesterol, and high-sugar diet.

[0060] In some embodiments, the *Akermansia myxophilus* described herein can be used to treat, prevent, or alleviate liver function impairment-related diseases, including at least one of the following: fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, or hepatocyte damage.

[0061] In particular, the *Akermansia myxophilus* or compositions containing it of the present invention can be used to treat or prevent liver injury. The liver injury can be caused by prolonged strenuous work; liver injury caused by long-term alcohol consumption; fatty liver, drug-induced liver disease, or liver injury caused by genetic metabolic factors.

[0062] In some embodiments, the myxotrophic Akkermansia of the present invention or compositions containing it can reduce ALT and / or AST levels.

[0063] In some embodiments, the myxotrophic Akkermansia of the present invention, or compositions containing it, can reduce liver weight.

[0064] In some embodiments, the kidney disease includes, but is not limited to, kidney injury and abnormal kidney function. The kidney disease includes, but is not limited to, primary glomerulonephritis, hypertensive arteriosclerosis, diabetic nephropathy, secondary glomerulonephritis, tubulointerstitial diseases (e.g., chronic pyelonephritis, chronic uric acid nephropathy, obstructive nephropathy, drug-induced nephropathy, etc.), and ischemic nephropathy. In some embodiments, the precipitating factors of the kidney disease include at least one of the following: a high-fat diet, a high-sugar diet, and a high-cholesterol diet. In some embodiments, the kidney disease is characterized by elevated serum creatinine.

[0065] In some implementations, the obesity and obesity-related diseases include, but are not limited to: overweight, obesity, metabolic syndrome, cardiovascular disease, cardiovascular and cerebrovascular diseases, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, and steatohepatitis.

[0066] In some implementations, the causes of obesity and obesity-related diseases include, but are not limited to, at least one of: high-fat diet, high-sugar diet, high cholesterol, high blood lipids, high blood sugar, NAFLD, or NASH.

[0067] In some implementations, the obesity and obesity-related diseases include, but are not limited to: obesity caused by a high-fat diet, obesity caused by high cholesterol, obesity caused by a high-sugar diet, obesity caused by high fat and high cholesterol, obesity caused by high fat and high sugar, obesity caused by high fat, high cholesterol and high sugar, or obesity in patients with NAFLD or NASH.

[0068] In some implementations, the obesity and obesity-related diseases include at least one of the following: obesity, metabolic syndrome, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, and steatohepatitis.

[0069] Compositions and methods are provided for treating, alleviating, controlling, reducing, or preventing symptoms, signs, or indicators of liver or kidney diseases, as well as the onset of the diseases themselves. The compositions and methods include microbial compositions selected to improve intestinal function in subjects administering them, thereby treating liver or kidney diseases and / or signs, symptoms, and indicators of these diseases.

[0070] 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, such as excessive visceral fat.

[0071] In some embodiments, the myxotrophic Akkermansia of the present invention or compositions containing it can reduce body fat and / or visceral fat and / or abdominal fat.

[0072] In some implementations, the diabetes includes, but is not limited to: type 1 diabetes, type 2 diabetes, gestational diabetes, HDAC activity-mediated diabetes, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic foot, diabetes caused by pancreatic beta cell damage, diabetes caused by insulin resistance, and diabetes caused by obesity.

[0073] In some implementations, the causes of diabetes include, but are not limited to: pancreatic islet cell dysfunction, decreased insulin secretion, increased insulin resistance, high-fat diet, high-sugar diet, high cholesterol, high blood lipids, high blood sugar, NAFLD, or NASH.

[0074] In some implementations, the causes of diabetes include at least one of the following: high-fat diet, high-sugar diet, and high-cholesterol diet.

[0075] In some implementations, the diabetes is characterized by hyperglycemia resulting from low levels of insulin and / or peripheral insulin resistance.

[0076] In some embodiments, the metabolic disorder includes, but is not limited to: (1) diabetes mellitus caused by glucose metabolism disorder, or (2) diabetes mellitus caused by impaired glucose tolerance or decreased glucose tolerance, or (3) diabetes mellitus caused by damage to insulin β cells, or (4) diabetes mellitus caused by insulin resistance.

[0077] In some embodiments, the Akkermansia myxophila of this disclosure can increase the secretion level of glucagon-like peptide-1 (GLP-1), thereby regulating the body's blood glucose balance, improving the body's glucose tolerance, and further improving the body's insulin sensitivity and leptin sensitivity, thereby achieving the effect of preventing and / or treating diabetes and / or hyperlipidemia.

[0078] In some implementations, the cardiovascular disease or cerebrovascular disease includes, but is not limited to: atherosclerosis, coronary heart disease, hypertension, cardiovascular disease in patients with NAFLD or NASH, cerebrovascular disease in patients with NAFLD or NASH, and high cholesterol disease.

[0079] In some implementations, the causes of cardiovascular disease or cerebrovascular disease include, but are not limited to, at least one of: atherosclerosis, NAFLD, NASH, hyperlipidemia, hyperglycemia, or hypercholesterolemia.

[0080] In some embodiments, the inflammatory disease is selected from diseases related to bronchial inflammation, such as bronchitis; diseases related to cervical inflammation, such as cervicitis; diseases related to conjunctival inflammation, such as conjunctivitis; diseases related to esophageal inflammation, such as esophagitis; diseases related to myocardial inflammation, such as myocarditis; diseases related to rectal inflammation, such as proctitis; diseases related to scleral inflammation, such as scleritis; diseases related to gum inflammation; and also includes diseases related to bone inflammation, lung inflammation (alveolitis), respiratory tract inflammation (e.g., asthma, such as bronchial asthma), acute respiratory distress syndrome (ARDS); inflammatory skin diseases, such as contact allergy, atopic dermatitis; fibrotic diseases (e.g., pulmonary fibrosis); and encephalitis.

[0081] In some embodiments, the *Akermansia myxophilus* strain of this disclosure has a therapeutic / preventive effect on intestinal barrier damage. In some embodiments, the intestinal barrier damage is caused by at least one of the following: a high-fat diet, a high-sugar diet, or a high-cholesterol diet.

[0082] In some implementations, examples of the tumor or cancer include, but are not limited to, solid tumors and non-solid tumors.

[0083] In some embodiments, the drug has at least one effect selected from the following: effect: reducing liver weight; treating early-stage fatty liver disease lesions; slowing down fat accumulation in liver cells; reducing serum AST and ALT; reducing inflammatory lesions of abdominal white fat; reducing body weight in mammals; reducing food intake in mammals; reducing body fat in mammals; reducing the level of at least one of the following indicators in mammalian serum: total cholesterol, low-density lipoprotein, and triglyceride levels; increasing the level of high-density lipoprotein in mammalian serum; improving oral glucose tolerance impairment in mammals; reducing fasting blood glucose in mammals; reducing HOMA-IR in mammals; reducing at least one of epididymal fat weight, perirenal fat weight, visceral fat weight, and inguinal fat weight in mammals; repairing gastrointestinal mucosal damage; increasing... Colonic mucus layer thickness; regulation of the body's immunity; increase IFNβ expression; promote IFNβ transcriptional activity; treatment, prevention, or relief of coronary heart disease; treatment, prevention, or relief of atherosclerosis; treatment, prevention, or relief of hyperglycemia; treatment, prevention, or relief of hyperlipidemia; treatment, prevention, or relief of hypercholesterolemia; treatment, prevention, or relief of liver function damage; treatment, prevention, or relief of fatty liver; treatment, prevention, or relief of NAFLD or NASH; treatment, prevention, or relief of hypertension; treatment, prevention, or relief of diabetes, preferably gestational diabetes, type II diabetes, or HDAC-mediated diabetes; treatment, prevention, or relief of obesity; treatment, prevention, or relief of metabolic syndrome; treatment, prevention, or relief of localized excessive sebum, excessive inguinal fat, excessive epididymal fat, and / or excessive brown fat; treatment, prevention, or relief of tumors.

[0084] The Akkermansia muciniphila MNH19250 isolated in this disclosure has strong autoaggregation and hydrophobicity, making it easy to adhere to epithelial cells. It also has good tolerance to pH, NaCl, and bile salts, thus enabling it to rapidly and stably colonize the intestines of humans or animals.

[0085] The Akkermansia muciniphila MNH19250 isolated in this publication can also synthesize large amounts of short-chain fatty acids, thus effectively treating or preventing metabolic diseases, reducing body weight, body fat, blood sugar, blood lipids, and cholesterol, improving or alleviating non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), repairing intestinal mucosa, promoting the expression of type I interferon IFNβ, enhancing immunity, and having the potential to prevent or treat viral infections and inhibit tumor growth.

[0086] Therefore, in some implementations, the drug can regulate both metabolism and immunity.

[0087] In some implementations, the drug can reduce abdominal fat while also repairing intestinal barrier damage.

[0088] In some implementations, the drug can improve liver damage while also reducing weight.

[0089] In some implementations, the drug can reduce weight while also reducing inflammation.

[0090] In some implementations, the drug has both lipid-lowering and anti-inflammatory effects. The lipid-lowering effect refers to reducing fat in cases of abdominal obesity or visceral fat.

[0091] In some implementations, the drug can repair intestinal barrier damage while lowering lipids.

[0092] In some embodiments, the drug lowers lipids while simultaneously reducing serum AST and / or ALT. The lipid-lowering effect is for abdominal obesity or for reducing visceral fat.

