Isolated Akkermansia muciniphila, composition containing same and use for preventing weight regain

Abstract

The present invention provides isolated Akkermansia muciniphila, a composition containing the same, and its use in preventing weight regain. Akkermansia muciniphila ) has high autoaggregation ability and strong surface hydrophobicity, can effectively adhere to intestinal epithelial cells, prolong intestinal residence time, and improve therapeutic effects. This strain adapts to the intestinal environment and has high stability. The present invention's Akkermansia muciniphila ( Farmhouse mucinophila ) can significantly inhibit weight rebound after discontinuation (reducing weight gain and fat accumulation), improve blood sugar homeostasis (lowering postprandial and fasting blood sugar levels), and reduce visceral fat accumulation, with long-lasting effects. This provides a highly effective and safe microbial therapy solution for maintaining weight after obesity treatment and preventing obesity progression in individuals prone to obesity.

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 isolation, a composition containing the same, 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] Screening for new strains of Akkermansia muciniphila faces significant challenges because it primarily resides in the human and animal intestines and is a strictly anaerobic microorganism with extremely high nutritional and culturing environment requirements and a long growth cycle. Furthermore, when validating drug efficacy, this genus is difficult to verify through in vitro cell-based assays due to its anaerobic nature. In vivo animal experiments also struggle to maintain a stable viable count due to the difficulty of culturing and the high degree of anaerobicity, and there are also issues with reproducibility. These in vitro and in vivo technical challenges have limited the discovery and application of new strains of Akkermansia muciniphila.

[0004] The hydrophobicity of the AKK PROBIO strain disclosed in CN116925975B reaches 31% after 60 minutes; its self-aggregation activity stabilizes at around 52% after 20 hours. Some literature suggests that AKK PROBIO has a weak ability to form biofilms, and in vitro testing results indicate that AKK PROBIO has a low ability to colonize in the intestine (https: / / doi.org / 10.3390 / foods13030442, Section 3.1). Poor colonization and adhesion abilities of the AKK strain would limit its commercial development.

[0005] With improved living standards and changing lifestyles, the incidence of obesity has rapidly increased. According to the World Health Organization, the number of obese people has doubled since 1980. In 2014, there were 1.9 billion overweight people worldwide. Obesity poses a constant threat to health. Consequently, drug treatments for obesity and other related conditions have garnered widespread attention. Commonly used weight loss medications today include liraglutide, orlistat, rimonabant, and semaglutide. While GLP-1 analogs such as liraglutide and semaglutide do have significant short-term weight loss effects, studies have found that GLP-1 agonists such as semaglutide and liraglutide are associated with a higher incidence of four gastrointestinal adverse reactions than other weight loss medications, including biliary tract disease, pancreatitis, intestinal obstruction, and gastroschisis. Furthermore, GLP-1 agonists are prone to rebound weight loss after discontinuation. One clinical trial showed that participants treated with semaglutide regained over 50% of their lost weight one year after stopping the drug. In other words, persistent use of semaglutide can indeed ensure weight loss; but once the intervention stops, the possibility of rebound is likely to be higher.

[0006] A population-based study found that from enrollment to one year after discontinuation, the liraglutide group regained a total of 8.7 kg, the most among all intervention groups. The combined liraglutide and moderate-to-high-intensity exercise group regained the least weight, a full 5.1 kg less (P=0.04). Researchers believe that one reason for the increased risk of weight regain after discontinuation of liraglutide is that liraglutide suppresses appetite and delays gastric emptying, effectively forcing us to control our appetite. However, once the drug is discontinued, we may be unable to cope with the return of appetite, ultimately leading to weight regain (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 randomized placebo-controlled trial[J]. eClinicalMedicine, 2024).

[0007] That is, due to human intervention, namely the increase in exogenous GLP-1, the body detects higher levels of GLP-1, and the synthesis of endogenous GLP-1 decreases accordingly. After abruptly stopping the drug, the body's previously high levels of GLP-1 drop sharply, and hunger and appetite "return with a vengeance," leading to a rapid rebound in weight, blood sugar, blood lipids, and body fat.

[0008] As can be seen, most existing weight loss products on the market only treat symptoms, not the root cause. Some even harm health, disrupt intestinal flora balance, disrupt metabolism, easily cause rebound or dependency, and even cause serious side effects. Therefore, there is a need for a product that can reduce drug side effects, prevent weight regain, or fundamentally improve obesity-prone physiques.

[0009] Akkermansia muciniphila shows potential in treating metabolic disorders, including obesity, diabetes, and metabolic-associated fatty liver disease (MAFLD). However, since appetite "returns 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 new strain of Akkermansia muciniphila ( Akkermansia muciniphila The live and pasteurized strains of the disclosed strains can effectively prevent weight regain and maintain healthy weight, blood sugar, and body fat after weight loss. The significant anti-weight regain effect of the pasteurized strains indicates that the bacteria contain a large amount of active substances or proteins, and the pasteurized strains improve the effectiveness and safety of Akkermansia muciniphila. The metabolites of the disclosed strains have anti-inflammatory effects and are beneficial for alleviating side effects induced by weight loss drugs, such as diarrhea, enteritis, pancreatitis, and gastrointestinal inflammation.

