Isolated ackermann muciniphile, composition containing same and application of isolated ackermann muciniphile

Abstract

The invention provides separated Ackermann muciniphile, a composition containing the same and application of the Ackermann muciniphile. The Ackermann muciniphile can be used for treating, preventing or relieving obesity, metabolic disorder caused by obesity, diabetes, inflammation, liver and kidney diseases, liver diseases, cardiovascular and cerebrovascular diseases, tumors and the like.

Description

Technical Field

[0001] The present disclosure relates to the field of microorganisms, and more specifically to a novel strain of Akkermansia muciniphila obtained by separation, a composition containing the strain, and uses thereof. Background Art

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

[0003] The screening of new strains of Akkermansia muciniphila faces great difficulties because it mainly exists in the intestines of humans or animals, is a strictly anaerobic microorganism, has extremely high requirements for nutrition and culture environment, and has a long growth cycle. In addition, when verifying the efficacy of the drug, the bacteria of this genus are difficult to verify through in vitro cell experiments because they are anaerobic; during in vivo animal experiments, the bacteria of this genus are difficult to maintain a stable number of viable bacteria due to the difficulty of culture and high degree of anaerobiosis, and there is a problem of insufficient repeatability. These in vivo and in vitro related technical difficulties have limited the discovery and application of new strains of Akkermansia muciniphila.

[0004] In addition, Akkermansia muciniphila grows slowly and has difficulty competing with other microbial strains, so there is a problem of difficulty in accessibility during the screening process.

[0005] The hydrophobicity of the AKK PROBIO strain disclosed in CN116925975B reaches 31% at 60 minutes; the self-aggregation tends to stabilize at 52% at 20 hours. Some literature believes that AKK PROBIO has a weak ability to form biofilms, and in vitro test results show that AKK PROBIO has a low ability to colonize in the intestine (https: / / doi.org / 10.3390 / foods13030442, Section 3.1). If the AKK strain has a weak ability to colonize and adhere, it will limit its commercial product development.

[0006] The prevalence of metabolic diseases such as obesity, diabetes, hypertension, hyperlipidemia, and fatty liver is increasing. At the same time, studies have shown that these diseases are related to the dysbiosis of microbial flora. In many populations, Akkermansia muciniphila is negatively correlated with obesity, diabetes, cardiovascular disease, and low-grade inflammation. Akkermansia muciniphila can protect the integrity of intestinal epithelial cells and mucus layer, thereby playing a metabolic protective role.

[0007] In the prior art, a mixture of multiple microorganisms is usually used to treat metabolic and obesity-related diseases or disorders, such as probiotics or fecal microbiota transplantation (FMT). However, the mixture of multiple microorganisms makes the mechanism more complicated, and there is no mature research on the mutual influence between the microorganisms, and the use of microbial mixtures usually disrupts the homeostasis of the intestinal flora. In addition, the use of microbial mixtures also requires additional consideration of whether each microorganism will affect the activity of each other. At the same time, it is also unclear whether it is the synergistic effect of the microbial mixture that makes it play a role in the treatment or prevention of the disease, or whether a single bacterium plays a role in the treatment or prevention of the disease.

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

[0009] The present invention discloses the isolation of a new muciniphilic Akkermansia strain (Akkermansia muciniphila). Compared with the existing muciniphilic Akkermansia strain, the new strain disclosed in the present invention has a stronger epithelial cell adhesion ability, and can be well colonized in the human or animal intestines, thereby being able to prolong the residence time in the intestines and better exert its efficacy in the intestines; at the same time, due to the excellent colonization and adhesion ability of the strain of the present invention, it can effectively prevent the adhesion of pathogens, which is beneficial to maintaining intestinal homeostasis and health. In addition, the strain disclosed in the present invention can synthesize a large amount of short-chain fatty acids, thereby effectively playing the role of treating or preventing metabolic diseases, reducing the subject's weight, body fat, blood sugar, blood lipids, and cholesterol, improving or alleviating non-alcoholic fatty liver disease (NAFLD) and non-alcoholic fatty hepatitis (NASH), and repairing the intestinal tissue mucosa. It can also promote the expression of type I interferon IFNβ in the subject and improve immunity, so it has the potential to prevent or treat viral infections and inhibit tumor growth.

[0010] In a first aspect, the present disclosure provides an isolated Akkermansia muciniphila having an average nucleotide identity (ANI) value of at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4% or at least 97.5% with the Akkermansia muciniphila (GCF_000020225.1) strain, and / or having a 16S rRNA sequence that is at least 98.65% identical to the sequence shown in SEQ ID NO.1.

[0011] In some embodiments, the alignment score (AF) of Akkermansia muciniphila to 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 that is 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, at least 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identical to the sequence shown in SEQ ID NO.1.

[0013] In some embodiments, the Akkermansia muciniphila is a novel strain of the species Akkermansia muciniphila, and the novel strain has an average nucleotide identity (ANI) value of at least 95% with Akkermansia muciniphila (GCF_000020225.1), 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%, 96.1%, 96.2%, 96.9%, 96.1%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 96.1%, 96.2 ... .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 the muciniphilic Akkermansia include short-chain fatty acids, and the short-chain fatty acids include at least one of acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, caproic acid and decanoic acid; preferably, the muciniphilic Akkermansia can produce high acetic acid and / or propionic acid. In some embodiments, the acetic acid content secreted by the muciniphilic Akkermansia 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 muciniphilic Akkermansia body 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 of the Akkermansia muciniphila 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 Akkermansia muciniphila 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 in Guangdong Microbiological Culture Collection Center (GDMCC) with a deposit number of GDMCC No: 63782 and a deposit date of June 21, 2024.

[0016] In a second aspect, the present disclosure provides a composition comprising Akkermansia muciniphila or its culture or its metabolites or secreted proteins as described in the first aspect.

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

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

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

[0020] In some embodiments, the composition comprises 1×10 4 Up to 1×10 12 cfu / mL or 1×10 4 Up to 1×1012 cfu / mg of live Akkermansia muciniphila.

[0021] In some embodiments, the composition comprises 1×10 5 Up to 1×10 11 cfu / mL or 1×10 5 Up to 1×10 11 cfu / mg of live Akkermansia muciniphila.

[0022] In some embodiments, the composition comprises 1×10 6 Up to 1×10 10 cfu / mL or 1×10 6 Up to 1×10 10 cfu / mg of live Akkermansia muciniphila.

[0023] In some embodiments, the composition comprises 1×10 7 Up to 1×10 9 cfu / mL or 1×10 7 Up to 1×10 9 cfu / mg of live Akkermansia muciniphila.

[0024] In some embodiments, each gram of the composition comprises 1×10 3 Up to 1×10 17 colony forming units (CFU); for example, 1×10 4 Up to 1×10 12 pcs, 1×10 5 Up to 1×10 11 or 1×10 6 Up to 1×10 10 Colony forming units (CFU) of bacteria, specifically, for example, 1×10 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 or any value therebetween of colony forming units (CFU).

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

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

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

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

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

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

[0031] In some embodiments, the auxiliary material may be at least one selected from a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, and a flavor.

[0032] In some embodiments, the composition comprises one or more of a buffer (e.g., sodium bicarbonate, infant formula or sterilized human milk or other agents that allow bacteria to survive and grow (e.g., survive in the acidic environment of the stomach and grow in the intestinal environment)), a lyoprotectant, a preservative, a stabilizer, a binder, a compacting agent, 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 preventing or treating metabolic diseases and / or tumors.

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

[0035] In some embodiments, suppressing appetite comprises reducing food intake and / or reducing appetite.

[0036] In some embodiments, the other active agent is selected from: a GLP-1 receptor agonist, a dual agonist of a GLP-1 receptor and a GCG receptor, a triple agonist of a GLP-1 receptor, a GIP receptor and a GCG receptor, an AMPK agonist or an active drug that promotes GLP-1 secretion.

[0037] In some embodiments, the other active agent is selected from the group consisting of metformin, sulfonylureas, meglitinide, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin, pioglitazone, rosiglitazone, pentoxifylline, omega-3-fatty acids, statins, ezetimibe, ursodeoxycholic acid, semaglutide, liraglutide, exenatide, and benaglutide.

[0038] In some embodiments, the other active agent may be an agent for preventing or treating tumors.

[0039] In some embodiments, the other active agent may be one or more of a probiotic, a prebiotic, or a combination thereof;

[0040] Preferably, the probiotics are selected from one or more of lactic acid bacteria, lactobacillus, lactococci, butyrate-producing bacteria, bifidobacteria, thermophilic streptococci, faecal streptococci, and mesenteric leuconostoc.

[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, nutritional compounds, biotin, polydextrose, fructooligosaccharide (FOS), galacto-oligosaccharide (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, manno-oligosaccharide, manno-oligosaccharide (MOS), inulin rich in fructooligosaccharide, oligo-glucose, tagatose, trans-galacto-oligosaccharide, pectin, resistant starch, xylo-oligosaccharide (XOS) and any combination thereof.

[0042] In some embodiments, the composition can be formulated as a frozen composition, such as a frozen composition prepared by quick freezing and drying, or lyophilization, for storage and / or transportation.

[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 alive. In some embodiments, the strain in the composition is freeze-dried or spray-dried and can partially or completely colonize the intestine. In some embodiments, the strain is reconstituted before administration. In some cases, the reconstitution is carried out by using a diluent as described herein.

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

[0046] In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric preparation. In some embodiments, the enteric preparation is a dosage form with an enteric coating. For example, the enteric preparation can be an enteric granule, an enteric tablet, or an enteric capsule. In some embodiments, the composition is a capsule. In some embodiments, the capsule is a hard capsule or a soft capsule; or the capsule is a sustained release capsule, a controlled release capsule, or an enteric capsule, or the capsule can be a microencapsulated capsule, or a microcapsule.

[0047] In some embodiments, the composition is a medicine, or a health product or a food.

[0048] In some embodiments, the composition is in an infant-suitable dosage form, a child-suitable dosage form, or an adult-suitable dosage form.

[0049] In some embodiments, the composition is in a parenteral dosage form.

[0050] In the third aspect, there is provided the use of the Akkermansia muciniphila described in the first aspect or the composition described in the second aspect in the preparation of drugs, health products or foods 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.

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

[0052] In some embodiments, the liver and kidney diseases include liver diseases, diseases related to liver damage, kidney diseases such as kidney damage and abnormal kidney function.

