Isolated ackermansiella muciniphila, composition containing same and application of isolated ackermansiella muciniphila in preventing re-fatting

Abstract

The invention provides isolated Ackermania muciniphila, a composition containing the same and application of the Ackermania muciniphila in preventing re-fatting. According to the present invention, the Akkermansia muciniphila has characteristics of high automatic aggregation ability and strong surface hydrophobicity, can effectively adhere to the intestinal epithelial cells, can prolong the intestinal retention time, and can improve the treatment effect. The strain adapts to the intestinal environment and is high in stability. The Akkermansia muciniphila disclosed by the invention can be used for remarkably inhibiting body weight rebound (reducing body weight growth rate and fat accumulation) after drug withdrawal, improving blood sugar steady state (reducing postprandial blood sugar and fasting blood sugar) and reducing visceral fat accumulation, and the action is lasting and does not rebound. An efficient and safe microbial therapy solution is provided for maintaining the body weight and preventing an easily-fat object from developing obesity after obesity treatment.

Description

Technical Field

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

[0002] Akkermansia muciniphila ( Akkermansia muciniphila ) is a mucin-degrading bacterium that normally 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] The hydrophobicity of the AKK PROBIO strain disclosed in CN116925975B reached 31% at 60 min; the self-aggregation tended to stabilize at 52% at 20 h. Some literature suggests 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.

[0005] With the improvement of people's living standards and changes in lifestyle, the incidence of obesity has risen rapidly. According to the World Health Organization, the number of obese people has doubled since 1980. In 2014, there were 1.9 billion overweight people in the world. Obesity is always a threat to physical health. Therefore, drug treatments related to obesity have received widespread attention. Modern commonly used weight loss drugs include liraglutide, orlistat, rimonabant, semaglutide, etc. Although the weight loss effect of GLP-1 analogs such as liraglutide and semaglutide is indeed significant in a short period of time, studies have found that the incidence of four gastrointestinal adverse reactions induced by GLP-1 agonists such as semaglutide and liraglutide is higher than that of other weight loss drugs, including biliary tract disease, pancreatitis, intestinal obstruction and gastric spasm. In addition, GLP-1 agonists are prone to rebound after discontinuation of medication. A clinical trial showed that participants treated with semaglutide recovered more than 50% of their lost weight one year after discontinuation of medication. In other words, persistent use of semaglutide can indeed ensure weight loss; but once the intervention stops, the possibility of rebound may be higher.

[0006] Studies in the population found that from enrollment to one year after discontinuation, the liraglutide group rebounded a total of 8.7 kg, the most among all intervention measures, while the combined intervention group of liraglutide + moderate to high intensity exercise had the least weight rebound, 5.1 kg less than the liraglutide group (P=0.04). Researchers believe that one of the reasons why liraglutide is easy to gain weight again after discontinuation of liraglutide is that liraglutide achieves weight loss by suppressing appetite and delaying gastric emptying, which is equivalent to forcing us to control our mouths. However, once we stop taking the medicine, we may not be able to cope with the restored appetite, which eventually leads to weight rebound (Jensen SBK, Blond MB, Sandsdal RM, et al. Healthy weight loss maintenance with exercise, GLP-1 receptor agonist, or both combined followed by one year without treatment: a post-treatment analysis of a randomised placebo-controlled trial[J]. eClinicalMedicine, 2024.).

[0007] That is to say, due to human intervention, that is, the increase of exogenous GLP-1, the body detects higher levels of GLP-1, and the synthesized endogenous GLP-1 will decrease accordingly. After the drug is suddenly stopped, the originally high level of GLP-1 in the body drops suddenly, and hunger and appetite will "return with a vengeance", leading to a rapid rebound in weight, blood sugar, blood lipids, and body fat.

[0008] It can be seen that most of the existing weight loss products on the market only treat the symptoms but not the root cause. Some even affect physical health, destroy the balance of intestinal flora, disrupt metabolism, easily cause rebound or dependence, and even cause serious side effects. Therefore, there is a need for a product that can reduce the side effects of drugs, prevent weight regain, or fundamentally improve the body's proneness to obesity.

[0009] Akkermansia muciniphila shows potential in treating metabolic disorders, including obesity, diabetes, and metabolic-associated fatty liver disease (MAFLD). However, since appetite will "return with a vengeance" after discontinuation of existing weight-loss drugs, making weight loss more difficult, screening out new species or strains from the genus Akkermansia muciniphila that can be effectively used to prevent weight regain remains a huge challenge, but also represents a huge unmet need. Summary of the invention

[0010] The present invention is based on the isolation of a novel strain of Akkermansia muciniphila ( Akkermansia muciniphila ). The live bacteria and pasteurized bacteria of the strain disclosed in the present invention can effectively prevent weight regain and maintain healthy weight, blood sugar and body fat after weight loss; the pasteurized bacteria have a significant effect in preventing weight regain, indicating that the bacteria contain a large amount of active substances or proteins, and the pasteurized bacteria improve the effectiveness and safety of the application of Akkermansia muciniphila. The metabolites of the strain disclosed in the present invention have anti-inflammatory effects, which are beneficial to alleviate the side effects induced by weight loss drugs, such as diarrhea, enteritis, pancreatitis, gastrointestinal inflammation, etc.