[0093] In some implementations, the drug can improve both liver and kidney damage.

[0094] In some implementations, the drug improves both diabetes and liver damage.

[0095] In some implementations, the drug improves both diabetes and kidney damage.

[0096] In some implementations, the drug can improve liver damage while also improving atherosclerosis.

[0097] In some implementations, the drug improves atherosclerosis while also lowering lipids. This lipid-lowering refers to reducing abdominal obesity or reducing visceral fat.

[0098] In some embodiments, the Akkermansia muciniphila of the present invention, or the composition thereof, has at least one of the following effects: influencing or regulating immune signaling in the desired subject, influencing intestinal barrier function, influencing or regulating fasting glucose homeostasis, influencing or regulating cholesterol homeostasis, triglyceride homeostasis, repairing liver damage, kidney damage, and promoting local adipose tissue metabolism.

[0099] In some embodiments, the Akkermansia muciniphila of the present invention, or the composition thereof, has an effect of reducing liver and / or kidney damage. The reduction of liver damage is achieved by reducing one or more elevated liver damage 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; the reduction of kidney damage is achieved by reducing serum blood urea nitrogen (BUN) and / or serum creatinine (CRE) indicators in the subject. Attached Figure Description

[0100] The embodiments will now be described in conjunction with the accompanying drawings, thereby making the above and other aspects and advantages of the present invention apparent and readily understood.

[0101] Figure 1 The image shows a photograph of the colony morphology of strain MNH19250.

[0102] Figure 2 The image shows a Gram staining photograph of strain MNH19250.

[0103] Figure 3 The image shows an electron microscope image of strain MNH19250.

[0104] Figure 4 The results show the tolerance of strain MNH19250 to different pH values. The horizontal axis represents pH value, and the vertical axis represents the survival percentage.

[0105] Figure 5 The results show the tolerance of strain MNH19250 to different concentrations of NaCl.

[0106] Figure 6 The results show the tolerance of strain MNH19250 to different concentrations of bile salts.

[0107] Figure 7 The results showed that strain MNH19250 had the ability to autoaggregate over 6 hours.

[0108] Figure 8 The phylogenetic tree of strain MNH19250 is shown.

[0109] Figure 9 The bar chart shows the relative fluorescence values ​​of IFNβ expression in the presence of strain MNH19250.

[0110] Figure 10 The results showed that strain MNH19250 significantly reduced liver function indicators (a: ALT; b: AST).

[0111] Figure 11 The results show that strain MNH19250 significantly improved non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NAFLD / NASH) (a: liver weight; b: liver weight as a percentage of body weight; c: hepatic steatosis; d: hepatic lobular inflammation; e: hepatocyte ballooning degeneration; f: non-alcoholic fatty liver disease activity score (NAS); g: liver HE tissue section).

[0112] Figure 12 The results showed that strain MNH19250 could improve renal function indicators (a: blood urea nitrogen (BUN); b: serum creatinine (CRE)).

[0113] Figure 13 The results showed that strain MNH19250 significantly reduced oral glucose tolerance in high-fat diet-induced type 2 diabetic mice (a. oral glucose tolerance results; b. area under the oral glucose tolerance curve; data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using Student's t-test; *, p < 0.05 compared with HFD-Control group; **, p < 0.01 compared with HFD-Control group).

[0114] Figure 14 The results showed that strain MNH19250 significantly reduced fasting blood glucose in high-fat diet-induced type 2 diabetic mice (data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the Student's t-test; *, p < 0.05 compared with the HFD-Control group).

[0115] Figure 15 The results showed that strain MNH19250 significantly reduced the body weight of mice with high-fat diet-induced obesity (a. body weight results; b. percentage change in body weight results; data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the t-test (Student's t-test); *, p < 0.05 compared with the HFD-Control group; **, p < 0.01 compared with the HFD-Control group).

[0116] Figure 16The results showed that strain MNH19250 significantly reduced blood lipids in high-fat diet-induced obese mice (a. serum total cholesterol (TCHO); b. serum triglycerides (TG); c. serum low-density lipoprotein cholesterol (LDL-C); d. LDL-C / HDL-C ratio; data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the t-test (Student's t-test); *, p < 0.05 compared with the HFD-Control group).

[0117] Figure 17 The results showed that strain MNH19250 significantly reduced fat weight and visceral fat ratio in high-fat diet-induced obese mice (a. epididymal fat; b. perirenal fat; c. mesenteric fat; d. visceral fat; e. visceral fat percentage; f. inguinal fat; data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the t-test (Student's t-test); *, p < 0.05 compared with the HFD-Control group).

[0118] Figure 18 The results showed that strain MNH19250 significantly increased the mucus layer thickness in high-fat diet-induced obese mice (a. Alicin blue staining of the colon; b. colonic mucus layer thickness; data are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the t-test (Student's test); *, p < 0.05 compared with the HFD-Control group).

[0119] Preservation of strains

[0120] The strain Akkermansia muciniphila MNH19250 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC No: 63782, deposited on June 21, 2024, at the Guangdong Institute of Microbiology, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit name is Akkermansia muciniphila MNH19250. Detailed Implementation

[0121] This disclosure discloses the isolation of a novel strain of *Ackermania* species, with accession number GDMCC No: 63782, and its identification using conventional classification and molecular biological methods. The identification results indicate that this strain is a novel strain belonging to the *Ackermania* species. Furthermore, this disclosure investigates the biochemical properties and therapeutic applications of this strain.

[0122] It is known in the art that bacterial species can be classified and identified using both traditional classification methods and molecular biological methods. Traditional classification methods include, but are not limited to, cell morphology observation, Gram staining, flagellar staining, and various metabolic experiments. Molecular biological methods include, but are not limited to, ribosomal RNA sequencing and whole-genome sequencing-based methods.

[0123] As used in this article, the term "prebiotic" can be a general term referring to chemical substances and / or components that can affect the growth and / or activity of microorganisms in a host (e.g., that can allow specific changes in the composition and / or activity of the microbiome).

[0124] The terms “subject,” “object,” “individual,” “host,” and “patient” are used interchangeably in this document to refer to any animal subject, including: humans, mammals, laboratory animals, livestock, and domestic pets.

[0125] The compositions or formulations disclosed herein can be administered as pharmaceutical preparations, therapeutic compositions, or medical probiotics. In some cases, the compositions are administered as pharmaceutical preparations. In some cases, the compositions are administered as medical probiotics. In some cases, the compositions (e.g., medical probiotics) can be administered orally, such as as capsules, pills, or tablets.

[0126] 16S rRNA is a type of ribosomal RNA found in prokaryotes. The 16S rRNA gene consists of variable and conserved regions. The conserved regions are common to all bacteria, while the variable regions vary to varying degrees among different bacteria. By comparing the 16S rRNA gene sequences of bacteria and analyzing their evolutionary distances based on sequence differences, an evolutionary tree can be constructed. When the 16S rRNA gene sequence identity between two strains is less than 98.65%, they can be identified as belonging 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), p. 23).

[0127] The "identity" between two nucleic acid molecule sequences 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 include, for example, the DNAStar "MegAlign" program.

[0128] Second-generation sequencing technology can also be used to identify bacterial species based on whole-genome sequencing, making the identification results more accurate. Average nucleotide identity (ANI) of bacterial genomes refers to the similarity of homologous genes between two bacterial genomes. ANI values ​​can be calculated using methods such as BLAST. In the field of bacterial taxonomy, it is generally believed that an ANI value of more than 95% is required to identify them as belonging to the same bacterial species (Jain C, Rodriguez-R LM, Phillippy AM, et al. High throughput ANI analysis of 90K prokaryotic genomes reveals clear speciesboundaries[J]. Nature Communications, 2018, 9(1): 5114.).

[0129] You can use various existing mature ANI value calculation tools, such as local calculation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), and online calculation tools ANI caculator (http: / / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani) and ANItools.

[0130] Using the methods described above, those skilled in the art can determine whether an isolated strain belongs to the Akkermansia muciniphila species identified by the 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%, 96.9 ... When the percentages are 7.2%, 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 they belong to the same bacterial species.

[0131] For example, when its 16S rRNA sequence has at least 98.65% identity with the sequence shown in SEQ ID NO.1, 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 100%, it can be determined that they belong to the same bacterial species.

[0132] A “strain” refers to a member of a bacterial species that possesses genetic characteristics that distinguish it from closely related members of the same bacterial species. These genetic characteristics can be the complete or partial absence of at least one gene, the complete or partial absence of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), absence (“cure” of at least one natural plasmid), the presence of at least one recombinant gene, the presence of at least one mutant gene, the presence of at least one exogenous gene (a gene from another species), at least one mutated regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the presence of at least one non-natural plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. 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 cases where a strain (compared to another strain of the same species) acquires or loses antibiotic resistance or acquires or loses biosynthetic capacity (e.g., auxotrophic strains), the strain or nutrient / metabolite can be distinguished by selection or anti-selection using antibiotics.

[0133] As used herein, “supernatant” or “supernatant” means the culture supernatant of a bacterial strain according to the present disclosure, optionally containing compounds and / or cell debris of the strain, and / or metabolites and / or molecules secreted by the strain.