[0011] Specifically, the effects of the strain disclosed herein on preventing weight regain include: 1. improving weight regain after drug discontinuation and maintaining weight loss; 2. improving blood sugar homeostasis imbalance after drug discontinuation and maintaining blood sugar balance; 3. improving local fat accumulation after drug discontinuation and maintaining the local fat weight loss effect; 4. metabolites promote the transcriptional activity of IFNβ, anti-inflammatory and regulate the body's immunity, and alleviate the side effects of GLP-1 agonist drugs.

[0012] At the same time, the new strain disclosed in the present invention has a stronger epithelial cell adhesion ability than the existing muciniphilic Akkermansia strain, and can colonize well in the human or animal intestines, thereby prolonging its residence time in the intestines and better exerting its medicinal effect 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 being GDMCC No: 63782, wherein the Akkermansia muciniphila includes live bacteria, freeze-dried bacteria, or inactivated bacteria of the Akkermansia muciniphila, and the culture includes any one of the following A) to D):

[0014] A) a fermentation broth of the Akkermansia muciniphila;

[0015] B) the supernatant of the fermentation broth of Akkermansia muciniphila;

[0016] C) an inactivated fermentation broth of the Akkermansia muciniphila;

[0017] D) The concentrated or dried product of any one of A) to C) above.

[0018] 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 medicament for preventing weight regain or maintaining weight after weight loss.

[0019] 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 weight for non-disease treatment purposes.

[0020] 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%.

[0021] In some embodiments, the Akkermansia muciniphila ( ​ ) and 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%.

[0022] In some embodiments, the 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 set forth in SEQ ID NO. 1.

[0023] In some embodiments, the Akkermansia muciniphila is an Akkermansia muciniphila species ( ​ )) is a new strain that is different from 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%. 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%.

[0024] In some embodiments, the metabolic products of the Akkermansia muciniphila include short-chain fatty acids, including at least one of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid, and decanoic acid. Preferably, the Akkermansia muciniphila can produce high levels of acetic acid and / or propionic acid. In some embodiments, the acetic acid content secreted by the Akkermansia muciniphila 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 Akkermansia muciniphila 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.

[0025] 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.

[0026] In some embodiments, the Akkermansia muciniphila ( ​ )MNH19250, deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC), the deposit number is GDMCCNo: 63782, and the deposit date is June 21, 2024.

[0027] 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.

[0028] In some embodiments, the fermentation culture or fermentation culture supernatant is a fermentation culture, a fermentation culture supernatant, an inactivated fermentation broth obtained using a liquid culture medium under anaerobic culture conditions, or a concentrated or dried product of any of the foregoing.

[0029] In some embodiments, the composition is provided in liquid form or solid form.

[0030] In some embodiments, the composition comprises 1×10 4 to 1×10 12 cfu / mL or 1×10 4 to 1×10 12 cfu / mg of the viable Akkermansia muciniphila bacteria.

[0031] In some embodiments, the composition comprises 1×10 5 to 1×10 11 cfu / mL or 1×10 5 to 1×10 11 cfu / mg of the viable Akkermansia muciniphila bacteria.

[0032] In some embodiments, the composition comprises 1×10 6 to 1×10 10 cfu / mL or 1×10 6 to 1×10 10 cfu / mg of the viable Akkermansia muciniphila bacteria.

[0033] In some embodiments, the composition comprises 1×10 7 to 1×10 9 cfu / mL or 1×10 7 to 1×10 9 cfu / mg of the viable Akkermansia muciniphila bacteria.

[0034] In some embodiments, each mg of the composition comprises 1×10 3 to 1×1017 colony-forming units (CFU) of bacteria; for example, 1×10 4 to 1×10 12 1×10 5 to 1×10 11 or 1×10 6 to 1×10 10 colony forming units (CFU), 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 , 2×108 , 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).

[0035] 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 pasteurized.

[0036] In some embodiments, the composition is in the form of a liquid, foam, cream, spray, powder (eg, lyophilized powder), or gel.

[0037] In some embodiments, the composition is in the form of a powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral solution, suspension, emulsion, liquid formulation, sustained-release formulation, nanoformulation, or microencapsulated capsule.

[0038] In some embodiments, the composition is in the form of an oral dosage or an injection.

[0039] In some embodiments, the composition further comprises one or more pharmaceutically acceptable carriers, excipients or adjuvants. The pharmaceutically acceptable adjuvants are well known to those skilled in the art.

[0040] 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.

[0041] In some embodiments, the composition comprises one or more of a buffering agent (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.

[0042] In some embodiments, the composition further comprises one or more additional active agents.

[0043] The other active agent has the function of suppressing appetite.

[0044] In some embodiments, suppressing appetite comprises reducing food intake and / or reducing appetite.

[0045] In some embodiments, the other active agent is selected from: a GLP-1 receptor agonist, a dual agonist of the GLP-1 receptor and the GCG receptor, a triple agonist of the GLP-1 receptor, the GIP receptor and the GCG receptor, an AMPK agonist or an active drug that promotes GLP-1 secretion.