[0011] Specifically, the effects of the strain disclosed in the present invention in preventing weight regain include: 1. Improving weight regain after drug withdrawal and maintaining weight loss; 2. Improving blood sugar homeostasis imbalance after drug withdrawal and maintaining blood sugar balance; 3. Improving local fat accumulation after drug withdrawal and maintaining local fat weight loss effect; 4. Metabolites promote the transcription activity of IFNβ, anti-inflammatory and regulate the body's immunity, and reduce the side effects of GLP-1 agonist drugs.

[0012] At the same time, compared with the existing muciniphilic Akkermansia strains, 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 its 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.

[0013] In a first aspect, the present disclosure provides a composition for preventing weight regain, the composition comprising an effective amount of Akkermansia muciniphila or a culture of the Akkermansia muciniphila, the Akkermansia muciniphila deposit number is GDMCC No: 63782, wherein the Akkermansia muciniphila includes live bacteria, lyophilized bacteria or inactivated bacteria of the Akkermansia muciniphila, and the culture includes any one of the following A) to D): A) a fermentation broth of the Akkermansia muciniphila; B) the supernatant of the fermentation broth of the Akkermansia muciniphila; C) a fermentation broth of the Akkermansia muciniphila that has been inactivated; D) The concentrated or dried product of any one of A) to C) above.

[0014] In a second aspect, the present disclosure provides the Akkermansia muciniphila ( Akkermansia muciniphila ) or the culture of Akkermansia muciniphila, or the composition of the first aspect in the preparation of a medicine, health product or food for preventing weight regain or maintaining weight after weight loss.

[0015] In a third aspect, the present disclosure provides the Akkermansia muciniphila ( Akkermansia muciniphila ) or the culture of Akkermansia muciniphila, or the composition of the first aspect for preventing weight regain or maintaining a healthy body weight for non-disease treatment purposes.

[0016] In a fourth aspect, the present disclosure provides a non-therapeutic method for preventing obesity or maintaining a healthy weight, wherein a subject in need thereof takes an effective amount of the Akkermansia muciniphila ( Akkermansia muciniphila ) or the culture of Akkermansia muciniphila, or the composition of the first aspect. Non-therapeutic methods for preventing weight regain or maintaining a healthy weight can be health care, health preservation, etc. In some embodiments, the Akkermansia muciniphila ( Akkermansia muciniphila ) and Akkermansia muciniphila ( Akkermansia muciniphila ) (GCF_000020225.1) strain has an alignment score (AF) of at least 90%, at least 90.5%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100%.

[0017] In some embodiments, the Akkermansia muciniphila ( Akkermansia muciniphila) and Akkermansia muciniphila ( Akkermansia muciniphila ) The average nucleotide identity (ANI) values ​​of the MNH19250 strain were 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%, 9 7.3%, 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%.

[0018] In some embodiments, the Akkermansia muciniphila ( Akkermansia muciniphila ) having a 16S rRNA sequence that is 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.

[0019] In some embodiments, the Akkermansia muciniphila is an Akkermansia muciniphila species ( Akkermansia muciniphila )) and the new strain is different from Akkermansia muciniphila ( Akkermansia muciniphila) (GCF_000020225.1) has an average nucleotide identity (ANI) value of at least 95%, such as at least 95.1%, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, at least 96%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1%, at least 97.1%. %, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 99%, 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%.

[0020] In some embodiments, the metabolites of the muciniphilic Akkermansia include short-chain fatty acids, which include at least one of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric 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 1500 μg / mL. In some embodiments, the acetic acid content of the muciniphilic Akkermansia is not less than 50 μg / mg, 60 μg / mg, 70 μg / mg, 80 μg / mg, 90 μg / mg, 95 μg / mg or 100 μg / mg.

[0021] 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.

[0022] In some embodiments, the Akkermansia muciniphila ( Akkermansia muciniphila )MNH19250, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCCNo: 63782, and the deposit date is June 21, 2024.

[0023] In some embodiments, the culture of Akkermansia muciniphila includes a solid culture of Akkermansia muciniphila, a fermentation culture or a supernatant of a fermentation culture, an inactivated fermentation broth, or a concentrated or dried product of any of the foregoing.

[0024] In some embodiments, the fermentation culture or fermentation culture supernatant is a fermentation culture obtained under anaerobic culture conditions using a liquid culture medium, a fermentation culture supernatant, a fermentation broth inactivated product, or a concentrated or dried product of any of the foregoing.

[0025] In some embodiments, the composition is provided in liquid form or in solid form.

[0026] In some embodiments, the composition comprises 1×10 4 Up to 1×10 12 cfu / mL or 1×10 4 Up to 1×10 12 cfu / mg of live Akkermansia muciniphila.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] In some embodiments, each mg of the composition comprises 1×10 3 Up to 1×1017 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), 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 , 2×108 , 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 , 2×10 11 , 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 in between of colony forming units (CFU).

[0031] 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.

[0032] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (eg, lyophilized powder), or gel.

[0033] 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.

[0034] In some embodiments, the composition is in the form of an oral dosage or an injection.

[0035] In some embodiments, the composition further comprises one or more pharmaceutically acceptable carriers or excipients or auxiliary materials. The pharmaceutically acceptable auxiliary materials are well known to those skilled in the art.

[0036] 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.

[0037] 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.