[0134] Compositions can be prepared using the *Ackermannius myxotropicus* described herein, for example, by using pharmaceutically acceptable excipients. The pharmaceutical composition comprises a pharmaceutically effective amount of the *Ackermannius myxotropicus*, such as *Ackermannius myxotropicus* with accession number GDMCC NO: 63782. Similarly, *Ackermannius myxotropicus* with accession number GDMCC NO: 63782 can also be prepared as a pharmaceutical composition, for example, by using pharmaceutically acceptable excipients, comprising a pharmaceutically effective amount of the *Ackermannius myxotropicus*.

[0135] Suitable pharmaceutically acceptable excipients include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings.

[0136] The compositions described herein can be formulated in any form suitable for enhancing the abundance of *Akermansia myxophilus* in the body. The compositions can be administered orally (e.g., via oral gavage), intramuscularly, by inhalation, intracranially, intralymphaticly, intraocularly, intraperitoneally, intrapleurally, intrathecally, intratracheally, intrauterinely, intravascularly, intravenously, intravesically, intranasally, intragastrically, intra-gastrointestinally, via bile infusion, via cardiac infusion, via preanal, rectal, subcutaneous, sublingual, local, intravaginal, percutaneously, via the ureter, or via the urethra.

[0137] Examples of dosage forms to which the compositions described herein are applicable include, but are not limited to, tablets, aerosols, chewing sticks, capsules, capsules containing coated granules, capsules containing sustained-release granules, capsules containing sustained-release granules, and concentrates.

[0138] In some embodiments, the composition is a sugar-coated tablet, gel capsule, tablet, sheet capsule, powder, etc.

[0139] In some implementations, administration may also be carried out by including the drug in the subject's diet.

[0140] The compositions provided herein may contain pharmaceutically acceptable excipients, diluents, or carriers. Such pharmaceutically acceptable excipients, diluents, or carriers are well known in the art.

[0141] In some embodiments, the *Ackermannius myxotropicus* in the compositions of this disclosure is lyophilized. In some embodiments, the *Ackermannius myxotropicus* in the compositions of this disclosure is spray-dried. In some embodiments, the *Ackermannius myxotropicus* in the compositions of this disclosure is lyophilized or spray-dried and is viable. In some embodiments, the *Ackermannius myxotropicus* in the compositions of this disclosure is lyophilized or spray-dried and is capable of partially or completely colonizing the intestine. In some embodiments, the lyophilized *Ackermannius myxotropicus* is reconstituted prior to administration. In some embodiments, the reconstitution is performed using the diluents described herein.

[0142] In some embodiments, the compositions of this disclosure are administered orally. Oral administration may involve swallowing, thereby allowing the composition to enter the gastrointestinal tract, and / or administration via the mouth, tongue, or sublingual route.

[0143] In some embodiments, the composition is prepared by freeze-drying or spray-drying.

[0144] The compositions disclosed herein include pharmaceutical compositions.

[0145] The subject of this disclosure may be a human or an animal, including but not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, etc.

[0146] The pharmaceutical compositions disclosed herein can be used to treat, prevent, or alleviate metabolic diseases or diseases caused by metabolic disorders.

[0147] The pharmaceutical compositions disclosed herein can be used to treat, prevent, or alleviate tumors.

[0148] In some implementations, the metabolic diseases, metabolic disorders, or diseases caused by metabolic disorders include, but are not limited to, at least one of the following: liver diseases, obesity and obesity-related diseases, cardiovascular diseases, diabetes, dyslipidemia, cardiovascular and cerebrovascular diseases, 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 steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic diseases, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), atherosclerosis, and kidney disease.

[0149] diabetes

[0150] Diabetes includes type 1 diabetes (T1D), type 2 diabetes (T2D), and gestational diabetes mellitus (GDM). Type 1 diabetes is caused by autoimmune damage or idiopathic factors, characterized by absolute destruction of pancreatic function. It mostly occurs in children and adolescents and requires insulin treatment to achieve satisfactory results; otherwise, it can be life-threatening. Type 2 diabetes is a multifactorial syndrome characterized by abnormal carbohydrate / lipid metabolism, typically including hyperglycemia, hypertension, and abnormal cholesterol. Type 2 diabetes is caused by the ineffective function of insulin (low binding to receptors), therefore, not only fasting blood glucose but also 2-hour postprandial blood glucose should be monitored, and pancreatic function tests should be performed in particular. There are two types of diabetes during pregnancy: one is diabetes diagnosed before pregnancy, called "diabetes mellitus complicated by pregnancy"; the other is diabetes that appears or is diagnosed during pregnancy but occurs during pregnancy, also known as "gestational diabetes mellitus (GDM)," which accounts for more than 80% of diabetes cases in pregnant women.

[0151] Four types of metabolic disease-related models—high-fat diet (HFD) induced mouse obesity model, high-fat, high-glucose, and high-cholesterol induced mouse NASH model, high-fat diet combined with streptozotocin (HFD-STZ) induced mouse type II diabetes model, and leptin receptor gene defective mouse model (db / db)—are commonly used mouse models of metabolic diseases. These model mice are usually accompanied by metabolic diseases such as obesity, insulin resistance, hyperglycemia, hyperlipidemia, hypercholesterolemia, and NAFLD / NASH.

[0152] Insulin resistance refers to a decrease in the efficiency of insulin in promoting glucose uptake and utilization due to various reasons. The body compensates by secreting excessive insulin, resulting in hyperinsulinemia to maintain stable blood glucose levels. Insulin resistance can easily lead to metabolic syndrome and type II diabetes.

[0153] Oral glucose tolerance test (OGTT) is used to measure pancreatic β-cell function and the body's ability to regulate blood glucose. It is currently a recognized diagnostic indicator for diabetes. When glucose metabolism is disordered, blood glucose levels rise sharply after ingesting a certain amount of glucose, or the rise is not significant but cannot be reduced to fasting levels or the original level within a short period of time. This is called impaired glucose tolerance or decreased glucose tolerance. Impaired glucose tolerance indicates a decreased ability of the body to metabolize glucose and is commonly seen in type II diabetes and obesity.

[0154] HOMA-IR is an indicator used to assess an individual's insulin resistance level and is now widely used in clinical practice to evaluate insulin sensitivity in diabetic patients. It is calculated as: fasting plasma glucose level (FPG, mmol / L) × fasting insulin level (FINS, μU / mL) / 22.5. The HOMA-IR index for normal individuals is 1. As insulin resistance increases, the HOMA-IR index will be higher than 1.

[0155] L cells in the gut can secrete glucagon-like peptide-1 (GLP-1), which can promote insulin production by pancreatic β cells and inhibit glucagon production by pancreatic α cells, thereby regulating the body's blood glucose balance and improving the body's glucose tolerance.

[0156] Liver dysfunction

[0157] Liver dysfunction refers to abnormal or damaged liver function. Alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) are sensitive markers of liver dysfunction. In cases of abnormal liver function (e.g., liver injury, non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH)), blood ALT and / or AST levels are significantly elevated. Generally, ALT is more sensitive than AST in reflecting acute liver injury. Persistently elevated ALT suggests chronic liver injury. In cases of chronic hepatitis, cirrhosis, and liver cancer, AST levels are significantly elevated, sometimes exceeding ALT levels. AST levels indicate the chronicity, extent, and severity of liver disease, and may even suggest the prognosis of chronic liver disease.

[0158] Common liver diseases that cause 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); non-alcoholic fatty liver disease (NAFLD or NASH); drug-induced / toxic liver injury; cirrhosis; liver cancer; Wilson's disease; alpha-1 antitrypsin deficiency; hemochromatosis, etc.

[0159] In addition, fat accumulation in the liver is an important factor in the development of non-alcoholic fatty liver disease (NAFLD or NASH). Therefore, when ALT and / or AST levels decrease and liver weight is reduced after drug intervention, it can be seen that the drug has a certain therapeutic effect.

[0160] Nonalcoholic fatty liver disease (NAFLD) refers to the excessive accumulation of fat in the liver as triglycerides (TG) (steatodegeneration). Some NAFLD patients also have hepatocellular damage and inflammation in addition to excessive fat (steatohepatitis), i.e., nonalcoholic steatohepatitis (NASH). NASH is widely considered a liver manifestation of metabolic syndromes such as type 2 diabetes, insulin resistance, central obesity, hyperlipidemia (low high-density lipoprotein cholesterol, high triglycerides), and hypertension.

[0161] Liver and kidney dysfunction

[0162] Liver and kidney dysfunction refers to functional acute renal failure that occurs in severe liver disease. In decompensated cirrhosis, liver and kidney syndrome can occur due to insufficient effective circulating blood volume, reduced prostaglandins, and other reasons.

[0163] Cardiovascular disease

[0164] Triglycerides (TG) primarily participate in energy metabolism in the human body, generating heat. High TG levels in the blood can lead to increased blood viscosity, causing lipids to deposit on the blood vessel walls, gradually forming small plaques, i.e., atherosclerosis. Elevated LDL-C is a major, independent risk factor for the occurrence and development of atherosclerosis; elevated LDL-C levels are also an indicator of coronary heart disease. Because HDL-C can transport cholesterol from the blood vessel walls to the liver for breakdown and metabolism (i.e., reverse cholesterol transport), it can reduce cholesterol deposition on the blood vessel walls, thus playing an anti-atherosclerotic role.

[0165] inflammation

[0166] Lipopolysaccharide (LPS), also known as an endotoxin, is a phospholipid that forms the outer cell wall of Gram-negative bacteria. Besides ensuring the integrity of bacterial structure, LPS also protects these bacteria from being broken down by bile salts secreted by the gallbladder. Normally, LPS is blocked from the bloodstream by the tight junctions of intestinal wall cells. If LPS enters the bloodstream, it can induce a strong inflammatory response in an animal. Therefore, the level of LPS in the blood can reflect the level of inflammation.