[0046] In some embodiments, the other active agent may be one or more of a probiotic, a prebiotic, or a combination thereof;

[0047] 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 krumelans, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus mucilaginosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus sakei, Lactococcus lactis, and Lactococcus cremoris.

[0048] Preferably, the prebiotic is selected from the group consisting of 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, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), starch, cellulose, β-glucan, hemicellulose, lactulose, manno-oligosaccharides, manno-oligosaccharides (MOS), inulin rich in fructooligosaccharides, oligo-glucose, tagatose, trans-galacto-oligosaccharides, pectin, resistant starch, xylo-oligosaccharides (XOS) and any combination thereof.

[0049] In some embodiments, the composition can be formulated as a frozen composition, for example, by quick freezing and drying, or lyophilization, for storage and / or transportation.

[0050] In some embodiments, the composition is obtained by spray drying. In some embodiments, the composition is obtained by electrostatic spray drying.

[0051] In some embodiments, the strain in the composition is freeze-dried or spray-dried. In some embodiments, the strain in the composition is electrostatically spray-dried. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is viable. In some embodiments, the strain in the composition is freeze-dried or spray-dried and is capable of partially or completely colonizing the intestine. In some embodiments, the strain is reconstituted prior to administration. In some cases, the reconstitution is performed using a diluent as described herein.

[0052] 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.

[0053] In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is an enteric-coated formulation. In some embodiments, the enteric-coated formulation is a dosage form having an enteric coating. For example, the enteric-coated formulation can be an enteric granule, an enteric-coated tablet, or an enteric-coated 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-coated capsule, or the capsule can be a microencapsulated capsule or a microcapsule.

[0054] In some embodiments, the composition is a pharmaceutical.

[0055] In some embodiments, the composition is an infant-suitable dosage form, a pediatric-suitable dosage form, or an adult-suitable dosage form. In some embodiments, the composition is an enteral dosage form or a parenteral dosage form.

[0056] As used herein, microbial population generally refers to the microbial population that is basically composed of a single strain, species or genus, which may be the case when cultivating a group from a subgroup of the separation and purification of such strain, species or genus. Therefore, for a given microbial population, if cultivated from an isolated 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% 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 in this particular population. On the contrary, the level of undesirable 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 impurity level 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 be lower than the above-mentioned level. Where a composition comprises a consortium of multiple populations of microorganisms, each population may have the aforementioned purity, either prior to its incorporation into the composition, or when measured aggregately with respect to the consortium.

[0057] 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 can be obtained, for example, by recovering supernatant from an Akkermansia muciniphila culture or by extracting cellular components or cell fractions, metabolites, or secreted compounds from an Akkermansia muciniphila culture; these components can correspond to isolated forms of components from Akkermansia muciniphila, or any mixture of one or more components from Akkermansia muciniphila.

[0058] In some embodiments, 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.

[0059] In some embodiments, the obesity includes but is not limited to: overweight, obesity.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] In some embodiments, obesity treatment includes but is not limited to administration of weight loss medications or probiotics.

[0064] In some embodiments, weight loss medications include but are not limited to orlistat, phentermine, topiramate, and GLP-1 receptor agonists (e.g., semaglutide, liraglutide, and tirzepatide).

[0065] In some embodiments, the Akkermansia muciniphila of the present invention ( ​ ), or the composition is administered to an obese patient after treatment with a GLP-1 receptor agonist.

[0066] In some embodiments, the Akkermansia muciniphila of the present invention ​ , or the combination prevents weight gain 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.

[0067] In some embodiments, the Akkermansia muciniphila of the present invention ( ​), or a composition having at least one property selected from the following: maintaining weight after discontinuation of a weight-loss drug; preventing weight rebound induced by a high-fat diet; controlling weight gain caused by a high-sugar, high-fat diet; preventing blood sugar elevation and / or maintaining blood sugar balance, such as preventing blood sugar elevation and / or maintaining blood sugar balance after discontinuation of a weight-loss drug; 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 a weight-loss drug. In some embodiments, preventing weight regain includes preventing weight regain in patients receiving obesity treatment, for example, patients receiving treatment with a GLP-1 receptor agonist (e.g., semaglutide).

[0068] The Akkermansia muciniphila isolated and obtained by the present invention ( ​ ) MNH19250 has strong auto-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.

[0069] The Akkermansia muciniphila isolated and obtained by the present invention ( ​ ) MNH19250 can also synthesize large amounts of short-chain fatty acids, thereby effectively preventing weight regain in patients undergoing obesity treatment and / or preventing obesity-prone subjects from developing obesity.

[0070] Beneficial effects of the present invention

[0071] Akkermansia muciniphila of the present invention ( ​ ) has high autoaggregation ability and strong surface hydrophobicity, can effectively adhere to intestinal epithelial cells, prolong intestinal residence time, and improve therapeutic effects. This strain adapts to the intestinal environment and has high stability. The present invention's Akkermansia muciniphila ( ​ ) can significantly inhibit weight rebound after discontinuation (reducing weight gain and fat accumulation), improve blood sugar homeostasis (lowering postprandial and fasting blood sugar levels), and reduce visceral fat accumulation, with long-lasting effects. This provides a highly effective and safe microbial therapy solution for maintaining weight after obesity treatment and preventing obesity progression in individuals prone to obesity. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The following description of the embodiments will be made 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.