[0038] In some embodiments, the composition further comprises one or more additional active agents.

[0039] The other active agent has an appetite suppressing function.

[0040] In some embodiments, suppressing appetite comprises reducing food intake and / or reducing appetite.

[0041] 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.

[0042] In some embodiments, the other active agent may be one or more of a probiotic, a prebiotic, or a combination thereof; Preferably, the probiotics are selected from at least one of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus kefir-like, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus sakei, Lactococcus lactis, and Lactococcus cremoris; 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.

[0043] 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.

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

[0045] In some embodiments, the 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.

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

[0047] In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric 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.

[0048] In some embodiments, the composition is a medicine, or a health product or a food.

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

[0050] As used herein, microbial population generally refers to the microbial population consisting of a single strain, species or genus, which may be the case when the subgroup culture group is separated and purified from such strains, species or genus. Therefore, for a given microbial population, if cultivated from a separated microbial species or strain, such a population will be referred to as purified or substantially pure in this article. The resulting population can be at least 80% purity for the microbial species or strain, at least 90% purity, at least 95% purity, at least 98% purity, at least 99% purity, at least 99.5% purity, or at least 99.9% purity relative to other microbial species or strains in this particular population. On the contrary, the level of non-desired strains in any specific 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 impurity level in the purified population aggregate, such as other undesirable microbial strains or species, can be the above-mentioned level of each desired population in proportion or less than the above-mentioned level. Where a composition comprises a consortium of multiple populations of microorganisms, each population may have the above-described purity, either prior to its incorporation into the composition, or when the consortium is measured in aggregate.

[0051] 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 Akkermansia muciniphila culture or by extracting from Akkermansia muciniphila culture; may correspond to components in isolated form from Akkermansia muciniphila, or any mixture of one or more components from Akkermansia muciniphila.

[0052] In some embodiments, preventing weight regain includes preventing weight regain in a patient receiving obesity treatment and / or preventing a subject with a predisposition to obesity from developing obesity.

[0053] In some embodiments, the obesity includes but is not limited to: overweight, obesity.

[0054] In some embodiments, the causes of obesity include, but are not limited to, at least one of a high-fat diet and a high-sugar diet.

[0055] In some embodiments, the obesity includes but is 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.

[0056] 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.

[0057] In some embodiments, obesity treatment includes but is not limited to administration of weight loss medications or probiotics.

[0058] In some embodiments, weight loss drugs include but are not limited to orlistat, phentermine, topiramate, GLP-1 receptor agonists (such as semaglutide, liraglutide, tirzepatide).

[0059] In some embodiments, the Akkermansia muciniphila of the present invention ( Akkermansia muciniphila ), or the composition is administered to an obese patient after treatment with a GLP-1 receptor agonist.

[0060] In some embodiments, the Akkermansia muciniphila of the present invention (Akkermansia muciniphila) , or the combination prevents weight recurrence by promoting the secretion of glucagon-like peptide-1 (GLP-1) by intestinal L cells; regulating food intake and energy metabolism; increasing the thickness of the colonic mucus layer, repairing intestinal barrier function, and reducing serum endotoxin levels; upregulating the expression of type I interferon IFNβ, inhibiting adipose tissue inflammation; or inhibiting visceral fat accumulation, reducing liver weight and fatty degeneration score.

[0061] In some embodiments, the Akkermansia muciniphila of the present invention ( Akkermansia muciniphila), or the composition has at least one property selected from the following: maintaining weight after discontinuation of weight loss drugs; preventing weight rebound induced by a high-fat diet; controlling weight gain caused by a high-sugar, high-fat diet; preventing blood sugar from rising and / or maintaining blood sugar balance, such as preventing blood sugar from rising and / or maintaining blood sugar balance after discontinuation of weight loss drugs; improving obesity-related metabolic disorders, including insulin resistance, hyperglycemia or non-alcoholic fatty liver disease (NAFLD); and preventing fat accumulation, such as preventing fat accumulation after discontinuation of weight loss drugs. In some embodiments, the prevention of re-obesity includes preventing patients receiving obesity treatment from re-obesity, for example, patients receiving GLP-1 receptor agonists (e.g., semaglutide).

[0062] The Akkermansia muciniphila isolated and obtained in the present invention ( Akkermansia muciniphila ) MNH19250 has strong self-aggregation 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.

[0063] The Akkermansia muciniphila isolated and obtained in the present invention ( Akkermansia muciniphila ) MNH19250 can also synthesize a large amount of short-chain fatty acids, and therefore can effectively prevent patients receiving obesity treatment from regaining weight and / or prevent subjects with a prone physique from developing obesity.

[0064] Beneficial effects of the present invention Akkermansia muciniphila of the present invention ( Akkermansia muciniphila ) has high auto-aggregation ability and strong surface hydrophobicity, can effectively adhere to intestinal epithelial cells, prolong intestinal residence time, and improve treatment effect. The strain adapts to the intestinal environment and has high stability. The muciniphilic Akkermansia ( Akkermansia muciniphila ) can significantly inhibit weight rebound after drug withdrawal (reduce weight gain rate and fat accumulation), improve blood sugar homeostasis (reduce postprandial blood sugar and fasting blood sugar), reduce visceral fat accumulation, and have a lasting effect without rebound. It provides an efficient and safe microbial therapy solution for maintaining weight after obesity treatment and preventing obesity in subjects with a physique prone to obesity. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] 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.