[0167] obesity

[0168] Obesity refers to a significant degree of overweight and excessive fat accumulation, a state caused by excessive accumulation of body fat, especially triglycerides. It is an abnormal or excessive accumulation of fat that poses a risk to health. Excessive food intake or changes in metabolism lead to excessive fat accumulation, resulting in excessive weight gain and causing or inducing pathological and physiological changes in the body. A body mass index (BMI) over 25 is considered overweight, and over 30 is considered obese. Obesity increases the risk of many physical and mental illnesses. It is primarily associated with a combination of metabolic syndrome, including type 2 diabetes, hypertension, hypercholesterolemia, and hypertriglyceridemia. In general, the health effects of obesity fall into two main categories: diseases attributable to increased body fat (such as osteoarthritis, obstructive sleep apnea, etc.) and diseases with an increased number of fat cells (such as diabetes, dyslipidemia, cancer, cardiovascular disease, non-alcoholic fatty liver disease, or non-alcoholic steatohepatitis, etc.). The term "obesity-related diseases" can be selected from the following diseases: overeating, binge eating, bulimia, hypertension, diabetes, elevated plasma insulin levels, 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, lipid metabolism disorders, non-alcoholic steatohepatitis, cardiovascular disease, and polycystic ovary syndrome, as well as those with these obesity-related diseases who wish to lose weight.

[0169] Obesity-related diseases in this disclosure include at least one of the following: obesity, metabolic syndrome, cardiovascular disease, hyperlipidemia, hypercholesterolemia, hypertension, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, and steatohepatitis.

[0170] Interferon (IFN) receptor proteins are a class of cytokines secreted by host cells that regulate immune responses. Viruses, bacterial endotoxins, and synthetically produced double-stranded RNA can stimulate interferon production. Macrophages, lymphocytes, and somatic cells in the human body can all produce interferon. Among them, IFNβ belongs to type I interferon and can promote the activity of NK cells, macrophages, and T lymphocytes, thereby exerting antiviral, antitumor, and immunomodulatory effects.

[0171] The *Akkermansia muciniphila* strain or its products provided in this invention can significantly promote IFNβ transcriptional activity. Type I interferon IFNβ has been shown to modulate immunity and reconstruct the synergistic effect of innate and acquired immunity in the tumor microenvironment for the treatment of refractory drug-resistant cancers (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 indicate that strain MNH19250 or the *Akkermansia muciniphila* strain or its products provided in this invention can modulate immunity and achieve antitumor effects through immunomodulation, while also having potential functional roles in antiviral tumors.

[0172] Short-chain fatty acids (SCFAs) are important metabolites of gut microbiota, influencing a range of host activities as signaling molecules, primarily in the form of acetate, propionate, and butyrate. SCFAs can lower intestinal pH, inhibiting pathogen growth. They can activate target pathways such as GPR41, GPR43, GPR109A, and GPCR81, improving the integrity and function of colonic epithelial cells, enhancing intestinal barrier function, promoting the secretion of hormones such as GLP-1 and PYY from intestinal endocrine cells, increasing insulin sensitivity, increasing energy expenditure, promoting lipolysis, inhibiting the production of pro-inflammatory cytokines, and maintaining intestinal immune homeostasis. SCFAs have certain beneficial effects on metabolic diseases such as obesity, diabetes, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis, as well as ulcerative colitis, radiation proctitis, and Crohn's disease.

[0173] Propionic acid and butyric acid can be used as HDAC inhibitors. By inhibiting HDAC, they can alter the expression of various functional genes, regulate cell proliferation, apoptosis, and differentiation, thereby enhancing immunity while preventing the occurrence and development of inflammation.

[0174] Amuc-1100 and Amuc_1631(P9) proteins are outer membrane proteins derived from Akkermansia muciniphila. Amuc-1100 protein can interact with Toll-like receptor 2 (TLR2) in host cells, affecting intestinal health and immune regulation (Wang J, Xu W, Wang R, et al., The outer membrane protein Amuc_1100 of Akkermansia muciniphilapromotes intestinal 5-HT biosynthesis and extracellular availability through TLR2 signalling.[J].Food&function,2021,12(8):3597-3610.DOI:10.1039 / d1fo00115a.). Amuc-1100 promotes GLP-1 secretion, which helps improve glycemic control and metabolic disorders; "YOON HS,CHO CH,YUN MS,et al.,Akkermansiamuciniphila secretes a glucagon-like peptide-1-inducing protein that improves glucose homeostasis and ameliorates metabolic disease in mice[J].Nature Microbio-logy,2021,6:563-573"; Amuc-1100 remains stable during pasteurization and can inhibit the expression of CB1 receptor in the intestine and enhance the expression of tight junction proteins; "Ding,G.,Yang,X.,Li,Y.et al.,Gutmicrobiota regulates gut homeostasis,mucosal immunity and influences immune-related diseases.Mol Cell Biochem(2024). https: / / doi.org / 10.1007 / s11010-024- 05077-y ".

[0175] The Amuc_1631(P9) protein can promote GLP-1 secretion from 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.). The P9 protein also exhibits a strong anti-inflammatory effect and can regulate intestinal barrier function, thereby playing a role in inflammatory bowel disease. The P9 protein enhances the function of the intestinal barrier by increasing the number of goblet cells, mucus production, and the 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)".

[0176] Therefore, the Akkermansia muciniphila involved in this invention can affect or regulate immune signal transduction and / or affect intestinal barrier function and / or affect glucose homeostasis and / or cholesterol homeostasis and / or triglyceride homeostasis.

[0177] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are merely illustrative and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature or the product / instrument instruction manual. All reagents or instruments whose manufacturers are specified are commercially available.

[0178] Example

[0179] The liquid MM01 culture medium involved in the examples has the following composition: 5 g / L peptone, 5 g / L trypsin, 10 g / L yeast extract, 5 g / L beef extract, 5 g / L glucose, 2 g / L K2HPO4, 2 g / L sodium acetate, 1 mL / L Tween 80, 5 mg / L heme, 0.5 g / L L-cysteine ​​hydrochloride, 1 μL / L vitamin K1, and 8 mL / L inorganic salt solution (each 1 L includes 0.25 g calcium chloride, 1 g K2HPO4, 1 g KH2PO4, 0.5 g magnesium sulfate, 10 g sodium bicarbonate, and 2 g sodium chloride).

[0180] The solid MM01 culture medium involved in the examples has the following composition: 5 g / L peptone, 5 g / L trypsin, 10 g / L yeast extract, 5 g / L beef extract, 5 g / L glucose, 2 g / L K2HPO4, 2 g / L sodium acetate, 1 mL / L Tween 80, 5 mg / L heme, 0.5 g / L L-cysteine ​​hydrochloride, 1 μL / L vitamin K1, 8 mL / L inorganic salt solution (each 1 L includes 0.25 g calcium chloride, 1 g K2HPO4, 1 g KH2PO4, 0.5 g magnesium sulfate, 10 g sodium bicarbonate, and 2 g sodium chloride), and 15 g / L agar.

[0181] Anaerobic blood agar plates were purchased from Huankai Microbiology. The formula was as follows: 10 g / L casein pancreatic enzyme digest, 3 g / L cardiac pancreatic enzyme digest, 1 g / L corn starch, 5 g / L vesicular enzyme digest, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar, and 50–100 mL / L sterile defibrinated sheep blood. The pH was 7.3 ± 0.2.

[0182] The above-mentioned culture medium can be prepared using conventional preparation methods and sterilization methods.

[0183] Example 1. Isolation and Identification of Strains

[0184] 1.1 Isolation and purification of strain MNH19250

[0185] An intestinal strain, designated MNH19250, was isolated from a sample of a healthy male volunteer in Guangzhou, Guangdong Province, China. The isolation method employed standard procedures, including serial dilution, followed by single-colony isolation and culture. The strain was purified and anaerobically cultured at 37°C. The purified culture was then prepared into a 20% glycerol / water suspension and stored at -80°C.

[0186] Specifically, the method for isolating the strain is as follows:

[0187] Donors collect 5g of sample, place it in a sample collection and preservation tube, shake to homogenize, place the processed sample in an ice box, and deliver it to the laboratory within 24 hours for bacterial isolation.

[0188] Dispense physiological saline into tubes of 9 mL each in a biosafety cabinet; prepare anaerobic blood agar plates for bacterial isolation, and transfer them to the anaerobic workstation 24 hours in advance, labeling them with sample information, culture medium type, isolation date, etc.

[0189] Take a fresh sample and place it in an anaerobic workstation. Vortex the sample for 1 minute to mix thoroughly. Transfer 1 mL of the sample to 9 mL of physiological saline and mix well to a final volume of 10 mL. -1 Diluent, then serially diluted to 10. -6 Diluent, for later use.

[0190] Take 10 -6 The diluent was dropped into anaerobic blood agar plates at a rate of 100 μL / plate. The plates were spread evenly and allowed to dry on the surface. The plates were then inverted and incubated at 37°C for 3–5 days.

[0191] Observe the growth of the strains on the isolation medium (anaerobic blood agar plates) and pick single colonies with sterile toothpicks for strain purification. The purified strains are then cultured anaerobically at 37°C. The pure culture strains are prepared into 20% glycerol / water-bacterial suspensions and stored at -80°C.