[0073] ​ : shows the colony morphology of strain MNH19250.

[0074] ​ : Gram-stained photographs of strain MNH19250 are shown.

[0075] ​ : shows an electron micrograph of strain MNH19250.

[0076] ​ : 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.

[0077] ​ : Shows the results of the tolerance of strain MNH19250 to different concentrations of NaCl.

[0078] ​ : Shows the results of the tolerance of strain MNH19250 to different concentrations of bile salts.

[0079] ​ : Shows the 6h autoaggregation ability of strain MNH19250.

[0080] ​ : shows the phylogenetic tree of strain MNH19250.

[0081] ​ : A graph showing the body weight at the end point of an animal experiment to prevent weight regain in an obese mouse model.

[0082] ​ : Shows the percentage of body weight gain after discontinuation of semaglutide in an animal experiment to prevent weight regain in an obese mouse model.

[0083] ​ : Shows the percentage of body weight gain at the end point of the animal experiment to prevent weight regain in an obese mouse model.

[0084] ​ : Shows the body weight gain rate after discontinuation of semaglutide in an animal experiment to prevent weight regain in an obese mouse model.

[0085] ​ : Shows the area under the oral glucose tolerance curve in the oral glucose tolerance test experiment.

[0086] ​ : Shows fasting blood glucose in an oral glucose tolerance test.

[0087] ​ : Showed that MNH19250 can improve liver weight after cessation of semaglutide treatment.

[0088] ​ : Showed that MNH19250 can improve subcutaneous fat weight after cessation of semaglutide treatment.

[0089] ​: Showed that MNH19250 can improve epididymal fat weight after cessation of semaglutide treatment.

[0090] ​ : Showed that MNH19250 can improve perirenal fat mass after cessation of semaglutide treatment.

[0091] ​ : Showed that MNH19250 can improve mesenteric fat weight after cessation of semaglutide treatment.

[0092] ​ : Showed that MNH19250 can improve white fat mass after cessation of semaglutide treatment.

[0093] ​ : Showed that MNH19250 can improve visceral fat mass after discontinuation of semaglutide treatment.

[0094] ​ : A bar graph showing the relative fluorescence values ​​of IFNβ expression in the presence of strain MNH19250.

[0095] Strain preservation

[0096] strain Akkermansia muciniphila ( ​ ) MNH19250, deposited in Guangdong Microbial 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 ​ ​ MNH19250. DETAILED DESCRIPTION

[0097] This paper reports the isolation of a new strain of Akkermansia muciniphila, deposited with GDMCC No. 63782. The strain was identified using traditional taxonomic and molecular biological methods. The results indicate that the strain is a new strain of Akkermansia muciniphila. Furthermore, the present invention investigates the biochemical properties and therapeutic applications of this strain.

[0098] As is known in the art, bacterial species can be classified and identified using traditional classification methods and molecular biology methods. Traditional classification methods include, but are not limited to, cell morphology observation, Gram staining, flagellar staining, and various metabolic assays. Molecular biology methods include, but are not limited to, ribosomal RNA sequencing and whole genome sequencing-based methods.

[0099] 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).

[0100] 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.

[0101] Compositions or preparations disclosed herein can be used as pharmaceutical preparations, therapeutic compositions or medical probiotics. In some cases, compositions are used in the form of pharmaceutical preparations. In some cases, compositions are used in the form of medical probiotics. In some cases, compositions (such as medical probiotics) can be orally administered, for example, as capsules, pills or tablets.

[0102] 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.

[0103] The term "homeostasis" refers to the mechanisms of processes that contribute to maintaining a balanced internal state in an organism, such as glucose regulation homeostasis, triglyceride homeostasis, cholesterol homeostasis.

[0104] The term "preventing weight regain" includes preventing weight loss caused by the use of slimming drugs, diet control or exercise, and weight rebound after stopping the drug, resuming the diet or stopping the exercise; and preventing subjects with a physique prone to obesity from developing obesity.

[0105] 16S rRNA is a type of ribosomal RNA in prokaryotes. The 16S rRNA gene consists of a variable region and a conserved region. The conserved region is shared by all bacteria, while the variable region varies to varying degrees among different bacteria. By comparing bacterial 16S rRNA gene sequences and basing the number of sequence differences and their evolutionary distance, an evolutionary tree can be constructed. 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 betweenaverage 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).

[0106] 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.

[0107] 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 using methods such as BLAST. In the field of bacterial taxonomy, it is generally believed that the ANI value must reach above 95% to be considered 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.).

[0108] Various existing mature ANI value calculation tools can be used, such as the local calculation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), online calculation tools ANI caculator (http: / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani) and ANItools.