[0066] Figure 1 : shows the colony morphology photograph of strain MNH19250.

[0067] Figure 2 : Gram staining photographs of strain MNH19250 are shown.

[0068] Figure 3: shows the electron micrograph of strain MNH19250.

[0069] 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.

[0070] Figure 5 : Shows the tolerance results of strain MNH19250 to different concentrations of NaCl.

[0071] Figure 6 : Shows the tolerance results of strain MNH19250 to different concentrations of bile salts.

[0072] Figure 7 : Shows the 6h autoaggregation ability of strain MNH19250.

[0073] Figure 8 : The phylogenetic tree of strain MNH19250 is shown.

[0074] Figure 9A : Shows the endpoint body weight of an animal experiment to prevent weight regain in an obese mouse model.

[0075] Figure 9B : Shows the percentage of body weight gain after discontinuation of semaglutide in an animal experiment to prevent weight regain in an obese mouse model.

[0076] Figure 9C : Shows the percentage of body weight gain at the end point of the animal experiment to prevent re-obesity in an obese mouse model.

[0077] Figure 9D : Shows the body weight gain rate after discontinuation of semaglutide in an animal experiment to prevent weight regain in an obese mouse model.

[0078] Figure 10A : Shows the area under the oral glucose tolerance curve in an oral glucose tolerance test.

[0079] Figure 10B : Shows fasting blood glucose in an oral glucose tolerance test.

[0080] Figure 11A : MNH19250 was shown to improve liver weight after cessation of semaglutide treatment.

[0081] Figure 11B : MNH19250 was shown to improve subcutaneous fat weight after cessation of semaglutide treatment.

[0082] Figure 11C : MNH19250 was shown to improve epididymal fat weight after cessation of semaglutide treatment.

[0083] Figure 11D : MNH19250 was shown to improve perirenal fat mass after cessation of semaglutide treatment.

[0084] Figure 11E : MNH19250 was shown to improve mesenteric fat weight after cessation of semaglutide treatment.

[0085] Figure 11F : MNH19250 was shown to improve white fat mass after cessation of semaglutide treatment.

[0086] Figure 11G : MNH19250 was shown to improve visceral fat weight after cessation of semaglutide treatment.

[0087] Figure 12 : A bar graph showing the relative fluorescence values ​​of IFNβ expression in the presence of strain MNH19250.

[0088] Deposit of strains

[0089] Akkermansia muciniphila ( Akkermansia muciniphila ) MNH19250, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC No: 63782, the deposit date is June 21, 2024, the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, the deposit name is Akkermansia muciniphila MNH19250. DETAILED DESCRIPTION

[0090] 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 is a new strain of Akkermansia muciniphila. Furthermore, the present invention studies the biochemical properties and therapeutic uses of the strain.

[0091] 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.

[0092] 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 (e.g., can allow for specific changes in the composition and / or activity of a microbiome).

[0093] 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.

[0094] 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.

[0095] In the context of the present invention, the term "polypeptide" is equivalent to "protein". A polypeptide has a specific amino acid sequence. A "variant" of a polypeptide of the present invention preferably has an amino acid sequence that has at least 50% sequence identity with a polypeptide of the present invention.

[0096] The term "homeostasis" refers to the mechanisms of processes that contribute to maintaining a balanced internal state in an organism, eg, glucose regulation homeostasis, triglyceride homeostasis, cholesterol homeostasis.

[0097] The term "preventing weight regain" includes preventing weight loss caused by the use of slimming drugs, controlling diet or exercise, and weight rebound after stopping the drug, resuming diet or stopping exercise; as well as preventing subjects with a physique prone to obesity from developing obesity.

[0098] 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%, they can be judged to belong to different species (see Kim, M., Oh, H.-S., Park, S.-C., & Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary Microbiology, 64(Pt 2), 346–351, and Liu, C., Du, M.-X., Abuduaini,R., Yu, H.-Y., Li, D.-H., Wang, Y.-J., Liu, S.-J. (2021). Enlightening the taxonomy darkness of human gut microbiomes with a cultured biobank. Microbiome, 9(1), P23).

[0099] 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.

[0100] 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.).

[0101] 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.

[0102] 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 inventors. Akkermansia muciniphila ) (GCF_000020225.1) has an average nucleotide identity ANI value of 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%, 97.9 When the content of the bacterial strains is 7.3%, 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.

[0103] 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%, at least 98.8%, at least 99%, 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%, it can be determined to belong to the same bacterial species.

[0104] The alignment fraction (AF) refers to the fraction of orthologous genes shared between two genomes. Based on ANI>95%, the researchers proposed further using the alignment fraction (AF, i.e. the fraction of orthologous genes shared between two genomes) as an additional threshold to restrict species to ensure that the ANI value is not based on a small set of conserved genes. There is no unified standard for the AF threshold. Some literature uses 65% (Parks, DH, Chuvochina, M., Chaumeil, PA. et al. A complete domain-to-species taxonomy for Bacteria and Archaea. Nat Biotechnol 38, 1079–1086(2020). ), and some literature uses 30% (Zeng, S., Patangia, D., Almeida, A. et al. Acompendium of 32,277 metagenome-assembled genomes and over 80 million genesfrom the early-life human gut microbiome. Nat Commun 13, 5139 (2022). ). In short, the AF value can be used as an auxiliary additional threshold.