[0192] 1.2 Morphological characteristics of strain MNH19250

[0193] Cultivation and morphological characteristics

[0194] Strain MNH19250 was inoculated onto MM01 medium and anaerobically cultured at 37°C for 72 hours. Visible colonies formed on MM01 plates. The colonies were round, with regular and smooth edges, approximately 0.5 mm in diameter, pale yellow, and translucent. The strain was Gram-negative. Microscopic morphology showed no flagella, was non-motile, rod-shaped, and approximately 0.5–1 μm × 1.5–3 μm in size. See the photograph of the colony morphology of strain MNH19250 after 72 hours of culture on MM01 plates. Figure 1 See the Gram staining photograph of strain MNH19250. Figure 2 See electron microscope images. Figure 3 .

[0195] 1.3 Physiological characteristics of strain MNH19250

[0196] Strain MNH19250 does not grow under aerobic conditions, but only under anaerobic conditions. It can grow in a pH range of 6.0–9.0, with the optimal growth pH around 8.0 (see [link to strain's pH tolerance results]). Figure 4 Growth was significantly inhibited on media containing more than 2% (w / v) NaCl (see results for strain tolerance to different NaCl concentrations). Figure 5Strain MNH19250 can survive and grow in a bile salt concentration range of 0%–0.25% (w / v), but its growth is significantly inhibited at bile salt concentrations greater than or equal to 0.3% (see [link to strain's tolerance to different bile salt concentrations] for results). Figure 6 ).

[0197] 1.4 Results of biochemical identification of strain MNH19250 using API 20A

[0198] Biochemical identification of strain MNH19250 was performed using API 20A (purchased from bioMérieux, CN2030025). Specific experimental procedures were performed according to standard API reagent handling guidelines. The culture conditions for strain MNH19250 were 37°C, anaerobic. Experimental results are shown in Table 1.

[0199] Table 1. API 20A test results for strain MNH19250

[0200]

[0201] MNH19250 can ferment glucose, lactose, and mannose to produce acid. Therefore, glucose, lactose, mannose, and their derivatives can serve as carbon sources during the fermentation or cultivation of strain MNH19250.

[0202] MNH19250 can hydrolyze aesculin (ESC), thus synthesizing β-glucosidase. β-glucosidase belongs to the cellulase class and can hydrolyze cellobiose and short-chain cellooligosaccharides to produce glucose.

[0203] MNH19250 can hydrolyze gelatin (GEL), which means it can synthesize proteases. The function of proteases is to convert non-absorbable proteins into polypeptides or amino acids that can penetrate the bacterial cell. These enzymes are mainly extracellular enzymes, which can first hydrolyze gelatin into polypeptides, and then further hydrolyze it into amino acids, causing it to lose its gel properties and liquefy, allowing the black substance in the reaction well to diffuse, thus showing a positive reaction.

[0204] 1.5 Minimum inhibitory concentration test of antibiotics for strain MNH19250

[0205] The minimum inhibitory concentration of antibiotics for strain MNH19250 was determined using E-test (purchased from Liofilchem) discs, and the test results are shown in Table 2.

[0206] Table 2. Results of antibiotic minimum inhibitory concentration tests for strain MNH19250

[0207]

[0208] The results showed that MNH19250 was sensitive to ampicillin, chloramphenicol, clindamycin, amoxicillin, rifampin, imipenem, penicillin, and cefquinoxime. This indicates that MNH19250 is sensitive to most types of antibiotics, and the risk of developing antibiotic resistance with long-term use of MNH19250 is low.

[0209] 1.6 Determination of the autoaggregation ability of strain MNH19250

[0210] Strain MNH19250 was inoculated into MM01 liquid medium and cultured anaerobically at 37°C for 48 hours. 20 mL of the fermentation broth was taken, centrifuged (4500 rpm, 4°C, 10 min) to collect the cells, washed twice with sterile PBS (pH 7.2), resuspended in PBS buffer, and the OD600 value was adjusted to 0.5, denoted as A0.

[0211] The bacterial culture was placed at 37°C (directly in a cuvette) and its OD600 value was measured every 30 minutes and recorded as At. The measurement was repeated for 6 hours, with three replicates.

[0212] The autoaggregation ability of the strain is calculated using the following formula: Autoaggregation ability of strain (%) = [1 - (At / A0)] × 100%.

[0213] The 6-hour autoaggregation efficiency of MNH19250 is 54.21% (see [reference]). Figure 7 Compared with other strains in the literature, its autoaggregation ability is more than 20% stronger (Xin Ma 1, Meng Tian 2, Xueping Yu, et al., Foods. 2024, Jan 30; 13(3):442. Characterization and Preliminary Safety Evaluation of Akkermansiamuciniphila PROBIO). https: / / doi.org / 10.3390 / foods13030442 Figure 1d shows that the autoaggregation ability of A. muciniphila PROBIO is about 30% (8h). Strains with strong autoaggregation ability have a strong ability to adhere to epithelial cells and can better colonize the human intestine, thus exerting a probiotic effect.

[0214] 1.7 Surface hydrophobicity determination of strain MNH19250

[0215] Strain MNH19250 was inoculated into MM01 liquid medium and cultured anaerobically at 37°C for 48 hours. 20 mL of fermentation broth was taken, centrifuged (4500 rpm, 4°C, 10 min) to collect the cells, washed twice with sterile PBS (pH 7.2), resuspended in PBS buffer, and the OD600 value was adjusted to 0.5, denoted as A0.

[0216] Add 4 mL of xylene to 4 mL of the resuspension, vortex the two-phase system for 5 min, incubate at room temperature for 1 h, then carefully remove the xylene phase. Measure the absorbance (A) of the aqueous phase at 600 nm, with three replicates. Calculate the surface hydrophobicity of the test strain using the following formula:

[0217] H%=[(A0-A) / A0]×100, where A0 and A represent the absorbance values ​​before and after organic solvent extraction, respectively.

[0218] The surface hydrophobicity of strain MNH19250 was 57.06%, which is more than 25% 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 1c, A. muciniphila PROBIO has a surface hydrophobicity of 31% (1h)). Strains with strong surface hydrophobicity have a strong ability to adhere to epithelial cells, which can better colonize the human intestine and thus exert a probiotic effect. At the same time, they can inhibit the adhesion of other pathogens and play a role in intestinal probiotic function.

[0219] 1.8 Amplification of the 16S rRNA gene in strain MNH19250

[0220] Fresh culture of strain MNH19250 was used to extract genomic DNA. The extracted genomic DNA was then used as a template for 16S rRNA gene amplification.

[0221] The primer pair used for PCR of the 16S rRNA gene is:

[0222] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID No. 2)

[0223] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID No. 3).

[0224] The PCR reaction procedure is as follows:

[0225] Pre-denaturation: 94℃, 4 min; denaturation: 94℃, 50 sec; annealing: 52℃, 40 sec; extension: 72℃, 70 sec; final extension: 72℃, 10 min (36 cycles).

[0226] 1.916S rRNA gene sequencing

[0227] The PCR product was purified and the 16S rRNA gene was sequenced by Sangon Biotech, yielding the 16S rRNA gene sequence (1355 bp), as shown in SEQ ID No. 1.

[0228] 1.10 Identification of strain MNH19250

[0229] The 16S rRNA gene sequence obtained above, as shown in SEQ ID No. 1, was analyzed using the NCBI Basic Local Alignment Search Tool to confirm the strain's classification information.

[0230] The sequence obtained was analyzed with data in GenBank using BLAST. The comparison results showed that the strain with the highest similarity to MNH19250 was Akkermansia muciniphila, with a similarity of 100%. Therefore, strain MNH19250 was determined to be a strain of Akkermansia muciniphila.

[0231] A phylogenetic tree was constructed by comparing the 16S rRNA gene sequences of strain MNH19250 with those of related Akkermansia sp. strains retrieved from databases such as GenBank.

[0232] The 16S rRNA gene sequence of strain MNH19250 was compared with the sequences of type strains with high 16S rRNA gene sequence similarity in the NCBI database using multiple sequence alignment. A phylogenetic tree was then constructed using MEGA5 software (the phylogenetic tree was constructed using the maximum likelihood method) (see [link to documentation]). Figure 8 ), Figure 8 The growth tree nodes only display values ​​where the Bootstrap value is greater than 50%.

[0233] The phylogenetic tree shows that strain MNH19250 clusters with Akkermansia sp. and Akkermansia muciniphila Muc AY271254. Therefore, strain MNH19250 is identified as a new strain of Akkermansia muciniphila.

[0234] 1.11 Genome analysis of strain MNH19250

[0235] Genome analysis of strain MNH19250

[0236] The genome of strain MNH19250 was fragmented using ultrasound, and then an Illumina sequencing library was constructed using a standard DNA library preparation kit (NEB Ultra™). The constructed sequencing library was then sequenced at 150 bp paired ends using NovaSeq (Illumina). Sequencing yielded 3.01 Gbp of data, with Q20 accounting for 97.42%.

[0237] The raw genome sequencing data was filtered using FastP (version 0.20.0). The filtered raw data was then assembled using SPAdes (version v3.14.0). The assembled genome yielded a total gene length of 2.82 Mbp, an N50 length of 378.4 kbp, and a GC content of 55.19%.