[0109] Using the above-described method, a person skilled in the art can determine whether an isolated strain belongs to the species Akkermansia muciniphila identified by the present inventors. For example, when compared with Akkermansia muciniphila ( ​ ​ )(GCF_000020225.1) has an average nucleotide identity ANI value of at least 95%, e.g., 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%, 97.2 ...1%, 97.2%, 97.9%, 97.1%, 97.1%, 97.2%, 97.9%, 97.1%, 97.1%, 97.2%, 97.9%, 97.1%, 97.1%, 97.2%, 97.9%, 97.1%, 97.1%, 97.2%, 97.9 When the expression levels of the bacterial strains are 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.

[0110] For another example, when its 16S rRNA sequence is at least 98.65% identical to 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.

[0111] The alignment fraction (AF) refers to the fraction of orthologous genes shared between two genomes. Based on an ANI > 95%, the researchers proposed using the AF as an additional threshold to restrict species, ensuring 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). ), while others use 30% (Zeng, S., Patangia, D., Almeida, A. et al. Acompendium of 32,277 metagenome-assembled genomes and over 80 million genes from the early-life human gut microbiome. Nat Commun 13, 5139 (2022). ). In short, the AF value can be used as an auxiliary additional threshold.

[0112] A "strain" is a member of a bacterial species that has genetic characteristics that allow it to be distinguished from closely related members of the same bacterial species. A genetic characteristic can be the complete or partial absence of at least one gene, the complete or partial absence of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), 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 cases where one strain (compared to another strain of the same species) acquires or loses antibiotic resistance or a biosynthetic capacity (e.g., an auxotrophic strain), the strains can be distinguished by selection or counterselection using antibiotics or nutrients / metabolites.

[0113] "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.

[0114] 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, Akkermansia muciniphila deposited with GDMCC NO: 63782. Similarly, Akkermansia muciniphila deposited with GDMCC NO: 63782 can also be prepared into a pharmaceutical composition, for example, by using a pharmaceutically acceptable excipient, comprising a pharmaceutically effective amount of the Akkermansia muciniphila.

[0115] Suitable pharmaceutically acceptable excipients that may be used include, for example, carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavors.

[0116] 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, preanally, rectally, subcutaneously, sublingually, topically, intravaginally, transdermally, or by ureteral or urethral administration.

[0117] Examples of dosage forms suitable for the compositions 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.

[0118] 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 powder, a toothpaste, or the like.

[0119] The compositions provided herein may comprise a pharmaceutically acceptable excipient, diluent or carrier. Such pharmaceutically acceptable excipients, diluents or carriers are well known in the art.

[0120] 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 viable. 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.

[0121] 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.

[0122] In some embodiments, the composition is prepared by freeze drying or spray drying.The composition disclosed herein includes a pharmaceutical composition.

[0123] The subject of the present disclosure may be a human or an animal, including but not limited to cattle, sheep, cats, dogs, horses, rabbits, monkeys, mice, rats, alpacas, camels, etc.

[0124] The pharmaceutical composition disclosed herein can be used to prevent patients receiving obesity treatment from regaining weight and / or prevent subjects with a physique prone to obesity from developing obesity.

[0125] Amuc-1100 and Amuc_1631 (P9) proteins are derived from Akkermansia muciniphila ( ​ ​The outer membrane proteinAmuc_1100 of ​ 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., ​ secretes a glucagon-like peptide-1-inducing protein that improvesglucose homeostasis and ameliorates metabolic disease in mice[J]. Nature Microbio-logy, 2021, 6: 563-573". Amuc-1100 remains stable during pasteurization and can inhibit the expression of CB1 receptors in the intestine 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".

[0126] Amuc_1631 (P9) protein can promote GLP-1 secretion 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 ​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 the differentiation of stem cells into secretory cells." Patrice D. Cani, Clara Depommier, Muriel Derrien, et al., ​ : paradigm for next-generation beneficial microorganisms. Nature Reviews Gastroenterology&Hepatology volume 19, pages 625–637 (2022)".

[0127] Therefore, the Akkermansia muciniphila involved in the present invention ( ​ ) 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.

[0128] The scheme of the present invention will be explained below with reference to the examples. Those skilled in the art will appreciate that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature of the art or in accordance with the product or instrument specifications are used. All reagents or instruments are commercially available if their manufacturers are specified.

[0129] Example

[0130] The liquid MM01 medium involved in the embodiment is composed of: peptone 5g / L, trypticase 5g / L, yeast powder 10g / L, beef extract 5g / L, glucose 5g / L, K2HPO4 2g / L, sodium acetate 2g / L, Tween 80 1mL / L, hemoglobin 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).

[0131] The solid MM01 medium involved in the embodiment is composed of: peptone 5g / L, trypticase 5g / L, yeast powder 10g / L, beef extract 5g / L, glucose 5g / L, K2HPO4 2g / L, sodium acetate 2g / L, Tween 80 1mL / L, hemoglobin 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.

[0132] Anaerobic blood agar plates were purchased from Huankai Microorganisms. The formula was as follows: 10 g / L casein pancreatic digest, 3 g / L cardiac 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.

[0133] The above culture medium can be prepared by conventional preparation methods and sterilization methods.