[0105] A "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, ribosomal 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, ribosomal 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 that gains or loses antibiotic resistance (compared to another strain of the same species) or gains or loses a biosynthetic ability (e.g., an auxotrophic strain), the strains can be distinguished by selection or counter-selection using antibiotics or nutrients / metabolites.

[0106] "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.

[0107] 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.

[0108] Suitable pharmaceutically acceptable excipients that may be used include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and flavors.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] In some embodiments, the Akkermansia muciniphila in the composition of the present disclosure is lyophilized. In some embodiments, the Akkermansia muciniphila in the composition of the present disclosure is spray-dried. In some embodiments, the Akkermansia muciniphila in the composition of the present disclosure is lyophilized or spray-dried and is alive. In some embodiments, the Akkermansia muciniphila in the composition of the present disclosure is lyophilized or spray-dried and can partially or completely colonize in the intestine. In some embodiments, the lyophilized Akkermansia muciniphila is reconstituted before administration. In some embodiments, the reconstitution is performed using a diluent described herein.

[0115] 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.

[0116] In some embodiments, the composition is prepared by freeze drying or spray drying. The composition disclosed herein includes a pharmaceutical composition, a health product or a food.

[0117] 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.

[0118] The pharmaceutical composition disclosed herein can be used to prevent patients receiving obesity treatment from regaining weight and / or prevent subjects with obesity-prone physiques from developing obesity.

[0119] Amuc-1100 and Amuc_1631 (P9) proteins are derived from Akkermansia muciniphila ( Akkermansia muciniphilaThe outer membrane proteinAmuc_1100 of . 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 proteinAmuc_1100 of Akkermansia muciniphila promotes intestinal 5-HT biosynthesisand extracellular availability through TLR2 signaling.[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 a glucagon-like peptide-1-inducing protein that improves glucose homeostasis and ameliorates metabolic disease in mice[J]. Nature Microbio-logy, 2021, 6: 563-573";Amuc-1100 remains stable during pasteurization, and can inhibit the expression of intestinal CB1 receptors and enhance the expression of tight junction proteins;"Ding, G.,Yang, X., Li, Y.etal., Gut microbiota regulates gut homeostasis, mucosal immunity andinfluences immune-related diseases. Mol Cell Biochem (2024). https: / / doi.org / 10.1007 / s11010-024-05077-y".

[0120] 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 muciniphilaincreases the GLP-1 secretionof 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)".

[0121] Therefore, the Akkermansia muciniphila ( Akkermansia muciniphila ) can also affect or modulate immune signaling and / or affect intestinal barrier function and / or affect glucose homeostasis and / or cholesterol homeostasis and / or triglyceride homeostasis.

[0122] 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.

[0123] Example The liquid MM01 culture 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, KH2PO4 1g, magnesium sulfate 0.5g, sodium bicarbonate 10g, sodium chloride 2g).

[0124] 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, KH2PO4 1g, magnesium sulfate 0.5g, sodium bicarbonate 10g, sodium chloride 2g), agar 15g / L.

[0125] Anaerobic blood agar plates were purchased from Huankai Microorganisms, with the 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.

[0126] The above culture medium can be prepared by conventional preparation methods and sterilization methods.

[0127] Example 1: Isolation and identification of strains 1.1 Isolation and purification of strain MNH19250 An intestinal strain numbered MNH19250 was isolated from a sample of a healthy male volunteer in Guangzhou, Guangdong, China. The isolation method adopted the 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.

[0128] Specifically, the strain isolation method is as follows: 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.

[0129] Dispense physiological saline in a biosafety cabinet, 9 mL / tube; prepare anaerobic blood agar plates for strain isolation culture medium, and transfer them to the anaerobic workstation 24 hours in advance, marking the sample information, culture medium type, isolation date, etc.

[0130] Take a new sample and place it in the anaerobic workstation and shake it on a vortex shaker for 1 min to mix it. 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.

[0131] Take 10- 6Drop 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.

[0132] Observe the growth of the strain on the isolation 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.

[0133] 1.2 Morphological characteristics of strain MNH19250 Culture and morphological characteristics 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 0.5~1µm×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 .

[0134] 1.3 Physiological characteristics of strain MNH19250 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 (for the results of strain tolerance to different concentrations of NaCl, see Figure 5 ); strain MNH19250 can survive and grow in the bile salt concentration range of 0%~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 ).

[0135] 1.4 Results of biochemical identification of strain MNH19250 by API 20A API 20A (purchased from BioMérieux, CN2030025) was used to perform biochemical identification of strain MNH19250. For specific experimental operations, please refer to the conventional API reagent operation guide. The culture conditions of strain MNH19250 were: 37°C, anaerobic. The experimental results are shown in Table 1.

[0136]

[0137] 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.

[0138] 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.

[0139] 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 a positive reaction is shown.

[0140] 1.5 Minimum inhibitory concentration test of antibiotics for strain MNH19250 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.

[0141]

[0142] 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.

[0143] 1.6 Determination of the autoaggregation ability of strain MNH19250 The strain MNH19250 was inoculated in 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.

[0144] 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.