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

[0054] The composition comprises a consortium of isolated and purified live microbial populations to reduce serum levels of one or more enzymes of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in a subject by at least 2U / L, 5U / L or 10U / L, 25IU / L, 30IU / L, 50IU / L compared to the ALT and / or AST levels in the subject before administration of the isolated and purified microbial species consortium; the composition comprises a consortium of isolated and purified live microbial populations to reduce serum levels of urea nitrogen (BUN) and creatinine (CRE) in the serum of the subject by at least 2mmol / L, 5mmol / L, 10mmol / L compared to the serum levels of urea nitrogen (BUN) and creatinine (CRE) in the serum of the subject before administration of the isolated and purified microbial species consortium.

[0055] As used herein, microbial populations generally refer to microbial populations that are substantially composed of a single strain, species or genus, which may be the case when a population is cultured from a subpopulation of such strains, species or genus that is separated and purified. Therefore, for a given microbial population, if cultured from a separated microbial species or strain, such a population will be referred to herein as purified or substantially pure. The resulting population may be at least 80% pure for the microbial species or strain, at least 90% pure, at least 95% pure, at least 98% pure, at least 99% pure, at least 99.5% pure, or at least 99.9% pure relative to other microbial species or strains within the particular population. On the contrary, the level of non-desirable strains in any particular desired microbial population 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 population aggregate, such as other undesirable microbial strains or species, can be proportional to the above-mentioned level of each desired population or less than the above-mentioned level. Less than 2%, less than 1%, less than 0.5% or less than 0.1%. In the case where the composition comprises a consortium of multiple microbial populations, each population can have the above-mentioned purities, either before it is incorporated into the composition, or when the consortium is measured in an aggregate. For example, the level of impurities in the purified population consortium, such as other undesirable microbial strains or species, can be proportional to the above-mentioned levels of each desired population or below the above-mentioned levels. Less than 2%, less than 1%, less than 0.5% or less than 0.1%. In the case where the composition comprises a consortium of multiple microbial populations, each population can have the above-mentioned purities, either before it is incorporated into the composition, or when the consortium is measured in an aggregate. For example, the level of impurities in the purified population consortium, such as other undesirable microbial strains or species, can be proportional to the above-mentioned levels of each desired population or below the above-mentioned levels.

[0056] The pharmaceutical compositions of the present disclosure may also include cellular components, metabolites, secreted molecules and compounds metabolized by Akkermansia muciniphila, and the like. Cellular components or cell fractions, metabolites or secreted compounds may be obtained, for example, by recovering the supernatant of a culture of Akkermansia muciniphila or by extracting from a culture of Akkermansia muciniphila; may correspond to components in isolated form from Akkermansia muciniphila, or any mixture of one or more components from Akkermansia muciniphila.

[0057] In some embodiments, the Akkermansia muciniphila disclosed herein can be used to treat or prevent diseases associated with liver function damage, wherein the diseases associated with liver function damage include at least one of the following diseases: fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, liver cancer, or liver cell damage.

[0058] In some embodiments, 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 embodiments, the liver disease includes that caused by a high-fat diet, caused by a high-cholesterol diet, caused by a high-sugar diet, caused by high fat and high cholesterol, caused by high fat and high sugar, and / or caused by high fat, high cholesterol and high sugar.

[0060] In some embodiments, the Akkermansia muciniphila described in the present disclosure can be used to treat, prevent or alleviate diseases related to liver function damage, wherein the diseases related to liver function damage include at least one of the following: fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis or liver cell damage.

[0061] In particular, the Akkermansia muciniphila of the present invention or a composition comprising the same can be used to treat or prevent liver damage. The liver damage can be liver damage caused by long-term hard work; liver damage caused by long-term drinking; liver damage caused by fatty liver, drug-induced liver disease, and genetic metabolic factors.

[0062] In some embodiments, the Akkermansia muciniphila of the present invention or a composition comprising the same can reduce ALT and / or AST levels.

[0063] In some embodiments, the Akkermansia muciniphila of the present invention or a composition comprising the same can reduce liver weight.

[0064] In some embodiments, the kidney disease includes but is not limited to kidney damage, abnormal renal function, etc. The kidney disease includes but is not limited to primary glomerulonephritis, hypertensive arteriosclerosis, diabetic nephropathy, secondary glomerulonephritis, tubulointerstitial lesions (e.g., chronic pyelonephritis, chronic uric acid nephropathy, obstructive nephropathy, drug-induced nephropathy, etc.), ischemic nephropathy, etc. In some embodiments, the cause of the kidney disease includes at least one of the following: a high-fat diet, a high-sugar diet, a high-cholesterol diet. In some embodiments, the kidney disease is manifested by an increase in blood creatinine.

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

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

[0067] In some embodiments, 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 embodiments, 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] Provided are compositions and methods for treating, alleviating, controlling, reducing or preventing symptoms, signs or indicators of liver and kidney diseases and the onset of the disease itself. The compositions and methods include microbial compositions selected to improve intestinal function of subjects to whom they are administered, thereby treating liver and 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 dietary obesity and / or metabolic obesity. In particular, the obesity is abdominal obesity or apple-shaped obesity, such as excess visceral fat.

[0071] In some embodiments, the Akkermansia muciniphila of the present invention or a composition comprising the same can reduce body fat and / or visceral fat and / or abdominal fat.

[0072] In some embodiments, the diabetes includes but is not limited to type I diabetes, type II diabetes, gestational diabetes, diabetes mediated by HDAC activity, diabetic nephropathy, diabetic neuropathy, diabetic eye disease, diabetic retinopathy, diabetic foot, diabetes caused by damage to pancreatic B cells, diabetes caused by insulin resistance, and diabetes caused by obesity.

[0073] In some embodiments, the causes of diabetes include, but are not limited to, at least one of islet cell dysfunction, decreased insulin secretion, increased insulin resistance, a high-fat diet, a high-sugar diet, high cholesterol, hyperlipidemia, hyperglycemia, NAFLD, or NASH.

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

[0075] In some embodiments, the diabetes mellitus is manifested 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 caused by disordered glucose metabolism, or (2) diabetes caused by abnormal glucose tolerance or decreased glucose tolerance, or (3) diabetes caused by damaged insulin B cells, or (4) diabetes caused by insulin resistance.

[0077] In some embodiments, the Akkermansia muciniphila disclosed herein has the effect of increasing the secretion level of glucagon-like peptide-1 (GLP-1), thereby regulating the body's blood sugar 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 embodiments, the cardiovascular disease or cardiovascular and cerebrovascular disease includes, but is not limited to, atherosclerosis, coronary heart disease, hypertension, cardiovascular disease in patients with NAFLD or NASH, cardiovascular and cerebrovascular disease in patients with NAFLD or NASH, and high cholesterol disease.

[0079] In some embodiments, the causes of the cardiovascular disease or cardiovascular and 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 associated with bronchial inflammation, such as bronchitis; diseases associated with cervical inflammation, such as cervicitis; diseases associated with conjunctival inflammation, such as conjunctivitis; diseases associated with esophageal inflammation, such as esophagitis; diseases associated with myocardial inflammation, such as myocarditis; diseases associated with rectal inflammation, such as proctitis; diseases associated with scleral inflammation, such as scleritis; diseases associated with gum inflammation; also including diseases associated with bone inflammation, lung inflammation (alveolitis), respiratory inflammation (such as asthma, such as bronchial asthma), acute respiratory distress syndrome (ARDS); inflammatory skin diseases, such as contact allergies, atopic dermatitis; fibrotic diseases (e.g., pulmonary fibrosis); encephalitis.

[0081] In some embodiments, the Akkermansia muciniphila disclosed herein has the effect of treating / preventing intestinal barrier damage. In some embodiments, the inducement of intestinal barrier damage includes at least one of the following: a high-fat diet, a high-sugar diet, and a high-cholesterol diet.

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

[0083] In some embodiments, the medicine, health product or food has at least one effect selected from the following: effect: reducing liver weight; treating early fatty hepatitis lesions; slowing down fat accumulation in liver cells; reducing serum AST and ALT; reducing abdominal white fat inflammatory lesions; reducing the weight of mammals; reducing the food intake of mammals; reducing body fat in mammals; reducing the level of at least one of the following indicators in the serum of mammals: total cholesterol level, low-density lipoprotein and triglyceride level; increasing the level of serum high-density lipoprotein in mammals; improving impaired oral glucose tolerance in mammals; reducing fasting blood sugar in mammals; reducing the HOMA-IR index in mammals; reducing at least one of the epididymal fat weight, perirenal fat weight, visceral fat weight and inguinal fat weight in mammals; repairing digestive tract mucosal damage ; increase the thickness of the colon mucus layer; regulate the body's immunity; increase the expression of IFNβ; promote the transcriptional activity of IFNβ; treat, prevent or relieve coronary heart disease; treat, prevent or relieve atherosclerosis; treat, prevent or relieve hyperglycemia; treat, prevent or relieve hyperlipidemia; treat, prevent or relieve high cholesterol; treat, prevent or relieve liver damage; treat, prevent or relieve fatty liver; treat, prevent or relieve NAFLD or NASH; treat, prevent or relieve hypertension; treat, prevent or relieve diabetes, preferably gestational diabetes or type II diabetes or diabetes mediated by HDAC activity; treat, prevent or relieve obesity; treat, prevent or relieve metabolic syndrome; treat, prevent or relieve local excess sebum, excess inguinal fat, excess epididymal fat and / or excess brown fat; treat, prevent or relieve tumors.

[0084] The Akkermansia muciniphila MNH19250 isolated in the present invention has strong self-aggregation ability and hydrophobicity, easily adheres to epithelial cells, and has good pH, NaCl and bile salt tolerance, so it can quickly and stably colonize in the human or animal intestines.

[0085] The Akkermansia muciniphila MNH19250 isolated and obtained in the present invention can also synthesize a large amount of short-chain fatty acids, and thus can effectively treat or prevent metabolic diseases, reduce the subject's body weight, body fat, blood sugar, blood lipids, and cholesterol, improve or alleviate non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), repair intestinal tissue mucosa, and promote the expression of type I interferon IFNβ in the subject, improve immunity, and has the potential to prevent or treat viral infections and inhibit tumor growth.

[0086] Therefore, in some embodiments, the medicine, health product or food can regulate immunity while regulating metabolism.