[0134] Example 1: Isolation and identification of strains

[0135] 1.1 Isolation and purification of strain MNH19250

[0136] An intestinal strain, designated MNH19250, was isolated from a sample collected from a healthy male volunteer in Guangzhou, Guangdong Province, China. Conventional strain isolation methods were used, including gradient dilution, followed by isolation and culture of single colonies. Purification was performed and anaerobically cultured at 37°C. The pure culture was prepared into a 20% glycerol / water suspension and stored at -80°C.

[0137] Specifically, the strain isolation method is as follows:

[0138] The donor takes 5g of sample, puts it into the sample collection and storage tube, shakes it to homogenize it, and then places the processed sample in an ice box. It is then sent to the laboratory for strain isolation within 24 hours.

[0139] Aliquot physiological saline solution in a biosafety cabinet, 9 mL / tube; prepare anaerobic blood agar plates for strain isolation and transfer them to the anaerobic workstation 24 h in advance. Label the plates with sample information, culture medium type, isolation date, etc.

[0140] Take a new sample and place it in an 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, set aside.

[0141] Take 10- 6 Drop the diluted solution onto the anaerobic blood agar plate at a rate of 100 μL / plate. Spread evenly. After the plate surface is dry, invert the plate and culture at 37°C for 3-5 days.

[0142] Observe the growth of the strain on the isolation medium (anaerobic blood agar plate) and pick a single colony with a sterile toothpick for strain purification. Incubate the purified strain anaerobically at 37°C. Prepare a 20% glycerol / water solution of the pure culture and store it at -80°C.

[0143] 1.2 Morphological characteristics of strain MNH19250 Culture and morphological characteristics

[0144] Strain MNH19250 was inoculated into MM01 medium and incubated anaerobically at 37°C for 72 hours. Visible colonies formed on the MM01 plate. These colonies were round, with regular, smooth edges, approximately 0.5 mm in diameter, pale yellow, and translucent. The strain was Gram-negative and, under microscopic observation, lacked flagella, was nonmotile, and had rod-shaped shapes, approximately 0.5-1 µm x 1.5-3 µm in size. See the photo of the colony morphology of strain MNH19250 after 72 hours of incubation on MM01 plates for details. ​ For Gram staining photos of strain MNH19250, see ​ , electron microscope photos see ​ .

[0145] 1.3 Physiological characteristics of strain MNH19250

[0146] Strain MNH19250 does not grow under aerobic conditions, but only under anaerobic conditions. It can grow in the pH range of 6.0-9.0, with the optimal growth pH being around 8.0 (for the strain's tolerance to different pH values, see ​ ); Growth was significantly inhibited on media containing more than 2% (w / v) NaCl (for strain tolerance to different concentrations of NaCl, see ​ The 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 the strain's tolerance to different concentrations of bile salts, see ​ ).

[0147] 1.4 Biochemical identification results of strain MNH19250 using API 20A

[0148] Biochemical characterization of strain MNH19250 was performed using API 20A (purchased from bioMérieux, CN2030025). Specific experimental procedures are described in the standard API reagent instructions. MNH19250 was cultured at 37°C under anaerobic conditions. Results are shown in Table 1.

[0149]

[0150] MNH19250 can ferment glucose, lactose, and mannose to produce acid. Therefore, during the fermentation or cultivation of strain MNH19250, glucose, lactose, mannose, and their derivatives can be used as carbon sources.

[0151] MNH19250 can hydrolyze esculin (ESC), which means it can synthesize β-glucosidase. β-glucosidase belongs to the cellulase family and can hydrolyze cellobiose and short-chain cellooligosaccharides to produce glucose.

[0152] MNH19250 can hydrolyze gelatin (GEL), which in turn allows it to synthesize proteases. Proteases convert indigestible proteins into peptides or amino acids that can penetrate the bacterial cell. These enzymes, primarily exoenzymes, hydrolyze gelatin into peptides and then amino acids, causing it to lose its gelling properties and liquefy. This allows the black substance in the reaction well to diffuse, resulting in a positive reaction.

[0153] 1.5 Antibiotic Minimum Inhibitory Concentration Test for Strain MNH19250

[0154] 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.

[0155]

[0156] The results showed that MNH19250 was sensitive to ampicillin, chloramphenicol, clindamycin, amoxicillin, rifampicin, imipenem, penicillin, and cefquinome. This suggests that MNH19250 is sensitive to most antibiotics, and the risk of developing antibiotic resistance in subjects with long-term MNH19250 use is low.

[0157] 1.6 Determination of the autoaggregation ability of strain MNH19250

[0158] Strain MNH19250 was inoculated into MM01 liquid medium and cultured anaerobically at 37°C for 48 h. 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.

[0159] Place the bacterial solution at 37°C (directly in a cuvette), measure its OD600 value every 30 minutes, record it as At, and measure for 6 hours, with three replicates.

[0160] The auto-aggregation ability of the strain was calculated according to the following formula: auto-aggregation ability of the strain (%) = [1-(At / A0)] × 100%.

[0161] The 6h autoaggregation ability of MNH19250 was 54.21% (see ​ ), compared with other strains in the literature, the auto-aggregation ability is more than 20% stronger (Xin Ma, Meng Tian, ​​Xueping Yu, et al., Foods. 2024, Jan 30;13(3): 442.Characterization and Preliminary Safety Evaluation of ​ ​ 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.