[0238] Genomic gene prediction analysis was performed using the prokaryotic analysis software genome annotation workflow Prokka (version 1.14.5). A total of 2376 CDS sequences were predicted, with an average CDS sequence length of 1039 bp.

[0239] Potential antibiotic resistance genes in the genome were analyzed using RGI (version 4.2.2), with the antibiotic resistance gene database being CARD (version 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). Detailed comparison information is shown in Table 3.

[0240] Table 3. List of drug resistance gene information

[0241] strain genes resistance gene Gene name Comparison consistency (%) MNH19250_02133 ARO:3000777 adeF 41.42

[0242] Analysis of potential virulence factors and related genes in the genome was performed using NCBI blastp (version 2.7.1+) to align with the virulence factor database VFDB (http: / / www.mgc.ac.cn / cgibin / VFs / v5 / main.cgi, updated September 19, 2019). Detailed alignment results are shown in Table 4.

[0243] Table 4. List of Potential Toxicity Genes of MNH-19250

[0244] strain genes VFDB gene Gene name Comparison consistency (%) MNH19250_00898 VFG001855 htpB 61.932 MNH19250_02218 VFG002225 gmd 65.607

[0245] The analysis of potential secondary metabolic gene clusters in the genome was performed using antiSMASH6 (version 6.0.1). Detailed alignment results are shown in Table 5.

[0246] Table 5. List of potential secondary metabolic gene clusters of MNH-19250

[0247] Gene cluster range type from arrive Most similar known gene cluster Similarity Region2.1 terpene 295638 316573 - - Region 2.2 arylpolyene 378291 419466 herboxidiene 3％ Region 3.1 terpene 99023 119922 - -

[0248] The analysis of potential primary metabolic gene clusters in the genome was performed using gutSMASH5 (version 1.0.0). Detailed alignment results are shown in Table 6.

[0249] Table 6. List of potential primary metabolic gene clusters in MNH-19250

[0250]

[0251]

[0252] Analysis of the Amuc_1100 and P9 protein coding genes of strain MNH19250. The Amuc_1100 protein sequence (WP_197738471) and the Amuc_1631(P9) protein sequence (ACD05451) were compared using BLAST.

[0253] Table 7. Analysis of protein-coding genes of MNH19250 Amuc_1100

[0254] qseqid sseqid pident qcovs MNH19250_01469 WP_197738471 99.369 100

[0255] sequence:

[0256] >MNH19250_01469hypothetical protein

[0257] MSNWITDNKPAAMVAGVGLLLFLGLSATGYIVNSKRSELDKKISIAAKEIKSANAAEITPSRSSNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILITDTSSWLGFQVYSTQAPSVQAASTLGFELKAINSLVNKLAECGLSKFIKVYRPQLPIETPANNPEESDEADQAPWTPMPLEIAFQGDRESVLKAMNAITGMQDYLFTVNSIRIRNERMMPPPIANPAAAKPAAAQPATGAASLTPADEATAPAAPAIQQVIKPYMGKEQVFVQVSLNLVHFNQPKAQEPSED(SEQ ID NO.4)

[0258] Table 8. Analysis of the gene encoding the P9 protein of MNH19250

[0259] qseqid sseqid pident qcovs MNH19250_00667 ACD05451 99.865 100

[0260] Sequence:

[0261] >MNH19250_00667Tail-specific protease

[0262] (SEQ ID NO.5)

[0263] Example 2. Fatty acid composition analysis of strain MNH19250

[0264] 2.1 Fatty acid composition analysis of strain MNH19250

[0265] Strain MNH19250 was inoculated on MM01 plates and anaerobically cultured at 37°C for 72 hours. The bacterial cells were then collected for fatty acid extraction and methylation. The fatty acid composition of strain MNH19250 was analyzed using the fully automated bacterial identification system from MIDI (Microbial ID, Inc., Newark, Del).

[0266] The major fatty acid (>10%) of the experimental strain MNH19250 was C15:Oanteiso 48.73%. 2.2 Analysis of short-chain fatty acids (SCFA) of strain MNH19250.

[0267] Bacterial cell preparation

[0268] Strain MNH19250 was inoculated into 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 for later use.

[0269] Pretreatment of bacterial supernatant samples

[0270] (1) After the sample is thawed, vortex for 3 minutes to mix it.

[0271] (2) Transfer 50 μL of sample into a 1.5 mL centrifuge tube, add 100 mg of phosphoric acid solution (0.5%, v / v), and vortex at 2500 r / min for 3 min;

[0272] (3) Add 750 n of MTBE extractant containing internal standard, vortex at 2500 r / min for 3 min, sonicate at 4 m for 5 min, and centrifuge at 4 m and 12000 r / min for 10 min.

[0273] (4) Aspirate 200 μL of supernatant into the inner liner of the sample vial and store it in a -20°C freezer for GC-MS / MS analysis.

[0274] bacterial sample pretreatment

[0275] (1) After the sample is thawed, add 100 ml of frozen ultrapure water extract to resuspend it evenly;

[0276] (2) Transfer 50 μL of bacterial suspension sample into a 1.5 mL centrifuge tube, add 100 g of phosphate solution (0.5%, v / v), and vortex for 3 min to mix.

[0277] (3) Immerse in liquid nitrogen for 2 minutes, remove and thaw completely on ice, vortex at 2500 r / min for 3 minutes, and repeat 3 times;

[0278] (4) Add 150 nr of MTBE extractant containing internal standard, vortex at 2500 r / min for 3 min, sonicate at 4 m for 5 min, and centrifuge at 4 m and 12000 r / min for 10 min.

[0279] (5) Aspirate 90% of the supernatant into the inner liner of the injection vial for GC-MS / MS analysis.

[0280] (6) The remaining 50M bacterial suspension was repeatedly frozen and thawed in liquid nitrogen 3 times, centrifuged at 12000r / min for 10min, and the supernatant was taken to determine the protein concentration by BCA method.

[0281] Standard preparation: separately Standard solutions of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid were prepared at concentrations of 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, and 20 μg / mL. Chromatographic and mass spectrometric detection was performed using the conditions shown in Table 9.

[0282] Obtain the chromatographic peak intensity data of the corresponding quantitative signals for each concentration of standard. Plot standard curves for different substances with the external standard to internal standard concentration ratio (Concentration Ratio) as the x-axis and the external standard to internal standard peak area ratio (Area Ratio) as the y-axis.

[0283] Table 9. Main conditions for chromatography-mass spectrometry

[0284]

[0285] The ratio of the integrated peak area of ​​all detected samples was substituted into the linear equation of the standard curve for calculation. After being substituted into the sample calculation formula, the content data of the substance in the actual sample was finally obtained. The results showed that the supernatant of the bacterial solution and the bacterial cells contained short-chain fatty acids acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. The contents of acetic acid and propionic acid are shown in Table 10.

[0286] Table 10. Detection of short-chain and medium-chain fatty acid content in MNH19250 bacterial supernatant and bacterial cells

[0287]

[0288] The test results show that strain MNH19250 can synthesize short-chain fatty acids during its growth, including large amounts of acetic acid and propionic acid.

[0289] Example 3. Effect of strain MNH19250 on IFNβ expression

[0290] To verify whether MNH19250 can promote IFNβ expression, this study used a constructed THP-1 cell line carrying the IFNβ gene promoter reporter gene (THP-1-IFNβ-promoter reporter cell, a cell line built by Muen Company) to evaluate the effect of MNH19250 on IFNβ transcriptional activity.

[0291] 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 cell lines expressing the reporter gene (see the following references for details: Huashan Du, TianminXu, 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 IIFN-dependent monocyte reprogramming of the tumor microenvironment” Cell 184,5338-5356).

[0292] Preparation of culture supernatant of strain MNH19250: strain MNH19250 was inoculated into MM01 liquid medium and cultured anaerobically at 37℃ for 48 hours. The bacterial cells were removed by centrifugation. The culture supernatant was filtered through a 0.22μm filter, dispensed, and the collected material was stored at -80℃ for later use.

[0293] Control group: DMEM complete medium (Gibco, containing 10% FBS) containing 10% volume of MM01 liquid medium;

[0294] MSA-2 group (positive control group): DMEM complete medium containing 10 μM MSA-2 (purchased from Taoshu Biotechnology);

[0295] Group MNH19250: DMEM complete medium containing 10% volume of culture supernatant of strain MNH19250.

[0296] THP-1-IFNβ-promoter reporter cells were seeded in 96-well plates at 1 × 10⁶ cells per well. 5 Cells were collected and treated according to the established groups. After culturing for another 24 hours, the cells were centrifuged at 300g for 5 minutes, the culture supernatant was removed, and 50μL of 1×Luminescence (Promega) was added to normalize the cells to the control group. This was used to evaluate the effect of strain MNH19250 on IFNβ transcriptional activity.

[0297] Experimental results are as follows Figure 9 As shown, strain MNH19250 significantly promotes the transcriptional activity of IFNβ. Type I interferon IFNβ has been shown to regulate immunity and exert anti-tumor effects through immunomodulation, while also possessing potential functional roles against viral tumors. Therefore, these results indicate that MNH19250 and its metabolites can regulate the body's immunity and exert anti-tumor effects through immunomodulation, while also having potential preventive and inhibitory effects against sexually transmitted tumors.

[0298] 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.

[0299] This study investigated the effects of strain MNH19250 on liver and kidney function and related diseases using a mouse model of liver and kidney injury caused by a high-fat diet. The experimental protocol has been approved by the Muen Biotechnology Laboratory Animal Management and Use Committee's ethical review.