[0087] In some embodiments, the medicine, health product or food can repair intestinal barrier damage while reducing abdominal fat.

[0088] In some embodiments, the medicine, health product or food can reduce weight while improving liver damage.

[0089] In some embodiments, the medicine, health product or food can reduce weight and fight inflammation at the same time.

[0090] In some embodiments, the medicine, health product or food can reduce blood lipids and also fight inflammation. The blood lipid reduction is to reduce blood lipids in abdominal obesity or to reduce visceral fat.

[0091] In some embodiments, the medicine, health product or food can repair intestinal barrier damage while lowering lipids.

[0092] In some embodiments, the medicine, health product or food can reduce blood lipids while reducing serum AST and / or ALT. The lipid reduction is the reduction of abdominal obesity or the reduction of visceral fat.

[0093] In some embodiments, the medicine, health product or food can improve kidney damage while improving liver damage.

[0094] In some embodiments, the medicine, health product or food can improve liver damage while improving diabetes.

[0095] In some embodiments, the medicine, health product or food can improve kidney damage while improving diabetes.

[0096] In some embodiments, the medicine, health product or food can improve liver damage while improving atherosclerosis.

[0097] In some embodiments, the medicine, health product or food can reduce lipids while improving atherosclerosis. The lipid reduction is the reduction of abdominal obesity or the reduction of visceral fat.

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

[0099] In some embodiments, the Akkermansia muciniphila or the composition described in the present invention has the effect of reducing liver and / or kidney damage, wherein the reduction of liver damage is to reduce one or more elevated liver damage or liver disease indicators of the subject, such as reducing the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and / or liver weight indicators of the subject; the reduction of kidney damage is to reduce the blood urea nitrogen (BUN) in the serum of the subject and / or reduce the CRE (creatinine) index in the serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] The embodiments will be described below in conjunction with the accompanying drawings so that the above and other aspects and advantages of the present invention will become apparent and easily understood.

[0101] Figure 1 : shows the colony morphology photograph of strain MNH19250.

[0102] Figure 2 : Gram staining photographs of strain MNH19250 are shown.

[0103] Figure 3 : shows the electron micrograph of strain MNH19250.

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

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

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

[0107] Figure 7 : Shows the 6h autoaggregation ability of strain MNH19250.

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

[0109] Fig. 9 : A bar graph showing the relative fluorescence values ​​of IFNβ expression in the presence of strain MNH19250.

[0110] Fig.10 : It shows that strain MNH19250 can significantly reduce liver function indicators (a: ALT; b: AST).

[0111] Fig.11 : It was shown that strain MNH19250 can significantly improve non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NAFLD / NASH) (a: liver weight; b: percentage of liver weight to body weight; c: hepatic steatosis; d: lobular inflammation; e: hepatocyte ballooning; f: non-alcoholic fatty liver disease activity score (NAS); g: liver HE tissue section).

[0112] Fig.12 : It shows that strain MNH19250 can improve renal function indicators (a: urea nitrogen BUN; b: blood creatinine CRE).

[0113] Fig.13 : It shows that strain MNH19250 can significantly reduce the oral glucose tolerance of high-fat diet-induced type 2 diabetic mice (a. oral glucose tolerance results; b. oral glucose tolerance area under the curve results; data are shown 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; **, p < 0.01 compared with the HFD-Control group).

[0114] Fig.14 : It shows that strain MNH19250 can significantly reduce fasting blood glucose in high-fat diet-induced type 2 diabetic mice (data are shown 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] Fig.15 : It shows that strain MNH19250 can significantly reduce the body weight of high-fat diet-induced obese mice (a. body weight results; b. body weight change ratio results; data are shown 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; **, p < 0.01 compared with the HFD-Control group).

[0116] Fig.16:Showed that strain MNH19250 can significantly reduce blood lipids in high-fat diet-induced obese mice (a. serum total cholesterol results (TCHO); b. serum triglyceride results (TG); c. serum low-density lipoprotein cholesterol results (LDL-C); d. low-density lipoprotein cholesterol / high-density lipoprotein cholesterol results (LDL-C / HDL-C); data are shown 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).

[0117] Fig.17 :Shows that strain MNH19250 can significantly reduce the fat weight and visceral fat ratio of high-fat diet-induced obese mice (a. epididymal fat; b. perirenal fat; c. mesenteric fat; d. visceral fat; e. visceral fat ratio; f. inguinal fat; data are shown 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).

[0118] Fig.18 : It was shown that strain MNH19250 can significantly increase the mucus layer thickness of high-fat diet-induced obese mice (a. Colon Alcian blue staining; b. Colon mucus layer thickness; data are shown 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).

[0119] Deposit of strains

[0120] The strain Akkermansia muciniphila MNH19250 is deposited in the Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC No: 63782, the deposit date is June 21, 2024, and the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, and the deposit name is Akkermansiamuciniphila MNH19250. DETAILED DESCRIPTION

[0121] The present invention isolates a new strain of Akkermansia muciniphila, with a deposit number of GDMCC No: 63782, and identifies it using traditional classification methods and molecular biology methods. The identification results show that the strain belongs to a new strain of Akkermansia muciniphila. Further, the present invention studies the biochemical properties and therapeutic uses of the strain.

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

[0123] As used herein, the term "prebiotic" may be a general term referring to chemicals and / or ingredients that can affect the growth and / or activity of microorganisms in a host (eg, can allow for specific changes in the composition and / or activity of a microbiome).

[0124] The terms "subject," "subject," "individual," "host," and "patient" are used interchangeably herein to refer to any animal subject, including: humans, mammals, laboratory animals, livestock, and domestic pets.

[0125] Compositions or preparations disclosed herein can be used as pharmaceutical preparations, therapeutic compositions, dietary supplements, nutritional supplements, medical probiotics or medical foods. In some cases, the composition is applied in the form of a pharmaceutical preparation. In some cases, the composition is applied in the form of a nutritional supplement. In some cases, the composition is applied in the form of a dietary supplement. In some cases, the composition is applied in the form of a medical food. In some cases, the composition is applied in the form of a medical probiotic. In some cases, the composition (e.g., dietary supplements, nutritional supplements, medical probiotics or medical foods) can be orally administered, for example as a capsule, pill or tablet.

[0126] 16S rRNA is a ribosomal RNA of prokaryotes. The 16S rRNA gene consists of a variable region and a conserved region. The conserved region is common to all bacteria, while the variable region has different degrees of difference between different bacteria. By comparing the 16S rRNA gene sequences of bacteria, according to the base number of sequence differences and their evolutionary distance, an evolutionary tree can be drawn. When the identity between the 16S rRNA gene sequences of two strains is less than 98.65%, it can be judged that they belong to different species (see, Kim, M., Oh, H.-S., Park, S.-C., & Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary Microbiology, 64(Pt 2), 346–351, and Liu, C., Du, M.-X., Abuduaini, R., Yu, H.-Y., Li, D.-H., Wang, Y.-J., Liu, S.-J. (2021). Enlightening the taxonomy darkness of human gut microbiomes with a cultured biobank. Microbiome, 9(1), P23).

[0127] The "identity" between two nucleic acid molecule sequences can be determined using known computer algorithms, such as the "FASTA" program, the GCG program package, BLASTN or FASTA. Commercial or publicly available programs may also be, for example, the DNAStar "MegAlign" program.

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

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

[0130] Using the above method, a person skilled in the art can determine whether an isolated strain belongs to the species of Akkermansia muciniphila identified by the present inventors. For example, when the average nucleotide identity ANI value with Akkermansia muciniphila (GCF_000020225.1) is at least 95%, such as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 97.1 ... When the content of the bacterial strains in the bacterial strains is 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 species.

[0131] For another example, when its 16S rRNA sequence has at least 98.65% identity with the sequence shown in SEQ ID NO.1, for example, 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 to belong to the same bacterial species.

[0132] "Strain" refers to a member of a bacterial species that has a genetic characteristic that allows it to be distinguished from closely related members of the same bacterial species. The genetic characteristic can be the total or partial absence of at least one gene, the total or partial absence of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the absence ("cure" of at least one native plasmid), the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one exogenous gene (a gene from another species), the presence of at least one mutated regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic characteristics between different strains can be identified by PCR amplification, optionally followed by DNA sequencing of the genomic region of interest or the entire genome. In the case of a strain (compared to another strain of the same species) gaining or losing antibiotic resistance or gaining or losing biosynthetic capacity (e.g., auxotrophic strains), the strains or nutrients / metabolites can be distinguished by selection or counter-selection using antibiotics.

[0133] "Supernatant" or "supernatant" within the meaning herein refers to the culture supernatant of the bacterial strain according to the present disclosure, optionally comprising compounds and / or cell debris of said strain, and / or metabolites and / or molecules secreted by said strain.

[0134] Compositions can be prepared using the Akkermansia muciniphila described herein, for example, by using a pharmaceutically acceptable excipient. The pharmaceutical composition comprises a pharmaceutically effective amount of the Akkermansia muciniphila, for example, the Akkermansia muciniphila with a deposit number of GDMCC NO: 63782. Similarly, the Akkermansia muciniphila with a deposit number of GDMCC NO: 63782 can also be prepared into a pharmaceutical composition, for example, by using a pharmaceutically acceptable excipient, which comprises a pharmaceutically effective amount of the Akkermansia muciniphila.

[0135] Suitable pharmaceutically acceptable excipients that may be used include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and flavors.

[0136] The compositions herein can be formulated into any form suitable for enhancing the abundance of Akkermansia muciniphila in a subject. The compositions can be administered orally (e.g., by oral gavage), intramuscularly, by inhalation, intracranially, intralymphatically, intraocularly, intraperitoneally, intrapleurally, intrathecally, intratracheally, intrauterinely, intravascularly, intravenously, intravesically, intranasally, intragastrointestinally, by bile infusion, by cardiac infusion, anteriorly, rectally, subcutaneously, sublingually, topically, intravaginally, transdermally, by ureteral or urethral routes.

[0137] Examples of dosage forms suitable for the composition herein include, but are not limited to, tablets, aerosols, chewable 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, a gel capsule, a gel, an emulsion, a tablet, a sheet capsule, a hydrogel, a nanofiber gel, an electrospun fiber, a food bar, a candy, a fermented milk, a fermented cheese, a chewing gum, a powder or a toothpaste, etc.