[0162] 1.7 Determination of surface hydrophobicity of strain MNH19250

[0163] The strain MNH19250 was inoculated into MM01 liquid 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), and resuspended in PBS buffer. The OD600 value was adjusted to 0.5 and recorded as A0.

[0164] Add 4 mL of xylene to 4 mL of the resuspension. Vortex the two-phase system for 5 minutes. Incubate at room temperature for 1 hour, then carefully remove the xylene phase. Measure the absorbance (A) of the aqueous phase at 600 nm in triplicate. Calculate the surface hydrophobicity of the test strain according to the following formula:

[0165] H%=[(A0-A) / A0]×100%, where A0 and A represent the absorbance before and after organic solvent extraction, respectively.

[0166] The surface hydrophobicity of strain MNH19250 was 57.06%, which was more than 25% higher than that of other strains reported in the literature (Xin Ma, Meng Tian, ​​Xueping Yu, et al., Foods. 2024, Jan 30; 13(3): 442. ​ ​ 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, thereby exerting a probiotic effect, and at the same time can inhibit the adhesion of other pathogens, thereby playing an intestinal probiotic function.

[0167] 1.8 Amplification of the 16S rRNA gene of strain MNH19250

[0168] Fresh culture of strain MNH19250 was used to extract genomic DNA, which was then used as a template for 16S rRNA gene amplification.

[0169] The primer pairs used for PCR of the 16S rRNA gene are:

[0170] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 2)

[0171] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO. 3). The PCR reaction procedure is as follows:

[0172] 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).

[0173] 1.9 16S rRNA gene sequencing

[0174] The PCR product was purified and sequenced by Sangon Biotechnology Co., Ltd. to obtain the 16S rRNA gene sequence (1355 bp), as shown in SEQ ID NO. 1:

[0175]

[0176] 1.10 Identification of strain MNH19250

[0177] The 16S rRNA gene sequence obtained above, as shown in SEQ ID NO. 1, was analyzed using the NCBI Basic Local Alignment Search Tool to confirm the strain classification information.

[0178] The obtained 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 ( ​ ), the similarity is 100%, so the strain MNH19250 is judged to be Akkermansia muciniphila ( ​ ) strains planted.

[0179] The strain MNH19250 was compared with Akkermansia spp. retrieved from GenBank and other databases ( ​ ​ ) were compared with the 16S rRNA gene sequences of related strains to construct a phylogenetic tree.

[0180] The 16S rRNA gene sequence of strain MNH19250 was aligned 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 ​ ), ​ The nodes in the developmental tree only display the values ​​whose Bootstrap values ​​are greater than 50%.

[0181] From the phylogenetic tree, it can be seen that strain MNH19250 is closely related to Akkermansia spp. ​ )and ​ AY271254, therefore, strain MNH19250 was identified as Akkermansia muciniphila ( ​ ) new strains planted.

[0182] 1.11 Genome analysis of strain MNH19250

[0183] The genome of strain MNH19250 was fragmented using ultrasonication, and an Illumina sequencing library was constructed using a standard DNA library construction kit (NEB Ultra™). The constructed library was sequenced using a NovaSeq (Illumina) system for paired-end 150-bp sequencing. The sequencing yielded 3.01 Gbp of data, of which Q20 accounted for 97.42%.

[0184] The raw genome sequencing data was filtered using fastp (version 0.20.0). The filtered raw data was assembled using SPAdes (version v3.14.0). The assembled genome yielded a total gene length of 2.82 Mbp, an N50 length of 378.4 kbp, and a GC content of 55.19%.

[0185] The prokaryotic analysis software Genome Annotation Pipeline prokka (version 1.14.5) was used to predict and analyze genomic genes. A total of 2,376 CDS sequences were predicted, with an average CDS sequence length of 1,039 bp.

[0186] Potential antibiotic resistance genes in the genome were analyzed using RGI (version: 4.2.2), using the antibiotic resistance gene database CARD (version: 3.0.0, https: / / card.mcmaster.ca / analyze / rgi). Detailed comparison information is shown in Table 3.

[0187]

[0188] The genomes of potential virulence factors and related genes were analyzed using NCBI blastp (version 2.7.1+) against the virulence factor database (VFDB, http: / / www.mgc.ac.cn / cgibin / VFs / v5 / main.cgi, updated September 19, 2019). Detailed comparison results are shown in Table 4.

[0189]

[0190] GutSMASH5 (version 1.0.0) was used to analyze potential primary metabolic gene clusters in the genome. Detailed alignment results are shown in Table 5.

[0191]

[0192] Analysis of the genes encoding Amuc_1100 and P9 proteins in strain MNH19250. BLAST was used to align the Amuc_1100 protein sequence (WP_197738471) and the Amuc_1631 (P9) protein sequence (ACD05451).