[0300] 4.1 Experimental Methods

[0301] 1) Experimental animals: The mice used in the experiment were C57BL / 6J mice, 5-6 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0302] 2) Preparation of test samples from strain MNH19250: The glycerol cryovials of strain MNH19250 were thawed at 37°C and activated by inoculation onto MM01 plates in an anaerobic workstation. The activated strain was then inoculated into MM01 liquid medium and cultured anaerobically to obtain a sufficient quantity of culture. The cultured bacterial solution was concentrated by centrifugation and resuspended in PBS containing 0.05% cysteine ​​hydrochloride (L-Cys HCl) to obtain purity and viable count (2 × 10⁻⁶). 9 Test substances (CFU / mL) that meet the requirements for animal experiments.

[0303] 3) Negative control: PBS containing 0.05% L-Cys HCl was used as the negative control.

[0304] 4) Experimental Procedure: Male C57BL / 6J mice aged 5-6 weeks were fed a high-fat diet for 10 weeks after the quarantine period. Twelve mice weighing between 35.50g and 44.49g were randomly stratified and divided into two groups (experimental group and control group) of 6 mice per group. Drug administration began on day 1 (D1). The experimental group received the test substance, while the control group received a negative control. Drug administration was twice daily for 28 days. Mice had free access to water and food during the experiment, following a 12h / 12h diurnal cycle. A general clinical observation was conducted after each administration. The endpoint of the experiment was the day after the end of administration (D29). Dissection and tissue sampling were performed according to the protocol, and the data from each dissection and serum testing were analyzed. All data are expressed as Mean ± SD and plotted and statistically analyzed using GraphPadPrism software. Student's t-test was used for pairwise comparisons. Significance is indicated by *, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0305] See Figure 10 This showed that strain MNH19250 could significantly reduce liver function indicators. Specifically, Figure 10 The results showed that strain MNH19250 could significantly reduce serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), thus strain MNH19250 can improve liver function abnormalities caused by a high-fat diet and has the activity of treating or preventing liver damage.

[0306] See Figure 11 According to the American Association for the Study of Liver Diseases (NAS) scoring system (2005), strain MNH19250 showed activity in treating or preventing liver dysfunction, fatty liver, NAFLD / NASH. Specifically, Figure 11 The results showed that strain MNH19250 significantly improved non-alcoholic fatty liver disease (NAFLD / NASH): strain MNH19250 significantly reduced liver weight and the ratio of liver weight to body weight, significantly reduced hepatic steatosis and lobular inflammation, significantly alleviated hepatocellular ballooning degeneration, and significantly reduced the non-alcoholic fatty liver disease activity score (NAS). Therefore, strain MNH19250 can significantly improve non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, significantly improve hepatocellular damage, and has therapeutic or preventive activity against hepatocellular damage and NAFLD / NASH.

[0307] See Figure 12The results showed that strain MNH19250 significantly reduced serum BUN (blood urea nitrogen) and serum creatinine (CRE). Therefore, strain MNH19250 can significantly improve renal function abnormalities and has therapeutic or preventive activity against renal injury and renal function abnormalities.

[0308] Example 5: Use of strain MNH19250 for the prevention or treatment of diabetes

[0309] 5.1 Experimental Methods

[0310] 1) Experimental animals: The mice used in the experiment were C57BL / 6J mice, 5-6 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0311] 2) Preparation of test samples from strain MNH19250: The glycerol cryovials of strain MNH19250 were thawed at 37°C and activated by inoculation onto MM01 plates in an anaerobic workstation. The activated strain was then inoculated into MM01 liquid medium and cultured anaerobically to obtain a sufficient quantity of culture. The cultured bacterial solution was concentrated by centrifugation and resuspended in PBS containing 0.05% L-CysHCl to obtain purity and viable count (2 × 10⁻⁶). 9 Test substances (CFU / mL) that meet the requirements for animal experiments.

[0312] 3) Negative control: PBS containing 0.05% L-Cys HCl was used as the negative control.

[0313] 4) Experimental Procedure: Male C57BL / 6J mice aged 5-6 weeks were fed a high-fat diet for 10 weeks after the quarantine period. Twelve mice weighing between 35.50g and 44.49g were randomly stratified and divided into two groups of 6 mice each (experimental group and control group). Drug administration began on day 1 (D1). The experimental group received the test substance from strain MNH19250, while the control group received the negative control. Drug administration was twice daily for 28 days. The endpoint of the experiment was the day after the end of drug administration (D29). During the experiment, mice had free access to water and food, using a 12h / 12h diurnal cycle. General clinical observation was performed after each drug administration period. An oral glucose tolerance test (OGTT) was measured once during the last week of drug administration, and fasting blood glucose was measured at the endpoint (D29). All data are expressed as Mean ± SD and were plotted and statistically analyzed using GraphPadPrism software. Student's t-test was used for pairwise comparisons. Significance is indicated by *, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0314] 5.2 Experiment on oral glucose tolerance of strain MNH19250 in type 2 diabetic mice induced by a high-fat diet:

[0315] Oral glucose tolerance test (OGTT): During the last week of drug administration as described above, the OGTT was measured after a 12-hour fast (fasting from 20:30:00 to 08:30 the next day). The mice were weighed after fasting, and glucose was administered by gavage at a dose of 2 g / kg (glucose g / mouse fasting body weight kg). Fasting blood glucose was measured, and blood glucose levels were measured at 15 min, 30 min, 60 min, 90 min, and 120 min after glucose administration. Blood glucose levels were measured accurately at all six time points for each mouse, with strict timing.

[0316] 5.3 Results of the experiment on the effect of strain MNH19250 on oral glucose tolerance in type 2 diabetic mice induced by a high-fat diet:

[0317] See Figure 13 It can be seen that strain MNH19250 can significantly reduce oral glucose tolerance in mice with type 2 diabetes induced by a high-fat diet. Specifically, Figure 13 The results showed that strain MNH19250 could significantly inhibit the rise in blood glucose after glucose administration in type 2 diabetic mice, significantly improve oral glucose tolerance, and significantly reduce the area under the oral glucose tolerance curve in type 2 diabetic mice, indicating that strain MNH19250 can significantly control blood glucose and significantly improve glucose homeostasis in type 2 diabetic mice.

[0318] 5.4 Results of the experiment on the effect of strain MNH19250 on fasting blood glucose in type 2 diabetic mice induced by a high-fat diet:

[0319] See Figure 14 It can be seen that strain MNH19250 can significantly reduce fasting blood glucose in mice with type 2 diabetes induced by a high-fat diet. Therefore, strain MNH19250 has the activity of preventing or treating diabetes.

[0320] Example 6: Use of strain MNH19250 in the treatment and prevention of obesity and related diseases

[0321] 6.1 Experimental Methods:

[0322] 1) Experimental animals: The mice used in the experiment were C57BL / 6J mice, 5-6 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0323] 2) Preparation of test samples from strain MNH19250: The glycerol cryovials of strain MNH19250 were thawed at 37°C and activated by inoculation onto MM01 plates in an anaerobic workstation. The activated strain was then inoculated into MM01 liquid medium and cultured anaerobically to obtain a sufficient quantity of culture. The cultured bacterial solution was concentrated by centrifugation and resuspended in PBS containing 0.05% L-CysHCl to obtain purity and viable count (2 × 10⁻⁶). 9 Test substances (CFU / mL) that meet the requirements for animal experiments.

[0324] 3) Negative control: PBS containing 0.05% L-Cys HCl (cysteine ​​hydrochloride) was used as the negative control.

[0325] 4) Experimental Procedure: Male C57BL / 6J mice aged 5-6 weeks were fed a high-fat diet for 10 weeks after the quarantine period. Twelve mice weighing between 35.50g and 44.49g were randomly stratified and divided into two groups of 6 mice each (experimental group and control group). Day 1 was the day of drug administration after grouping. The experimental group was given the test substance of strain MNH19250, while the control group was given a negative control. Drug administration was twice daily for 28 days. Mice had free access to water and food during the experiment, using a 12h / 12h diurnal cycle. A general clinical observation was conducted once after the drug administration period. Animal weight was measured twice weekly during the drug administration period and again before dissection at the end of the experiment.

[0326] Mouse food intake was measured: Feed intake was measured on Day n, and remaining feed intake was measured on Day n+1 to obtain the food intake 24 hours before drug administration. During the drug administration period, feed intake and remaining feed intake 24 hours after administration were measured weekly. The endpoint of this experiment was the day after drug administration (D29). Dissection and tissue sampling were performed according to the protocol to determine the endpoint. Data were compiled and analyzed to determine body weight and percentage change in body weight, anatomical data, and serum test results. All data are expressed as Mean ± SD and were plotted and statistically analyzed using GraphPadPrism software. Student's t-test was used for pairwise comparisons. Significance was indicated by *: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0327] 6.2 Effects of strain MNH19250 on body weight in obese model mice:

[0328] See Figure 15 It can be seen that strain MNH19250 can significantly reduce the weight and weight gain of obese mice induced by a high-fat diet, and after four weeks of gavage intervention, the weight decreased by about 10% compared with that before the intervention, indicating that strain MNH19250 has a significant weight loss effect.