[0139] In some embodiments, administration can also be by inclusion in a subject's diet, such as inclusion in a functional food for humans or companion animals.

[0140] The compositions provided herein may include a pharmaceutically acceptable excipient, diluent or carrier. The pharmaceutically acceptable excipient, diluent or carrier is well known in the art.

[0141] In some embodiments, the Akkermansia muciniphila in the compositions of the present disclosure is lyophilized. In some embodiments, the Akkermansia muciniphila in the compositions of the present disclosure is spray-dried. In some embodiments, the Akkermansia muciniphila in the compositions of the present disclosure is lyophilized or spray-dried and is alive. In some embodiments, the Akkermansia muciniphila in the compositions of the present disclosure is lyophilized or spray-dried and is capable of partially or completely colonizing the intestine. In some embodiments, the lyophilized Akkermansia muciniphila is reconstituted prior to administration. In some embodiments, the reconstitution is performed using a diluent as described herein.

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

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

[0144] The compositions disclosed herein include pharmaceutical compositions, health products or foods.

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

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

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

[0148] In some embodiments, the metabolic disease, metabolic disorder, or disease caused by metabolic disorder includes but is not limited to: liver disease, obesity and obesity-related diseases, cardiovascular disease, diabetes, dyslipidemia, cardiovascular and cerebrovascular disease, glucose intolerance, atherosclerosis, coronary heart disease or hypertension, type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, alcoholic fatty hepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, uremia, ketoacidosis, hypoglycemia, thrombotic disease, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), atherosclerosis, and at least one of 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 reasons. It is characterized by absolute destruction of pancreatic islet function. It mostly occurs in children and adolescents. Insulin must be used to treat it to achieve satisfactory results, otherwise it will be life-threatening. Type 2 diabetes is a multifactorial syndrome characterized by abnormal carbohydrate / fat metabolism, usually including hyperglycemia, hypertension and abnormal cholesterol. Type 2 diabetes is caused by the inability of insulin to work effectively (low binding to receptors). Therefore, it is necessary not only to check fasting blood sugar, but also to observe blood sugar 2 hours after a meal, especially to do pancreatic islet function tests. There are two types of diabetes during pregnancy. One is that the patient has been diagnosed with diabetes before pregnancy, which is called "diabetes complicated with pregnancy"; the other is that the patient has normal glucose metabolism or potential glucose tolerance before pregnancy, and the diabetes appears or is diagnosed during pregnancy, which is also called "gestational diabetes (GDM)". More than 80% of pregnant women with diabetes have GDM.

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

[0152] Insulin resistance refers to the decrease in the efficiency of insulin in promoting glucose uptake and utilization due to various reasons. The body compensatory secretion of excessive insulin produces hyperinsulinemia to maintain blood sugar stability. Insulin resistance can easily lead to metabolic syndrome and type 2 diabetes.

[0153] Oral glucose tolerance is used to measure the function of pancreatic beta cells and the body's ability to regulate blood sugar, and is currently recognized as a diagnostic indicator for diagnosing diabetes. When glucose metabolism is disordered, blood sugar rises sharply after taking a certain amount of glucose orally, or the increase is not obvious, but it cannot drop to the fasting level or the original level in a short time. This is abnormal glucose tolerance or impaired glucose tolerance; abnormal glucose tolerance indicates that the body's ability to metabolize glucose is reduced, which is common in type II diabetes and obesity.

[0154] HOMA-IR is an index used to evaluate an individual's insulin resistance level. It is currently widely used in clinical evaluation of insulin sensitivity in diabetic patients. Its calculation method is: fasting blood glucose level (FPG, mmol / L) × fasting insulin level (FINS, μU / mL) / 22.5. The HOMA-IR index of a normal individual is 1. As the level of insulin resistance increases, the HOMA-IR index will be higher than 1.

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

[0156] Liver function disorders

[0157] Liver function diseases are abnormal liver function or liver function damage. Alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) are sensitive markers of liver function diseases. When the body's liver function is abnormal (for example, liver damage, non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH)), ALT and / or AST in the blood will increase significantly. In general, ALT is more sensitive than AST in reflecting acute liver damage. Continuous increase in ALT indicates chronic liver damage. In cases of chronic hepatitis, cirrhosis, liver cancer, etc., AST increases significantly and can exceed ALT. AST levels indicate the chronicity, extensiveness and severity of liver lesions, and even indicate the prognosis of chronic liver disease.

[0158] Common liver diseases with elevated ALT and / or AST include, but are not limited to: acute viral hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E); Epstein-Barr 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; α1-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 decreases after drug intervention, it can show that the drug has a certain therapeutic improvement effect.

[0160] Non-alcoholic fatty liver disease (NAFLD) refers to the accumulation of excess fat in the liver in the form of triglycerides (TG) (fatty degeneration). Some patients with NAFLD have liver cell damage and inflammation (steatohepatitis) in addition to excess fat, namely non-alcoholic steatohepatitis (NASH). NASH is widely considered to be the liver manifestation of metabolic syndrome, such as type 2 diabetes, insulin resistance, central obesity, hyperlipidemia (low high-density lipoprotein cholesterol, high triglycerides) and hypertension.

[0161] Liver and kidney disease

[0162] Liver and kidney function diseases refer to functional acute renal failure that occurs in severe liver disease and decompensated cirrhosis. Due to insufficient effective circulating blood volume and decreased prostaglandins, hepatorenal syndrome may occur.

[0163] Cardiovascular disease

[0164] TG (triglycerides) are mainly involved in energy metabolism in the human body and generate heat energy. Excessive TG content in the blood can lead to blood viscosity, causing lipids to deposit on the blood vessel walls, gradually forming small plaques, namely atherosclerosis. Increased LDL-C is the main and independent risk factor for the occurrence and development of atherosclerosis; the increased level of LDL-C is also an indicator for measuring coronary heart disease. Since HDL-C can transport cholesterol in the blood vessel wall to the liver for decomposition and metabolism (i.e., reverse cholesterol transport), it can reduce the deposition of cholesterol in the blood vessel wall and play an anti-atherosclerotic role.

[0165] Inflammation

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

[0167] obesity

[0168] Obesity refers to a certain degree of obvious overweight and excessive fat layer. It is 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 accumulation of body fat due to excessive food intake or changes in body metabolism causes excessive weight gain and causes pathological, physiological changes or lurking in the human body. A body mass index of more than 25 is considered overweight, and more than 30 is considered obese. Obesity increases the risk of many physical and mental diseases. It is mainly related to metabolic syndrome, a combination of diseases including type 2 diabetes, hypertension, hypercholesterolemia and hypertriglyceridemia. In general, the effects of obesity on health can be divided into two categories: diseases that can be attributed to increased body fat (such as osteoarthritis, obstructive sleep apnea, etc.) and diseases that increase the number of fat cells (diabetes, dyslipidemia, cancer, cardiovascular disease, non-alcoholic fatty liver or non-alcoholic steatohepatitis, etc.). The "obesity-related diseases" can be selected from the following diseases: overeating, binge eating, bulimia, hypertension, diabetes, elevated plasma insulin concentration, insulin resistance, hyperlipidemia, metabolic syndrome, insulin resistance syndrome, obesity-related gastroesophageal reflux disease, atherosclerosis, hypercholesterolemia, hyperuricemia, low back pain, cardiac hypertrophy and left ventricular hypertrophy, lipodystrophy, non-alcoholic steatohepatitis, cardiovascular disease, and polycystic ovary syndrome, as well as subjects with these obesity-related diseases including those who wish to lose weight.

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

[0170] Interferon (IFN) receptor proteins are a type of cytokine secreted by host cells that can regulate immune responses. Viruses, bacterial endotoxins, and synthetic double-stranded RNA can stimulate the production of interferon. Macrophages, lymphocytes, and somatic cells in the human body can all produce interferon. Among them, IFNβ belongs to type I interferon, which can promote the activity of NK cells, macrophages, and T lymphocytes, thereby playing antiviral, anti-tumor, and immune regulatory roles.

[0171] The Akkermansia muciniphila or its product provided by the present invention can significantly promote IFNβ transcription activity. Type I interferon IFNβ has been shown to be able to regulate immunity and rebuild the synergistic effect of innate immunity and acquired immunity in the tumor microenvironment, and is used to treat stubborn and 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 show that strain MNH19250 or the Akkermansia muciniphila or its product provided by the present invention can regulate immunity, and achieve anti-tumor through immunoregulation, and at the same time have potential functional effects on anti-viral tumors.

[0172] Short-chain fatty acids are one of the important metabolites of intestinal microorganisms. As signal molecules, they affect a series of host activities, mainly acetate, propionate and butyrate. Short-chain fatty acids can reduce intestinal pH and inhibit pathogen growth. Short-chain fatty acids can activate target pathways such as GPR41, GPR43, GPR109A, GPCR81, improve the integrity and function of colonic epithelial cells, enhance intestinal barrier function, promote the secretion of hormones such as GLP-1 and PYY in intestinal endocrine cells, increase insulin sensitivity, increase energy consumption, promote fat decomposition, inhibit the production of proinflammatory cytokines, and maintain intestinal immune homeostasis. Short-chain fatty acids have certain beneficial effects on metabolic diseases such as obesity, diabetes, non-alcoholic fatty liver, non-alcoholic steatohepatitis, ulcerative colitis, radiation proctitis, Crohn's disease, etc.

[0173] Propionic acid and butyric acid can act as HDAC inhibitors, changing the expression of multiple functional genes by inhibiting HDAC, regulating cell proliferation, apoptosis and differentiation, thereby preventing the occurrence and development of inflammation while improving immunity.

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

[0175] Amuc_1631 (P9) protein can promote the secretion of GLP-1 in intestinal L cells and has the potential to treat diabetes, obesity and other metabolic diseases (Wenxuan D, Yuchen Z, Xinyuan Z, et al., Heterologous expression of P9 from Akkermansia muciniphila increases the GLP-1 secretion of intestinal L cells [J]. World Journal of Microbiology & Biotechnology, 2024 (7): 40. DOI: 10.1007 / s11274-024-04012-z.). P9 protein also exhibits a strong anti-inflammatory effect and can regulate intestinal barrier function, thus playing a role in inflammatory bowel disease. P9 protein enhances intestinal barrier function by increasing the number of goblet cells, mucus production, and differentiation of stem cells into secretory cells." Patrice D. Cani, Clara Depommier, Muriel Derrien, et al., Akkermansia muciniphila: paradigm for next-generation beneficial microorganisms. Nature Reviews Gastroenterology & Hepatology volume 19, pages 625–637 (2022)".