[0145] The auto-aggregation ability of the strain was calculated according to the following formula: auto-aggregation ability of the strain (%) = [1-(At / A0)] × 100%.

[0146] 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, Meng Tian, ​​Xueping Yu, et al., Foods. 2024, Jan 30;13(3): 442. Akkermansia muciniphila 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, thereby exerting a probiotic effect.

[0147] 1.7 Determination of surface hydrophobicity of strain MNH19250 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.

[0148] 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: H%=[(A0-A) / A0]×100%, where A0 and A represent the absorbance values ​​before and after organic solvent extraction, respectively.

[0149] The surface hydrophobicity of strain MNH19250 was 57.06%, which was more than 25% higher than that of other strains in the literature (Xin Ma, Meng Tian, ​​Xueping Yu, et al., Foods. 2024, Jan 30; 13(3): 442. Akkermansia muciniphilaPROBIO (https: / / doi.org / 10.3390 / foods13030442); Figure 1c, the surface hydrophobicity of A. muciniphila PROBIO is 31% (1h)). Strains with strong surface hydrophobicity have stronger epithelial cell adhesion ability and can better colonize in the human intestine to play a probiotic role. At the same time, they can inhibit the adhesion of other pathogens and play an intestinal probiotic function.

[0150] 1.8 Amplification of the 16S rRNA gene of strain MNH19250 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.

[0151] The primer pairs used for PCR of 16S rRNA gene are: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 2) 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO. 3). The PCR reaction procedure is as follows: Pre-denaturation: 94℃, 4 min; denaturation: 94℃, 50 sec; annealing: 52℃, 40 sec; extension: 72℃, 70sec; final extension: 72℃, 10 min (36 cycles).

[0152] 1.916S rRNA gene sequencing The PCR product was purified and sequenced by Bioengineering to obtain the 16S rRNA gene sequence (1355 bp), as shown in SEQ ID NO. 1:

[0153] 1.10 Identification of strain MNH19250 The 16S rRNA gene sequence as shown in SEQ ID NO. 1 was analyzed using the NCBI Basic Local Alignment Search Tool to confirm the strain classification information.

[0154] The measured sequences were compared with the data in GenBank by BLAST analysis. The results showed that the strain with the highest similarity to MNH19250 was Akkermansia muciniphila ( Akkermansia muciniphila ), the similarity is 100%, so the strain MNH19250 is judged to be Akkermansia muciniphila ( Akkermansia muciniphila ) strains planted.

[0155] The strain MNH19250 was compared with Akkermansia genus obtained from GenBank and other databases ( Akkermansia sp. ) were compared with the 16S rRNA gene sequences of related strains to construct a phylogenetic tree.

[0156] 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.

[0157] From the phylogenetic tree, it can be seen that strain MNH19250 is closely related to Akkermansia spp. Akkermansia sp. )and Akkermansia muciniphila Muc AY271254, therefore, strain MNH19250 was identified as Akkermansia muciniphila ( Akkermansiamuciniphila ) new strains planted.

[0158] 1.11 Genome analysis of strain MNH19250 Genome analysis of strain MNH19250 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%.

[0159] 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%.

[0160] 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.

[0161] 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.

[0162]

[0163] 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.

[0164]

[0165] The analysis of potential primary metabolic gene clusters in the genome was performed using gutSMASH5 (version: 1.0.0). The detailed alignment results are shown in Table 5.

[0166]

[0167] 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.

[0168]

[0169] sequence: >MNH19250_01469 hypothetical protein MSNWITDNKPAAMVAGVGLLLFLGLSATGYIVNSKRSELDKKISIAAKEIKSANAAEITPSRSSNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILITDTSSWLGFQVYSTQAPSVQAASTLGFELKAINSLVNKLAECGLSKFIKVYRPQLPIETPANNPEESDEADQAPWTPMPLEIAFQGDRESVLKAMNAITGMQDYLFTVNSIRIRNERMMPPPIANPAAAKPAAAQPATGAASLTPADEATAPAAPAIQQVIKPYMGKEQVFVQVSLNLVHFNQPKAQEPSED (SEQ ID NO. 4)

[0170] Sequence: >MNH19250_00667 Tail-specific protease (SEQ ID NO. 5) Example 2. Analysis of fatty acid composition of strain MNH19250 2.1 Analysis of fatty acid composition of strain MNH19250 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 automated bacterial identification system from MIDI (Microbial ID, Inc., Newark, Del.).

[0171] The main fatty acid (>10%) of the experimental strain MNH19250 was C15:0 anteiso 48.73%. 2.2 Analysis of short-chain fatty acids (SCFA) of strain MNH19250 Bacteria preparation 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.

[0172] Pretreatment of bacterial supernatant samples (1) After thawing, vortex the sample for 3 min to mix; (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. (3) Add 750 n 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; (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.

[0173] Pretreatment of bacterial samples (1) After the sample is thawed, add 100% ultrapure water extract to resuspend it evenly; (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; (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; (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; (5) aspirating 90% of the supernatant into the liner tube of a sampling bottle for GC-MS / MS analysis;

[0174] (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.

[0175] Preparation of standard products: Prepare acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid at different concentrations of 0.005 μg / mL, 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 8 μg / mL, 10 μg / mL, and 20 μg / mL as standard solutions. Chromatographic mass spectrometry detection was performed using the conditions shown in Table 8.