[0329] 6.3 Effects of strain MNH19250 on blood lipids in a high-fat diet-induced obesity model mouse:

[0330] See Figure 16 The results showed that strain MNH19250 significantly reduced the concentrations of total cholesterol (TCHO), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in the serum of high-fat diet-induced obese mice, and significantly reduced the LDL-C to high-density lipoprotein cholesterol ratio. These data indicate that strain MNH19250 has therapeutic / preventive effects on hypercholesterolemia, hyperlipidemia, and cardiovascular and cerebrovascular diseases.

[0331] 6.4 Effects of strain MNH19250 on body fat in a high-fat diet-induced obesity model mouse:

[0332] See Figure 17 The results showed that strain MNH19250 could significantly reduce the weight of epididymal fat, perirenal fat, visceral fat and groin fat in mice with high-fat diet-induced obesity, and significantly reduce the ratio of visceral fat to body weight, indicating that strain MNH19250 has the effect of treating / preventing high visceral fat rate and treating / preventing obesity.

[0333] Example 7: Effects of strain MNH19250 on colonic mucosal repair in a high-fat diet-induced intestinal barrier dysfunction model mouse.

[0334] 7.1 Experimental Methods:

[0335] 1) Experimental animals: The mice used in the experiment were C57BL / 6J mice, 5-6 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0336] 2) Preparation of test samples from strain MNH19250: The glycerol cryovials of strain MNH19250 were thawed at 37°C and activated by inoculation onto MM01 plates in an anaerobic workstation. The activated strain was then inoculated into MM01 liquid medium and cultured anaerobically to obtain a sufficient quantity of culture. The cultured bacterial solution was concentrated by centrifugation and resuspended in PBS containing 0.05% L-CysHCl to obtain purity and viable count (2 × 10⁻⁶). 9 Test substances (CFU / mL) that meet the requirements for animal experiments.

[0337] 3) Negative control: PBS containing 0.05% L-Cys HCl was used as the negative control.

[0338] 4) Experimental Procedure: Male C57BL / 6J mice aged 5-6 weeks were fed a high-fat diet for 10 weeks after the quarantine period. Twelve mice weighing between 35.50g and 44.49g were randomly stratified and divided into two groups of 6 mice each (experimental group and control group). Drug administration began on day 1 (D1). The experimental group received MNH19250, while the control group received a negative control. Drug administration was twice daily for 28 days. Mice had free access to water and food during the experiment, following a 12h / 12h diurnal cycle. A general clinical observation was conducted after the drug administration ended. The endpoint of the experiment was the day after the drug administration ended (D29). At the endpoint, the colon was dissected, fixed in paraformaldehyde, and sections were stained with AB-PAS and Alixin Blue to stain the mucus layer, and the mucus layer thickness was measured. All data are expressed as Mean ± SD and were plotted and statistically analyzed using GraphPad Prism software. For pairwise comparisons, the Student's t-test was used. Significant differences are indicated by *, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0339] 7.2 Effects of strain MNH19250 on colonic mucosal repair in a high-fat diet-induced intestinal barrier dysfunction model mouse model:

[0340] See Figure 18 The results showed that strain MNH19250 could significantly increase the thickness of the colonic mucus layer in mice with a high-fat diet-induced intestinal barrier dysfunction model, indicating that strain MNH19250 has a therapeutic / preventive effect on intestinal barrier dysfunction.

[0341] Although the invention has been disclosed with reference to certain embodiments, it will be apparent that modifications and variations can be made without departing from the spirit and scope of the invention as disclosed herein and as set forth in the appended claims. Furthermore, it should be understood that while all embodiments disclosed illustrate implementations of the invention, they are provided only as non-limiting examples and should not be construed as limiting the various aspects of the invention thus illustrated. The invention is intended to have the full scope defined by the language of this disclosure, the following claims, and any equivalents thereof. Therefore, the drawings and detailed descriptions should be considered illustrative rather than restrictive.

Claims

1. An isolated strain of Akkermansia myxophila ( Akkermansia muciniphila The Akkermansia myxophila strain mentioned is Akkermansia myxophila strain MNH19250, which is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC NO: 63782 and deposit date of June 21, 2024.

2. A composition comprising: a1) the *Akermansia xylophilus* as described in claim 1; or a2) The fermentation culture of *Ackermania pseudomallei* as described in claim 1, or its dried product; or a3) The supernatant of the *Akermansia muciniphila* and its fermentation culture as described in claim 1; or a4) the Akkermansia myxophilus and its metabolites as described in claim 1; or a5) The Akkermansia mycotoxin strain of claim 1 and the protein isolated from the Akkermansia mycotoxin strain.

3. The composition as described in claim 2, The composition is a drug.

4. The composition of claim 3, wherein the *Ackermania* var. *misopsis* is a live bacterium.

5. The composition according to claim 3, The Ackermania gravidarum is a freeze-dried bacterium.

6. The composition according to any one of claims 2-5, wherein the composition comprises Akkermansia myxophilus as an active ingredient, wherein, The concentration of the *Ackermania* bacteriophyids was 10. 7 Up to 10 12 CFU / g.

7. The composition according to any one of claims 2-5, wherein the protein of the *Akermansia myxophila* is Amuc-1100 protein; and / or Amuc-1631 protein.

8. The composition according to any one of claims 2-5, wherein the composition further comprises one or more pharmaceutically acceptable carriers, excipients and / or excipients.

9. The composition according to any one of claims 2-5, wherein the composition further comprises one or more other active agents for the prevention or treatment of metabolic diseases, cardiovascular diseases, inflammatory diseases and / or tumors.

10. The composition of claim 9, wherein the other active agent is one or more of probiotics, prebiotics, and GLP-1 receptor agonists.

11. The composition of claim 10, The prebiotics are selected from inulin, mulberry leaf extract, berberine, Ganoderma lucidum, green coffee bean extract, oats, pectin, potato or its extract, citrus polyphenols, Ceylon cinnamon, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidins, resistant dextrin, ginseng or its extract, biotin, polydextrose, fructooligosaccharides (FOS), galactooligosaccharides (GOS), starch, cellulose, β-glucan, hemicellulose, lactulose, mannooligosaccharides, mannooligosaccharides (MOS), oligodextrose, tagatose, pectin, xylooligosaccharides (XOS), and any combination thereof; and / or The probiotics are selected from at least one of lactic acid bacteria, lactobacillus, and butyric acid-producing bacteria.

12. The composition of claim 10, The prebiotics are selected from inulin rich in fructooligosaccharides, yeast β-glucan, trans-galacto-oligosaccharides, resistant starch, and any combination thereof.

13. The composition of claim 2, wherein the composition is a health product having at least one of the following functions: (1) It helps control body fat; (2) It helps maintain healthy levels of blood lipids, cholesterol, or triglycerides; (3) It helps maintain healthy blood sugar levels.

14. The use of the Acetobacter xanthophyte as described in claim 1, or the composition of any one of claims 2-13, in the preparation of a medicament for the prevention, treatment, or relief of liver and kidney diseases, metabolic-related diseases, lipid-lowering diseases, or intestinal barrier damage-related diseases in patients with need; The liver and kidney diseases mentioned are selected from non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), abnormal liver function caused by a high-fat diet, liver fibrosis caused by a high-fat diet, liver cell damage caused by a high-fat diet, kidney damage caused by a high-fat diet, and abnormal kidney function caused by a high-fat diet; the metabolic-related diseases mentioned are selected from obesity, diabetes, hyperglycemia, hypercholesterolemia, hyperlipidemia, metabolic syndrome caused by a high-fat diet, glucose intolerance caused by a high-fat diet, lipid metabolism abnormalities caused by a high-fat diet, and dyslipidemia caused by a high-fat diet; the intestinal barrier damage-related diseases mentioned are selected from inflammatory bowel disease, Crohn's disease, and ulcerative colitis; the lipid-lowering measures are selected from reducing visceral fat and / or local fat in obese individuals.

15. The application as described in claim 14, wherein the *Akermansia myxophilus* or the composition reduces serum aspartate aminotransferase and / or alanine aminotransferase (ALT) and / or liver weight in the subject; and / or reduces serum levels of blood urea nitrogen (BUN) and / or creatinine (CRE); and / or The *Akermansia muciniphila*, or the composition thereof, increases the thickness of the colonic mucus layer; and / or The *Akermansia myxophilus* or the composition thereof has at least one of the following properties: regulating metabolism and immunity; reducing abdominal fat and repairing intestinal barrier damage; improving liver damage and reducing weight; reducing weight and reducing inflammation; lowering lipids and reducing inflammation; lowering lipids and repairing intestinal barrier damage; lowering serum AST and / or ALT and lowering lipids; improving liver damage and improving kidney damage; improving diabetes and improving liver damage; improving diabetes and improving kidney damage.

16. The application as described in claim 15, wherein the *Akermansia myxophilus* or the composition is capable of treating, preventing, alleviating, or improving at least one of the following: (1) Lose weight or control weight; (2) Prevention, treatment, improvement or reduction of liver damage caused by NAFLD or NASH, or high-fat diet; (3) To prevent, treat, improve or reduce kidney damage and abnormal kidney function caused by a high-fat diet; (4) Promotes the expression of type I interferon IFNβ in the target population, thereby enhancing immunity; (5) To prevent, treat, improve, or alleviate diabetes; (6) Reduce the subject's blood lipid and / or cholesterol levels; (7) Reduce the subject's local fat, body fat percentage, and / or visceral fat; (8) Repair the intestinal tissue mucosa of the object.

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