[0176] Therefore, the Akkermansia muciniphila involved in the present 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 scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature of the art or in accordance with the product or instrument instructions are used. All reagents or instruments are indicated by the manufacturer and can be purchased commercially.

[0178] Example

[0179] The liquid MM01 medium involved in the embodiment is composed of: peptone 5g / L, tryptic casein 5g / L, yeast powder 10g / L, beef extract 5g / L, glucose 5g / L, K2HPO4 2g / L, sodium acetate 2g / L, Tween 80 1mL / L, heme 5mg / L, L-cysteine ​​hydrochloride 0.5g / L, vitamin K1 1μL / L, inorganic salt solution 8ml / L (each 1L includes calcium chloride 0.25g, K2HPO4 1g, KH2PO41g, magnesium sulfate 0.5g, sodium bicarbonate 10g, sodium chloride 2g).

[0180] The solid MM01 medium involved in the embodiment is composed of: peptone 5g / L, tryptic casein 5g / L, yeast powder 10g / L, beef extract 5g / L, glucose 5g / L, K2HPO4 2g / L, sodium acetate 2g / L, Tween 80 1mL / L, heme 5mg / L, L-cysteine ​​hydrochloride 0.5g / L, vitamin K1 1μL / L, inorganic salt solution 8ml / L (each 1L includes calcium chloride 0.25g, K2HPO4 1g, KH2PO41g, magnesium sulfate 0.5g, sodium bicarbonate 10g, sodium chloride 2g), agar 15g / L.

[0181] Anaerobic blood agar plates were purchased from Huankai Microorganisms, with the following formula: 10 g / L casein pancreatic digest, 3 g / L heart pancreatic digest, 1 g / L corn starch, 5 g / L meat gastric digest, 5 g / L yeast extract powder, 5 g / L sodium chloride, 15 g / L agar, 50-100 mL / L sterile defibrinated sheep blood, pH 7.3±0.2.

[0182] The above culture medium can be prepared by 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 numbered MNH19250 was isolated from a sample of a healthy male volunteer in Guangzhou, Guangdong, China. The isolation method adopted a conventional strain isolation method, using gradient dilution, and then picking a single colony for isolation and culture, and then purifying the strain and anaerobic culture at 37°C. The pure culture strain was prepared into a 20% glycerol / water-bacteria solution and stored at -80°C.

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

[0187] The donor takes 5g of sample and puts it into a sample collection and storage tube. After shaking and homogenization, the processed sample is placed in an ice box and sent to the laboratory for strain isolation within 24 hours.

[0188] In the biosafety cabinet, dispense physiological saline solution into 9 mL / tube; prepare anaerobic blood agar plates for strain isolation culture medium, and transfer them to the anaerobic workstation 24 h in advance, marking the sample information, culture medium type, isolation date, etc.

[0189] Take a new sample and place it in the anaerobic workstation and shake it with a vortex shaker for 1 min. Pipette 1 mL of the sample into 9 mL of normal saline and mix it for 10 -1 dilution, and then serially diluted to 10 -6 Dilution solution for later use.

[0190] Take 10 -6 Drop the diluted solution onto the anaerobic blood agar plate at a rate of 100 μL / plate. Spread evenly. After the surface of the plate is dry, invert the plate and culture at 37°C for 3 to 5 days.

[0191] Observe the growth of the strain on the separation medium (anaerobic blood agar plate) and pick a single colony with a sterilized toothpick for strain purification. The purified strain is placed at 37°C for anaerobically cultured. The pure culture strain is prepared into a 20% glycerol / water-bacteria solution and stored at -80°C.

[0192] 1.2 Morphological characteristics of strain MNH19250

[0193] Culture and morphological characteristics

[0194] The strain MNH19250 was inoculated into MM01 medium and cultured anaerobically at 37°C for 72 hours. Visible colonies were formed on the MM01 plate medium. The colonies were round, with regular and smooth edges, about 0.5 mm in diameter, light yellow, and translucent. The strain was Gram-negative. Microscopic morphology showed that it had no flagella, was non-motile, rod-shaped, and about 0.5-1 μm × 1.5-3 μm in size. For photos of the colony morphology of the strain MNH19250 after 72 hours of culture on the MM01 plate, see Figure 1 For Gram staining photos of strain MNH19250, see Figure 2 , electron microscope photos see Figure 3 .

[0195] 1.3 Physiological characteristics of strain MNH19250

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

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

[0198] API 20A (purchased from BioMérieux, CN2030025) was used to perform biochemical identification on strain MNH19250. For specific experimental operations, see the conventional API reagent operation guide. Culture conditions of strain MNH19250: 37°C, anaerobic. The experimental results are shown in Table 1.

[0199] Table 1. Test results of strain MNH19250 API 20A

[0200]

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

[0202] MNH19250 can hydrolyze esculin (ESC), that is, it can synthesize 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), that is, it can synthesize protease. The function of protease is to convert the protein that cannot be absorbed into polypeptides or amino acids that can penetrate the bacteria. This enzyme is mainly an extracellular enzyme, which can hydrolyze gelatin into polypeptides first, and then further hydrolyze it into amino acids, losing the gel property and liquefying, allowing the black substance in the reaction well to diffuse, and showing a positive reaction.

[0204] 1.5 Antibiotic Minimum Inhibitory Concentration Test for Strain MNH19250

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

[0206] Table 2. Minimum inhibitory concentration test results of antibiotics for strain MNH19250

[0207]

[0208] The results showed that MNH19250 was sensitive to ampicillin, chloramphenicol, clindamycin, amoxicillin, rifampicin, imipenem, penicillin, and cefquinome. It can be seen that MNH19250 is sensitive to most types of antibiotics, and the risk of long-term use of MNH19250 leading to antibiotic resistance in subjects is low.

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

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

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

[0212] The auto-aggregation ability of the strain was calculated according to the following formula: auto-aggregation ability of the strain (%) = [1-(At / A0)] × 100%.

[0213] The 6h autoaggregation capacity of MNH19250 was 54.21% (see Figure 7 ), compared with other strains in the literature, the ability of automatic aggregation 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, the auto-aggregation ability of A.muciniphila PROBIO is about 30% (8h)). Strains with strong auto-aggregation ability have stronger epithelial cell adhesion ability and can better colonize in the human intestine to play a probiotic role.

[0214] 1.7 Determination of surface hydrophobicity of strain MNH19250

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

[0216] 4 mL of xylene was added to 4 mL of the resuspension, the two-phase system was vortexed for 5 min, incubated at room temperature for 1 h, and then the xylene phase was carefully removed. The absorbance value A of the aqueous phase was measured at 600 nm, and repeated three times. The surface hydrophobicity of the test strain was calculated according to the following formula:

[0217] H%=[(A0-A) / A0]×100, wherein 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% higher 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 Akkermansiamuciniphila PROBIO (https: / / doi.org / 10.3390 / foods13030442); Figure 1c, A. muciniphila PROBIO surface hydrophobicity is 31% (1h)). Strains with strong surface hydrophobicity have stronger epithelial cell adhesion ability, can better colonize in the human intestine and thus play a probiotic role, and at the same time can inhibit the adhesion of other pathogens and play an intestinal probiotic function.

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

[0220] A fresh culture of strain MNH19250 was taken to extract the genomic DNA of the strain, and the extracted genomic DNA of the strain was used as a template for 16S rRNA gene amplification.

[0221] The primer pairs used for PCR of 16S rRNA gene are:

[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°C, 4 min; denaturation: 94°C, 50 sec; annealing: 52°C, 40 sec; extension: 72°C, 70 sec; final extension: 72°C, 10 min (36 cycles).

[0226] 1.9 16S rRNA gene sequencing

[0227] The PCR product was purified and subjected to 16S rRNA gene sequencing by Sangon Company to obtain 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 as shown in SEQ ID No. 1 was analyzed by using NCBI Basic Local Alignment Search Tool to confirm the strain classification information.

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

[0231] The strain MNH19250 was compared with the 16S rRNA gene sequences of Akkermansia sp. related strains retrieved from GenBank and other databases to construct a phylogenetic tree.

[0232] The 16S rRNA gene sequence of strain MNH19250 was compared with the sequences of model strains with high 16S rRNA gene sequence similarity in the NCBI database, and then a phylogenetic tree was constructed using the software MEGA 5 (the phylogenetic tree was constructed using the maximum likelihood method) (see Figure 8 ), Figure 8 Only the Bootstrap values ​​greater than 50% are displayed in the developmental tree nodes.

[0233] From the phylogenetic tree, it can be seen that strain MNH19250 is clustered with Akkermansia sp. and Akkermansia muciniphila Muc AY271254. Therefore, strain MNH19250 is judged to be a new strain of Akkermansia muciniphila.

[0234] 1.11 Genome analysis of strain MNH19250

[0235] Genome analysis of strain MNH19250

[0236] The genome of the MNH19250 strain was fragmented by ultrasonic method, and then the Illumina sequencing library was constructed using a standard DNA library construction kit (NEB UltraTM). The constructed sequencing library was sequenced with NovaSeq (Illumina) for double-end 150bp. The sequencing obtained 3.01Gbp data, of which Q20 accounted for 97.42%.

[0237] The original genome sequencing data was filtered using fastp (version: 0.20.0). The filtered raw data was assembled using SPAdes (version: v3.14.0). The total gene length of the genome assembly was 2.82 Mbp, the N50 length was 378.4 kbp, and the GC content was 55.19%.

[0238] The genomic genes were predicted and analyzed using the prokaryotic analysis software genome annotation pipeline 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), where the antibiotic resistance gene database was CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). Detailed comparison information is shown in Table 3.

[0240] Table 3. Drug resistance gene information list

[0241] Strain Gene Resistance genes Gene name Comparison consistency (%) MNH19250_02133 ARO:3000777 F 41.42

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

[0243] Table 4. List of potential toxic genes of MNH-19250

[0244] Strain Gene VFDB gene Gene name Comparison consistency (%) MNH19250_00898 VFG001855 htb 61.932 MNH19250_02218 VFG002225 gmd 65.607

[0245] The analysis of potential secondary metabolism 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 metabolism gene clusters of MNH-19250

[0247] Gene cluster range type from arrive Most similar known gene cluster Similarity Region 2.1 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 of MNH-19250

[0250]

[0251] Analysis of the Amuc_1100 protein 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.