[0176] Obtain the chromatographic peak intensity data of the corresponding quantitative signal of each concentration standard. Use the external standard to internal standard concentration ratio (Concentration Ratio) 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.

[0177]

[0178] 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 9.

[0179]

[0180] 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.

[0181] Example 3: Use of strain MNH19250 to prevent obesity 3.1 Animal experiment of MNH19250 in preventing relapse of obesity in obese mouse model after cessation of semaglutide treatment Mice were fed a high-fat diet for 10 weeks, and then induced to be obese. After 4 weeks of semaglutide treatment, mice were treated with live MNH19250 and pasteurized MNH19250 to investigate the effect of MNH19250 on preventing obesity after semaglutide treatment was discontinued. This experimental protocol has been reviewed by the ethics committee of the Experimental Animal Care and Use Committee of Muen Biotechnology.

[0182] 3.1.1 Experimental methods Experimental animals: C57BL / 6J mice were purchased from Guangdong Yaokang Biotechnology Co., Ltd.

[0183] Test strains: After thawing the glycerol cryopreserved tube of MNH19250 strain at 37°C, inoculate it into MM01 medium in a biosafety cabinet for activation. The activated strain was inoculated into MM01 liquid medium for cultivation to obtain a sufficient amount of culture. The cultured bacterial solution was concentrated by centrifugation and resuspended in PBS containing 25% glycerol and 0.05% L-Cys HCl to obtain the purity and viable count (2×10 9 CFU / mL) of the test subject MNH19250 live bacteria (MNH19250-L) that met the requirements of animal experiments, among which some of the MNH19250 live bacteria were incubated at 70°C for 30 minutes to obtain pasteurized MNH19250 bacteria (MNH19250-P).

[0184] Negative control: PBS containing 25% glycerol and 0.05% L-Cys HCl was used as a negative control.

[0185] Experimental process: After the quarantine period of 5-6-week-old C57BL / 6J male mice, 8 mice were randomly selected to be fed with basal feed, and the remaining mice were all fed with high-fat diet for 10 weeks. After 10 weeks, all mice fed with basal feed were transferred to NCD group. 32 mice with a body weight range of 36g-44g were selected for mice fed with high-fat diet, and 8 mice / group were randomly stratified according to body weight. They were divided into 5 groups, namely NCD group, HFD group, HFD+Sema group, MNH19250-L group and MNH19250-P group. Among them, the NCD group continued to be fed with basal feed, and the other 4 groups continued to be fed with high-fat diet. After grouping (D1), drug administration began. The NCD group and HFD group were given negative control products, and the remaining three groups were given semaglutide (30nmol / kg, once every three days). Semaglutide administration was stopped after 4 weeks of administration. The HFD+Sema group began to be given the negative control substance, the MNH19250-L group and the MNH19250-P group began to be given MNH19250-L and MNH19250-P, and the NCD group and the HFD group continued to be given the negative control substance. The drug was administered once a day for a total of 28 days. During the administration period, the mice continued to be given a high-fat diet. During the experiment, the mice had free access to water and food, and a 12h / 12h day and night cycle was adopted.

[0186] During the experiment, general clinical observation was performed once after each administration. The endpoint of this experiment was the day after the end of administration (D56). The end point of the experiment was dissected and sampled according to the protocol, and the body weight and body weight change percentage, various dissected data results and serum test data results were summarized and analyzed; fasting blood sugar and blood sugar levels at 15min, 30min, 60min, 90min, and 120min after sugar administration were measured. Each mouse was strictly timed and blood sugar levels were accurately measured at 6 time points to obtain blood sugar indicators.

[0187] Subcutaneous fat, epididymal fat, perirenal fat, and mesenteric fat were directly dissected and weighed for measurement; visceral fat was the sum of epididymal fat, perirenal fat, and mesenteric fat; and white fat was the sum of all fat.

[0188] 3.2 Experiment on the effect of MNH19250 on oral glucose tolerance in high-fat diet-induced type 2 diabetic mice: Oral glucose tolerance test (OGTT): In the last week of administration, measure the OGTT after fasting for 12 hours (e.g. fasting from 20:30:00 in the evening to 08:30:00 the next day). Weigh the fasting weight of mice, and gavage glucose according to the fasting weight of mice. The gavage glucose dose is 2g / kg (g glucose / kg fasting weight of mice). Measure fasting blood sugar and blood sugar levels 15min, 30min, 60min, 90min, and 120min after sugar administration. Strictly time each mouse and accurately measure blood sugar levels at 6 time points.

[0189] 3.3 Experimental Results The experimental results are shown in Figure 9A - Figure 9D , Figure 10A - Figure 10B as well as Figure 11A - Figure 11G All data were expressed as Mean ± SD, and GraphPad Prism software was used for plotting and statistical analysis. For pairwise comparisons, Student's ttest was used for analysis. No significant difference was not indicated; significant difference was indicated by *, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0190] (1) MNH19250 can improve weight regain after cessation of semaglutide treatment. Figure 9A to Figure 9D shown. Figure 9A to Figure 9D The results showed that pasteurized MNH19250 significantly reduced weight gain and weight gain rate after discontinuation of semaglutide treatment, and live MNH19250 reduced weight gain and weight gain rate after discontinuation of semaglutide treatment, indicating that MNH19250 can improve weight regain after discontinuation of semaglutide treatment, maintain weight after weight loss, and has the use of preventing weight regain.