[0252] Table 7. Analysis of protein coding genes of MNH19250 Amuc_1100

[0253] qseqid sseqid pident qcovs MNH19250_01469 WP_197738471 99.369 100

[0254] sequence:

[0255] >MNH19250_01469hypothetical protein

[0256] MSNWITDNKPAAMVAGVGLLLFLGLSATGYIVNSKRSELDKKISIAAKEIKSANAAEITPSRSSNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILITDTSSWLGFQVYSTQAPSVQAASTLGFELKAINSLVNKLAECGLSKFIKVYRPQLPIETPANNPEESDEADQAPWTPMPLEIAFQGDRESVLKAMNAITGMQDYLFTVNSIRIRNERMMPPPIANPAAAKPAAAQPATGAASLTPADEATAPAAPAIQQVIKPYMGKEQVFVQVSLNLVHFNQPKAQEPSED(SEQ ID NO.4)

[0257] Table 8. Analysis of the gene encoding the P9 protein of MNH19250

[0258] qseqid sseqid pident qcovs MNH19250_00667 ACD05451 99.865 100

[0259] Sequence:

[0260] >MNH19250_00667Tail-specific protease

[0261] (SEQ ID NO.5)

[0262] Example 2. Analysis of fatty acid composition of strain MNH19250

[0263] 2.1 Analysis of fatty acid composition of strain MNH19250

[0264] The strain MNH19250 was inoculated on MM01 plates and cultured anaerobically at 37°C for 72 hours. The bacteria were then collected and their fatty acids were extracted and methylated. The fatty acid composition of the strain MNH19250 was analyzed using the fully automatic bacterial identification system of MIDI (Microbial ID, Inc., Newark, Del.).

[0265] The main fatty acid (>10%) of the experimental strain MNH19250 was C15:0anteiso 48.73%. 2.2 Analysis of short-chain fatty acids (SCFA) of strain MNH19250

[0266] Bacteria preparation

[0267] The strain MNH19250 was inoculated into MM01 liquid culture medium and cultured anaerobically at 37°C for 48 hours. The bacterial cells and the supernatant were collected by centrifugation and stored at -80°C for later use.

[0268] Pretreatment of bacterial supernatant samples

[0269] (1) After thawing, vortex the sample for 3 min to mix;

[0270] (2) Pipette 50 μL of sample into a 1.5 mL centrifuge tube, add 100 μL of phosphoric acid solution (0.5%, v / v), and vortex at 2500 rpm for 3 min.

[0271] (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;

[0272] (4) Pipette 200 ml of supernatant into the liner of the injection bottle and store it in a -20 °C refrigerator until GC-MS / MS analysis.

[0273] Pretreatment of bacterial samples

[0274] (1) After the sample is thawed, add 100% ultrapure water extract to resuspend it evenly;

[0275] (2) Pipette 50 μL of bacterial suspension sample into a 1.5 mL centrifuge tube, add 100 μL of phosphoric acid solution (0.5%, v / v), and vortex for 3 min to mix;

[0276] (3) Soak in liquid nitrogen for 2 min, take out and thaw completely on ice, vortex at 2500 rpm for 3 min, and repeat 3 times;

[0277] (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;

[0278] (5) Pipette 90% of the supernatant into the liner of the injection bottle for GC-MS / MS analysis.

[0279] (6) The remaining 50 mL of bacterial suspension was repeatedly frozen and thawed in liquid nitrogen three times. After centrifugation at 12,000 rpm for 10 min, the supernatant was collected and the protein concentration was determined by the BCA method.

[0280] Standard preparation: Prepare 0.005 μg / mL, 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 8 μg / mL, 10 μg / mL, 20 μg / mL of different concentrations of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid as standard solution. Use the conditions shown in Table 9 for chromatography-mass spectrometry detection.

[0281] Obtain the chromatographic peak intensity data of the corresponding quantitative signal of each concentration standard. Use the external standard to internal standard concentration ratio (ConcentrationRatio) as the horizontal axis and the external standard to internal standard peak area ratio (Area Ratio) as the vertical axis to draw standard curves for different substances.

[0282] Table 9. Main conditions of chromatography-mass spectrometry

[0283]

[0284] The integrated peak area ratios of all detected samples were substituted into the linear equation of the standard curve for calculation. After the sample calculation formula was entered, the content data of the substance in the actual sample was finally obtained. The results showed that both the bacterial supernatant and the bacteria contained short-chain fatty acids such as 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.

[0285] Table 10. Detection of short-chain fatty acids and medium-chain fatty acids in the supernatant and cells of MNH19250 bacterial liquid

[0286]

[0287] The test results show that strain MNH19250 can synthesize short-chain fatty acids during its growth, including a large amount of acetic acid and propionic acid.

[0288] Example 3. Effect of strain MNH19250 on IFNβ expression

[0289] To verify whether MNH19250 can promote the expression of IFNβ, this study used THP-1 cells carrying the IFNβ gene promoter reporter gene (THP-1-IFNβ-promoter reporter cells, a cell line built by Muen) to evaluate the effect of MNH19250 on the transcriptional activity of IFNβ.

[0290] 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 literature for details: Huashan Du, Tianmin Xu, Manhua Cui, "cGAS-STING signaling in cancer immunity and immunotherapy" Biomedicine & Pharmacotherapy 133 (2021) 110972; Jiang et al., "cGAS-STING, an important pathway in cancer immunotherapy" Journal of Hematology & Oncology (2020) 13: 81; Khiem C. Lam et al., "Microbiota triggers STING-type IIFN-dependent monocyte reprogramming of the tumor microenvironment" Cell 184, 5338-5356).

[0291] Preparation of culture supernatant of strain MNH19250: inoculate strain MNH19250 in MM01 liquid culture medium, culture anaerobically at 37°C for 48 hours, remove bacteria by centrifugation, filter the culture supernatant with a 0.22 μm filter, aliquot, and store the collected material at -80°C for later use.

[0292] Control group (Control): DMEM complete medium (Gibco, containing 10% FBS) containing 10% volume of MM01 liquid medium;

[0293] MSA-2 group (positive control group): DMEM complete medium containing 10 μM MSA-2 (purchased from Taoshu Biotechnology);

[0294] Strain MNH19250 group: DMEM complete medium containing 10% volume of the culture supernatant of strain MNH19250.

[0295] THP-1-IFNβ-promoter reporter cells were seeded in 96-well plates at 1 × 10 5 cells. Treat the cells according to the set groups. After culturing for 24 hours, the cells were centrifuged at 300g for 5 minutes, the culture supernatant was removed, and 50 μL 1×Luminescence (Promega) was added to normalize the control group (Control) to evaluate the effect of strain MNH19250 on IFNβ transcription activity.

[0296] The experimental results are as follows Fig. 9 As shown, strain MNH19250 can significantly promote the transcriptional activity of IFNβ. Type I interferon IFNβ has been shown to regulate immunity and achieve anti-tumor effects through immune regulation, and has potential functional effects on anti-viral tumors. Therefore, these results show that MNH19250 and its metabolites can regulate the body's immunity and achieve anti-tumor effects through immune regulation, and have potential preventive and inhibitory effects on viral tumors.

[0297] Example 4: Strain MNH19250 improves liver and kidney function and related diseases in a high-fat diet-induced liver and kidney injury mouse model

[0298] The high-fat diet liver and kidney injury mouse model was used to study the improvement of liver and kidney function and related diseases by strain MNH19250. This experimental protocol has been ethically reviewed by the Experimental Animal Care and Use Committee of Muen Biotechnology.

[0299] 4.1 Experimental methods

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

[0301] 2) Preparation of the test substance of strain MNH19250: After thawing the glycerol cryovial of strain MNH19250 at 37°C, inoculate on MM01 plate in anaerobic workstation for activation, inoculate the activated strain in MM01 liquid culture medium, culture anaerobically, obtain a sufficient amount of culture, concentrate the cultured bacterial solution by centrifugation, and resuspend the bacterial culture in PBS containing 0.05% cysteine ​​hydrochloride (L-Cys HCl) to obtain the purity and viable count (2×10 9 CFU / mL) of the test substances that meet the requirements of animal experiments.

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

[0303] 4) Experimental process: After the quarantine period, 5-6 week old C57BL / 6J male mice were fed a high-fat diet for 10 weeks. Twelve mice with a body weight range of 35.50g-44.49g were selected and randomly stratified according to body weight, 6 mice / group, and divided into 2 groups (experimental group and control group). After grouping (D1), drug administration began. The experimental group was given the above-mentioned test substance, and the control group was given a negative control substance. The drug was administered twice a day for a total of 28 days. During the experiment, the mice were free to drink water and eat, and a 12h / 12h day and night cycle was adopted. During the experiment, a general clinical observation was performed after each administration. The endpoint of this experiment was the day after the end of the administration (D29). The end point of the experiment was dissected according to the protocol, and the results of each dissection data and serum test data were summarized and analyzed. All data were expressed in the form of Mean±SD, and the GraphPad Prism software was used for drawing and statistical analysis. For pairwise comparisons, the t-test (Student's t-test) analysis method was used. Significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0304] See also Fig.10 , showing that strain MNH19250 can significantly reduce liver function indicators. Specifically, Fig.10 The results showed that strain MNH19250 could significantly reduce alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, so strain MNH19250 could improve abnormal liver function caused by high-fat diet and had the activity of treating or preventing liver function damage.

[0305] See also Fig.11 According to the NAS scoring system proposed by the American Association for the Study of Liver Diseases (2005), strain MNH19250 has the activity of treating or preventing liver damage and fatty liver NAFLD / NASH. Specifically, Fig.11 The strain MNH19250 was shown to significantly improve non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NAFLD / NASH): the strain MNH19250 could significantly reduce liver weight and the ratio of liver weight to body weight, significantly reduce hepatic steatosis and lobular inflammation, significantly reduce hepatocyte ballooning, and significantly reduce non-alcoholic fatty liver disease activity score (NAS). Therefore, the strain MNH19250 could significantly improve non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, significantly improve liver cell damage, and has the activity of treating or preventing liver cell damage and NAFLD / NASH.

[0306] See also Fig.12, showing that strain MNH19250 can significantly reduce BUN (urea nitrogen) in serum and reduce creatinine (CRE) in serum. Therefore, strain MNH19250 can significantly improve abnormal renal function and has the activity of treating or preventing renal injury and abnormal renal function.

[0307] Example 5: Use of strain MNH19250 to prevent or treat diabetes

[0308] 5.1 Experimental Methods

[0309] 1) Experimental animals: The experimental mice were C57BL / 6J mice, 5-6 weeks old, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0310] 2) Preparation of the test substance of strain MNH19250: After thawing the glycerol cryopreservation tube of strain MNH19250 at 37°C, inoculate it on the MM01 plate in an anaerobic workstation for activation, inoculate the activated strain into MM01 liquid culture medium, and culture it anaerobically to obtain a sufficient amount of culture. After centrifuging and concentrating the cultured bacterial solution, resuspend the bacterial cells in PBS containing 0.05% L-CysHCl to obtain the purity and viable count (2×10 9 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0311] 3) Negative control: PBS containing 0.05% L-Cys HCl was used as a negative control.

[0312] 4) Experimental process: After the quarantine period, 5-6 week old C57BL / 6J male mice were fed a high-fat diet for 10 weeks. Twelve mice with a body weight range of 35.50g-44.49g were selected and randomly stratified according to body weight, 6 mice / group, and divided into 2 groups (experimental group and control group). After grouping (D1), drug administration began. The experimental group was given the test substance of strain MNH19250, and the control group was given the negative control substance. The drug was administered twice a day for a total of 28 days. The endpoint of this experiment was the day after the end of drug administration (D29). During the experiment, mice were free to drink water and eat, and a 12h / 12h day and night cycle was adopted. During the experiment, a general clinical observation was performed after each drug administration. OGTT was measured once within the last week of drug administration, and fasting blood glucose was measured at the end of the experiment (D29). All data were expressed in the form of Mean±SD, and GraphPad Prism software was used for drawing and statistical analysis. For pairwise comparisons, the t-test (Student's t-test) analysis method was used. Significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0313] 5.2 Experiment on oral glucose tolerance of strain MNH19250 in high-fat diet-induced type 2 diabetic mice:

[0314] Oral glucose tolerance test (OGTT): As described above, in the last week of administration, the OGTT was measured after fasting for 12 hours (fasting from 20:30:00 in the evening to 08:30:00 the next day). The fasting weight of the mice was weighed, and glucose was administered orally according to the fasting weight of the mice. The dose of glucose administered orally was 2 g / kg (g glucose / kg fasting weight of the mouse). Fasting blood glucose and blood glucose levels at 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes after sugar administration were measured. Each mouse was strictly timed and blood glucose levels were accurately measured at 6 time points.

[0315] 5.3 Experimental results on the effect of strain MNH19250 on oral glucose tolerance of type 2 diabetic mice induced by high-fat diet:

[0316] See also Fig.13 , it can be seen that strain MNH19250 can significantly reduce the oral glucose tolerance of high-fat diet-induced type 2 diabetic mice. Specifically, Fig.13 The results showed that strain MNH19250 could significantly inhibit the increase in blood sugar in type 2 diabetic mice after taking glucose, significantly improve oral glucose tolerance, and significantly reduce the area under the oral glucose tolerance curve of type 2 diabetic mice, indicating that strain MNH19250 can significantly control blood sugar and significantly improve sugar homeostasis in type 2 diabetic mice.

[0317] 5.4 Experimental results of the effect of strain MNH19250 on fasting blood glucose in high-fat diet-induced type 2 diabetic mice:

[0318] See also Fig.14 It can be seen that strain MNH19250 can significantly reduce the fasting blood glucose of high-fat diet-induced type 2 diabetic mice. Therefore, strain MNH19250 has the activity of preventing or treating diabetes.

[0319] Example 6: Use of strain MNH19250 in treating and preventing obesity and related diseases

[0320] 6.1 Experimental methods:

[0321] 1) Experimental animals: The experimental mice were C57BL / 6J mice, 5-6 weeks old, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0322] 2) Preparation of the test substance of strain MNH19250: After thawing the glycerol cryopreservation tube of strain MNH19250 at 37°C, inoculate it on the MM01 plate in an anaerobic workstation for activation, inoculate the activated strain into MM01 liquid culture medium, and culture it anaerobically to obtain a sufficient amount of culture. After centrifuging and concentrating the cultured bacterial solution, resuspend the bacterial cells in PBS containing 0.05% L-CysHCl to obtain the purity and viable count (2×10 9 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0323] 3) Negative control: PBS containing 0.05% L-Cys HCl (cysteine ​​hydrochloride) was used as a negative control

[0324] 4) Experimental process: After the quarantine period, 5-6 week old C57BL / 6J male mice were fed a high-fat diet for 10 weeks. Twelve mice with a body weight range of 35.50g-44.49g were selected and randomly stratified according to body weight, 6 mice / group, and divided into 2 groups (experimental group and control group). The day of drug administration after grouping was D1. The experimental group was given the test substance of strain MNH19250, and the control group was given the negative control substance. The drug was administered twice a day for a total of 28 days. During the experiment, the mice had free access to water and food, and a 12h / 12h day and night cycle was adopted. During the trial, a general clinical observation was conducted once after the drug administration. The animals were weighed twice a week during the drug administration period; the animals were weighed before dissection at the end of the trial.

[0325] Determination of mouse food intake: The amount of feed given was weighed on Day n, and the amount of remaining feed was weighed on Day n+1 to obtain the food intake 24 hours before administration; the amount of feed given and the amount of remaining feed after 24 hours of feed intake were weighed once a week during the administration period. The endpoint of this experiment was the day after the end of administration (D29). The end point of the experiment was dissected and sampled according to the protocol, and the body weight and percentage of body weight change, the results of various dissected data, and the results of serum test data were summarized and analyzed. All data were expressed in the form of Mean±SD, and GraphPadPrism software was used for drawing and statistical analysis. For pairwise comparisons, the t test (Student's t test) analysis method was used. Significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0326] 6.2 Effect of strain MNH19250 on the body weight of obese mice:

[0327] See also Fig.15 It can be seen that strain MNH19250 can significantly reduce the body weight and weight gain of obese mice induced by high-fat diet, and after four weeks of gavage intervention, the body weight was reduced by about 10% compared with before intervention, indicating that strain MNH19250 has a significant weight loss effect.

[0328] 6.3 Effects of strain MNH19250 on blood lipids in high-fat diet-induced obesity model mice:

[0329] See also Fig.16 The results showed that strain MNH19250 could significantly reduce the concentrations of total cholesterol (TCHO), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) in the serum of mice with high-fat diet-induced obesity, and significantly reduce the ratio of low-density lipoprotein cholesterol to high-density lipoprotein cholesterol. The above data indicate that strain MNH19250 has the effect of treating / preventing hypercholesterolemia, hyperlipidemia, and cardiovascular and cerebrovascular diseases.

[0330] 6.4 Effects of strain MNH19250 on body fat in high-fat diet-induced obesity model mice:

[0331] See also Fig.17 The results showed that strain MNH19250 could significantly reduce the weight of epididymal fat, perirenal fat, visceral fat and inguinal fat in mice induced by high-fat diet, 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.

[0332] Example 7: Effect of strain MNH19250 on the repair of colon mucosa in mice with impaired intestinal barrier function induced by high-fat diet

[0333] 7.1 Experimental methods:

[0334] 1) Experimental animals: The experimental mice were C57BL / 6J mice, 5-6 weeks old, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0335] 2) Preparation of the test substance of strain MNH19250: After thawing the glycerol cryopreservation tube of strain MNH19250 at 37°C, inoculate it on the MM01 plate in an anaerobic workstation for activation, inoculate the activated strain into MM01 liquid culture medium, and culture it anaerobically to obtain a sufficient amount of culture. After centrifuging and concentrating the cultured bacterial solution, resuspend the bacterial cells in PBS containing 0.05% L-CysHCl to obtain the purity and viable count (2×10 9 CFU / mL) of the test substances that meet the requirements of animal experiments.

[0336] 3) Negative control: PBS containing 0.05% L-Cys HCl was used as a negative control.

[0337] 4) Experimental process: After the quarantine period, 5-6 week old C57BL / 6J male mice were fed a high-fat diet for 10 weeks. Twelve mice with a body weight range of 35.50g-44.49g were selected and randomly stratified according to body weight, 6 mice / group, and divided into 2 groups (experimental group and control group). After grouping (D1), drug administration began. The experimental group was given MNH19250, and the control group was given a negative control. The drug was administered twice a day for a total of 28 days. During the experiment, mice were free to drink water and eat, and a 12h / 12h day and night cycle was adopted. During the experiment, a general clinical observation was performed after the end of drug administration. The endpoint of this experiment was the day after the end of drug administration (D29). At the end of the experiment, the colon was dissected and the mucus layer was stained with AB-PAS staining solution and the mucus layer thickness was measured. All data were expressed in the form of Mean±SD, and GraphPad Prism software was used for drawing and statistical analysis. For pairwise comparison, the t test (Student's t test) was used for analysis. Significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0338] 7.2 Effect of strain MNH19250 on the repair of colon mucosa in mice with impaired intestinal barrier function induced by high-fat diet:

[0339] See also Fig.18 The results showed that strain MNH19250 could significantly increase the thickness of the colon mucus layer in mice with high-fat diet-induced intestinal barrier dysfunction model, indicating that strain MNH19250 has the effect of treating / preventing intestinal barrier dysfunction.

[0340] Although the present invention has been disclosed with reference to certain embodiments, it is apparent that modifications and variations may be made without departing from the spirit and scope of the present invention as disclosed herein and as provided in the appended claims. In addition, it should be understood that although all examples disclosed illustrate embodiments of the present invention, they are provided only as non-limiting examples and therefore should not be considered as limiting the various aspects of the present invention thus described. The present invention is intended to have the full scope defined by the language of the present disclosure, the following claims, and any equivalents thereof. Therefore, the drawings and detailed description should be considered illustrative rather than restrictive.

Claims

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

1.