[0191] (2) MNH19250 can improve the increase in blood glucose after cessation of semaglutide treatment. Figure 10A to Figure 10B shown. Figure 10A and Figure 10B The results showed that MNH19250 could significantly reduce AUC-OGTT, 2h postprandial blood glucose and fasting blood glucose after discontinuation of semaglutide treatment, indicating that MNH19250 could improve the imbalance of blood glucose homeostasis after discontinuation of semaglutide treatment, improve blood glucose increase, and prevent blood glucose increase and maintain blood glucose balance.

[0192] (3) MNH19250 can improve fat accumulation after cessation of semaglutide treatment. Figure 11A to Figure 11G shown. Figure 11A to Figure 11G The results showed that MNH19250 could significantly reduce liver weight, subcutaneous fat, epididymal fat, perirenal fat, mesenteric fat, white fat, and visceral fat after discontinuation of semaglutide treatment, indicating that MNH19250 could improve fat accumulation after discontinuation of semaglutide treatment and could prevent body fat accumulation and weight regain.

[0193] Example 4. Effect of strain MNH19250 on IFNβ expression Although the weight loss effect of GLP-1 agonists such as liraglutide and semaglutide is indeed significant in the short term, studies have found that the incidence of four gastrointestinal adverse reactions induced by GLP-1 agonists semaglutide and liraglutide is higher than that of other weight loss drugs, including biliary disease, pancreatitis, intestinal obstruction and gastric spasm. Diarrhea, gastrointestinal inflammation and other reactions are common side effects of GLP-1 agonists.

[0194] Type I interferon β (IFNβ) has been shown to have anti-inflammatory effects and can promote macrophage reprogramming to promote anti-inflammatory phenotypes, thereby helping to terminate inflammation (Kumaran Satyanarayanan, S., El Kebir, D., Soboh, S.et al. IFN-β is a macrophage-derived effector cytokine facilitating the resolution of bacterial inflammation. Nat Commun 10, 3471 (2019). https: / / doi.org / 10.1038 / s41467-019-10903-9). IFNβ can also regulate immunity and achieve antiviral and anti-tumor effects through immune regulation.

[0195] 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.

[0196] 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 I IFN-dependent monocyte reprogramming of the tumor microenvironment" Cell 184, 5338-5356).

[0197] 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.

[0198] Control group (Control): DMEM complete medium (Gibco, containing 10% FBS) containing 10% volume of MM01 liquid medium; MSA-2 group (positive control group): DMEM complete medium containing 10 μM MSA-2 (purchased from Taoshu Biological); Strain MNH19250 group: DMEM complete medium containing 10% volume of the culture supernatant of strain MNH19250.

[0199] 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.

[0200] The experimental results are as follows Figure 12 As shown, the metabolites of strain MNH19250 can significantly promote the transcriptional activity of IFNβ. Therefore, these results indicate that MNH19250 and its metabolites have anti-inflammatory and immune-regulating functions, thereby reducing the side effects of GLP-1 agonist drugs such as liraglutide and semaglutide.

[0201] 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. A composition for preventing weight regain, characterized in that: The composition comprises an effective amount of Akkermansia muciniphila or a culture of the Akkermansia muciniphila, the Akkermansia muciniphila deposit number is GDMCC No: 63782, wherein the Akkermansia muciniphila comprises live bacteria, lyophilized bacteria or inactivated bacteria of the Akkermansia muciniphila, and the culture comprises any one of the following A) to D): A) a fermentation broth of the Akkermansia muciniphila; B) the supernatant of the fermentation broth of the Akkermansia muciniphila; C) a fermentation broth of the Akkermansia muciniphila that has been inactivated; D) The concentrated or dried product of any one of A) to C) above.

2. The composition according to claim 1, characterized in that The inactivated bacteria are bacteria obtained by pasteurization or lysis; the inactivated fermentation broth is a product obtained by pasteurization or lysis of the fermentation broth of Akkermansia muciniphila.

3. The composition according to claim 1 or 2, characterized in that The composition further comprises one or more pharmaceutically acceptable carriers, food carriers, excipients and / or adjuvants.

4. The composition according to claim 1 or 2, characterized in that The composition may also contain one or more other active agents.

5. The composition according to claim 4, characterized in that The other active agents are one or more of probiotics and prebiotics.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of a medicine, health product or food for preventing weight regain or maintaining weight after weight loss in a subject in need thereof.

7. The use according to claim 6, characterized in that: The composition has at least one selected from the following: (1) Maintaining body weight after discontinuation of weight-loss medication; (2) prevent blood sugar increases and / or maintain blood sugar balance after discontinuation of weight loss medications; (3) Prevent fat accumulation after discontinuation of weight loss medication.

8. The use according to claim 7, characterized in that: The subject is a subject receiving a GLP-1 receptor agonist for weight loss.

9. The use according to claim 7, characterized in that: The weight loss drug is a GLP-1 receptor agonist, and the GLP-1 receptor agonist includes liraglutide, semaglutide or telpotide.

10. Use of the composition according to any one of claims 1 to 5 in the preparation of medicines, health products or foods for preventing blood sugar from rising or maintaining blood sugar balance after drug withdrawal for weight loss.

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