2. The Akkermansia muciniphila of claim 1, comprising an Amuc-1100 protein having at least 95% identity to the sequence shown in SEQ ID NO.4; and / or an Amuc_1631 protein having at least 95% identity to the sequence shown in SEQ ID NO.5; Preferably, the average nucleotide identity (ANI) value of the Akkermansia muciniphila and the Akkermansia muciniphila MNH19250 strain is 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.1%, 98.9%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 98.1%, 98. 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%; and / or the 16S rRNA sequence of Akkermansia muciniphila is at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical to the sequence shown in SEQ ID NO.1; and / or the Akkermansia muciniphila comprises an Amuc-1100 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO.4; and / or the Akkermansia muciniphila comprises an Amuc_1631 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO.5; Preferably, the Akkermansia muciniphila is the Akkermansia muciniphila MNH19250 strain, which is deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) with a deposit number of GDMCC NO: 63782 and a deposit date of June 21, 2024.

3. A composition comprising the Akkermansia muciniphila according to any one of claims 1 to 2, a culture thereof, a metabolite thereof, and / or a protein isolated from the Akkermansia muciniphila, wherein the Akkermansia muciniphila has a similar structure to that of Akkermansia muciniphila. muciniphila) MNH19250 strains having an average nucleotide identity (ANI) value of at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2% , 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, and / or, having the same 16S rRNA sequence having at least 98.65% identity to the sequence shown in SEQ ID NO.1, for example at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity; and / or the Akkermansia muciniphila comprises an Amuc-1100 protein having at least 96%, at least 97%, at least 98%, at least 99% identity to the sequence shown in SEQ ID NO.4; and / or the Akkermansia muciniphila comprises an Amuc_1631 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO.5; And / or the Akkermansia muciniphila is Akkermansia muciniphila MNH19250 strain, which is deposited in Guangdong Microbiological Culture Collection Center (GDMCC) with a deposit number of GDMCC NO: 63782 and a deposit date of June 21, 2024; Preferably, the composition is any one of a medicine, a health product or a food; Preferably, the health product or food is any one of a nutritional composition, a food, a candy, a food bar, a food additive, a drink additive, and a dietary supplement.

4. The composition according to claim 3, wherein the Akkermansia muciniphila is selected from any one of the following: live bacteria, attenuated bacteria, inactivated bacteria, and freeze-dried bacteria; Preferably, the inactivated bacteria are heat-inactivated bacteria, such as pasteurized bacteria; Preferably, the composition comprises Akkermansia muciniphila as an active ingredient at a concentration of 10 7 Up to 10 12 CFU / g; Preferably, the culture of Akkermansia muciniphila is selected from any one of the following: a fermentation culture, a supernatant of a fermentation culture, or a dried product thereof; Preferably, the protein secreted by Akkermansia muciniphila is Amuc-1100 protein; and / or Amuc_1631 protein; Preferably, the composition further comprises one or more pharmaceutically acceptable carriers, food carriers, excipients and / or adjuvants.

5. The composition according to claim 3 or 4, further comprising one or more other active agents for preventing or treating metabolic diseases, cardiovascular diseases, inflammatory diseases and / or tumors; Preferably, the other active agent is one or more of probiotics, prebiotics, and GLP-1 receptor agonists; Preferably, the prebiotic is selected from inulin, mulberry leaf extract, berberine, ganoderma lucidum, green coffee bean extract, oat, pectin, potato or its extract, citrus polyphenols, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidins, resistant dextrin, yeast β-glucan, ginseng or its extract, nutritional compounds, biotin, polydextrose, fructooligosaccharide (FOS), galacto-oligosaccharide (GOS), starch, cellulose, b-glucan, hemicellulose, lactulose, manno-oligosaccharide, manno-oligosaccharide (MOS), inulin rich in fructooligosaccharide, oligo-glucose, tagatose, trans-galacto-oligosaccharide, pectin, resistant starch, xylo-oligosaccharide (XOS) and any combination thereof; Preferably, the probiotics are selected from at least one of lactic acid bacteria, lactobacillus, and butyrate-producing bacteria.

6. Use of the Akkermansia muciniphila according to any one of claims 1 to 2, or the composition according to any one of claims 3 to 5, in the preparation of a drug, health product or food for preventing, treating or alleviating inflammatory diseases, liver and kidney diseases, cardiovascular and cerebrovascular diseases, metabolic-related diseases, lipid-lowering, intestinal barrier damage-related diseases, immune-related diseases, and tumors in a subject in need thereof, wherein the Akkermansia muciniphila has a similar structure to that of Akkermansia muciniphila. muciniphila) MNH19250 strains having an average nucleotide identity (ANI) value of at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2% , 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%, and / or, having the same 16S rRNA sequence having at least 98.65% identity to the sequence shown in SEQ ID NO.1, for example at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity; and / or the Akkermansia muciniphila comprises an Amuc-1100 protein having at least 96%, at least 97%, at least 98%, at least 99% identity to the sequence shown in SEQ ID NO.4; and / or the Akkermansia muciniphila comprises an Amuc_1631 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO.5; And / or the Akkermansia muciniphila is Akkermansia muciniphila MNH19250 strain, which is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with a deposit number of GDMCC NO: 63782 and a deposit date of June 21, 2024.

7. A method for preventing, treating or alleviating inflammatory diseases, liver and kidney diseases, cardiovascular and cerebrovascular diseases, metabolism-related diseases, intestinal barrier damage-related diseases, immune-related diseases, and tumors, the method comprising administering an effective amount of Akkermansia muciniphila according to any one of claims 1 to 2 or the composition according to any one of claims 3 to 5 to a subject in need thereof, The Akkermansia muciniphila has an average nucleotide identity (ANI) value of at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2% with the Akkermansia muciniphila MNH19250 strain. , 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%, and / or, having the same 16S rRNA sequence having at least 98.65% identity to the sequence shown in SEQ ID NO.1, for example at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity; and / or the Akkermansia muciniphila comprises an Amuc-1100 protein having at least 96%, at least 97%, at least 98%, at least 99% identity to the sequence shown in SEQ ID NO.4; and / or the Akkermansia muciniphila comprises an Amuc_1631 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO.5; And / or the Akkermansia muciniphila is Akkermansia muciniphila MNH19250 strain, which is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with a deposit number of GDMCC NO: 63782 and a deposit date of June 21, 2024.

8. The use according to claim 6 or the method according to claim 7, wherein the inflammatory disease is selected from the group consisting of bronchitis, cervicitis, conjunctival inflammation, esophageal inflammation, myocardial inflammation, rectal inflammation, scleral inflammation, gum inflammation, bone inflammation, lung inflammation, respiratory tract inflammation, inflammatory skin disease, fibrotic disease, and encephalitis; The liver and kidney diseases are selected from non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), liver fibrosis, cirrhosis, alcoholic liver disease and drug-induced liver injury, kidney injury, abnormal kidney function; preferably, the Akkermansia muciniphila or the composition reduces the serum aspartate aminotransferase and / or alanine aminotransferase ALT and / or liver weight of the subject; and / or reduces the level of urea nitrogen BUN and / or creatinine CRE in the subject's serum; The metabolism-related disease is selected from obesity, metabolic syndrome, insulin deficiency or insulin resistance-related disorders, diabetes, glucose intolerance, lipid metabolism disorders, hyperglycemia, dyslipidemia, irritable bowel syndrome, intestinal contraction disorders, hypercholesterolemia, hyperlipidemia; The cardiovascular and cerebrovascular disease is selected from hypertension, atherosclerosis, coronary heart disease and / or stroke; The intestinal barrier damage-related disease is selected from inflammatory bowel disease, Crohn's disease, and ulcerative colitis; preferably, the Akkermansia muciniphila or the composition increases the thickness of the colonic mucus layer; The lipid reduction is selected from reducing visceral fat and / or local fat; Preferably, the Akkermansia muciniphila or the composition has at least one property selected from the following: regulating immunity while regulating metabolism; repairing intestinal barrier damage while reducing abdominal fat; reducing weight while improving liver damage; fighting inflammation while losing weight; fighting inflammation while lowering lipids; repairing intestinal barrier damage while lowering lipids; lowering lipids while lowering serum AST and / or ALT; improving liver damage while improving kidney damage; improving diabetes while improving liver damage; improving diabetes while improving kidney damage; improving atherosclerosis while improving liver damage; and improving atherosclerosis while lowering lipids.

9. The use according to claim 6 or the method according to claim 7, wherein the Akkermansia muciniphila or the composition can treat, prevent, alleviate or improve at least one of the following: (1) Lose weight or control weight; (2) Prevent, treat, improve or alleviate liver damage, NAFLD or NASH; (3) Prevent, treat, improve or alleviate renal damage and renal dysfunction; (4) Promote the expression of type I interferon IFNβ in the subject and improve immunity; (5) Prevent, treat, improve or alleviate cancer; (6) Prevent, treat, improve or alleviate diabetes; (7) reducing blood lipid and / or cholesterol levels in a subject; (8) reducing local fat, body fat percentage, and / or visceral fat of a subject; (9) Repairing the intestinal tissue mucosa of the subject; (10) Prevent, treat and improve hypertension and atherosclerosis.

10. Use of Akkermansia muciniphila according to any one of claims 1 to 2, or the composition according to any one of claims 3 to 5 comprising the Akkermansia muciniphila strain, in affecting or regulating immune signal transduction in a subject in need thereof, affecting intestinal barrier function, affecting or regulating fasting glucose homeostasis, affecting or regulating cholesterol homeostasis, triglyceride homeostasis, repairing liver damage, kidney damage, promoting local adipose tissue metabolism, and reducing liver and / or kidney damage; The reduction of liver damage is to reduce one or more elevated liver damage or liver disease indicators of the subject, such as reducing the serum levels of aspartate aminotransferase AST, alanine aminotransferase ALT and / or liver weight indicators of the subject; the reduction of kidney damage is to reduce the blood urea nitrogen BUN in the serum of the subject and / or reduce the creatinine CRE indicator in the serum; The Akkermansia muciniphila has an average nucleotide identity (ANI) value of at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2% with the Akkermansia muciniphila MNH19250 strain. , 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%, and / or, having the same 16S rRNA sequence having at least 98.65% identity to the sequence shown in ID NO. 1, such as at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity; and / or the Akkermansia muciniphila comprises an Amuc-1100 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO.4; and / or the Akkermansia muciniphila comprises an Amuc_1631 protein that is at least 96%, at least 97%, at least 98%, at least 99% identical to the sequence shown in SEQ ID NO.5; And / or the Akkermansia muciniphila is Akkermansia muciniphila MNH19250 strain, which is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with a deposit number of GDMCC NO: 63782 and a deposit date of June 21, 2024.

Images