[0193]

[0194] sequence:

[0195] >MNH19250_01469 hypothetical protein

[0196] MSNWITDNKPAAMVAGVGLLLFLGLSATGYIVNSKRSELDKKISIAAKEIKSANAAEITPSRSSNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILITDTSSWLGFQVYSTQAPSVQAASTLGFELKAINSL VNKLAECGLSKFIKVYRPQLPIETPANNPEESDEADQAPWTPMPLEIAFQGDRESVLKAMNAITGMQDYLFTVNSIRIRNERMMPPPIANPAAAKPAAAQPATGAASLTPADEATAPAAPAIQQVIKPYMGKEQVFVQVSLNLVHFNQPKAQEPSED (SEQ ID NO. 4)

[0197]

[0198] sequence:

[0199] >MNH19250_00667 Tail-specific protease

[0200] MNMHSFRWIRLTAFSALAAAAITSCASAATDFNQVGKQMSLLLQNFHFSRKEFSDELSTKFLETYLRKVDPNKIFFTQQDVDALKRKYGKELDDYLMSGQMMDAAQAMHALYRQRAMQRISYARDLLKKGGFTFDKDKSIERSRRKTAAWPKDEAEMQQVWKDMVEEQLLSEILRRETVARLAKEQNKPDPLANEKPAEEKLLMRYERIQRNIQETDLEDVAETLLSAVALTYDPHTDYMGARQVDRFKISMGTELTGIGALLGSEDDGSTKITGIVVGGPADKSGELKLNDRIVAIDSDNSGEMVDILFMKLDKVVDMIRGAENTQMRLKVEPADAPGQAKIITLTRSKVPLKDELAKGEIIELTGAPEGRNRIGVLSLPSFYADMEGGDRRCAKDVKKILERMNKENVDGLVIDLRSNGGGSLEEVRLMTGFFTGNGPVVQIKDTRGNVDIKSAHNRQKLFNGPIVVLINKLSASASEILAAALQDYGRAVIVGDESTFGKGSVQQPVDIGQYLPFFAARDRAGLLKVTTQKFYRVAGGSTQLKGVESDIQLPTATAAFELGEDILDYAMPYDQITPCTNYKKDSSIAAMLPVLKDASAKRVEKDRDLQIAREDIAMMKQRIKDNKLSLNKKIREQENSALEERRKSINKERKIRFAEMAKEDATKYKIYRLTLDDVNAKELPLADPEKDNEQFMHLAEDPTAELDDSPEYPSGLDPELREGINIVQDMLKLESSGK (SEQ ID NO. 5)

[0201] Example 2. Fatty Acid Composition Analysis of Strain MNH19250

[0202] 2.1 Fatty Acid Composition Analysis of Strain MNH19250

[0203] Strain MNH19250 was inoculated on MM01 plates and cultured anaerobically at 37°C for 72 h. The cells were then harvested and subjected to fatty acid extraction and methylation. The fatty acid composition of strain MNH19250 was analyzed using the Microbial ID, Inc., Newark, Del., fully automated bacterial identification system.

[0204] 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

[0205] Bacteria preparation

[0206] The strain MNH19250 was inoculated into MM01 liquid culture medium and cultured anaerobically at 37°C for 48 hours. The bacteria and the supernatant were collected by centrifugation and stored at -80°C until use.

[0207] Pretreatment of bacterial supernatant samples

[0208] (1) After thawing, vortex the sample for 3 minutes to mix thoroughly;

[0209] (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.

[0210] (3) Add 750 nM MTBE extractant containing internal standard, vortex at 2500 rpm for 3 min, sonicate at 4 m for 5 min, and centrifuge at 4 m and 12000 rpm for 10 min;

[0211] (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.

[0212] Bacteria sample pretreatment

[0213] (1) After the sample is thawed, add 100% ultrapure water extract to resuspend it evenly;

[0214] (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;

[0215] (3) Soak in liquid nitrogen for 2 min, remove and thaw completely on ice, vortex at 2500 rpm for 3 min, and repeat 3 times;

[0216] (4) Add 150 nr of MTBE extractant containing internal standard, vortex at 2500 rpm for 3 min, sonicate at 4 rpm for 5 min, and centrifuge at 4 rpm and 12000 rpm for 10 min;

[0217] (5) Pipette 90% supernatant into the liner of the injection bottle for GC-MS / MS analysis;

[0218] (6) The remaining 50 μM bacterial suspension was repeatedly frozen and thawed in liquid nitrogen three times, centrifuged at 12,000 rpm for 10 min, and the supernatant was collected to determine the protein concentration using the BCA method.

[0219] Standard Preparation: Prepare standard solutions of 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, respectively. Chromatographic mass spectrometry detection was performed using the conditions shown in Table 8.

[0220] Obtain chromatographic peak intensity data for the quantitative signal corresponding to each concentration of the standard. Draw standard curves for different substances using 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.

[0221]

[0222] 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.

[0223]

[0224] 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.

[0225] Example 3: Use of strain MNH19250 to prevent obesity

[0226] 3.1 Animal Experiment on MNH19250 Preventing Obesity Relapse in Obese Mouse Model after Semaglutide Discontinuation

[0227] Mice were fed a high-fat diet for 10 weeks, induced to obesity, and then treated with semaglutide for 4 weeks before the drug was discontinued. Following semaglutide discontinuation, live and pasteurized MNH19250 bacteria were administered to investigate the effect of MNH19250 on preventing relapse after semaglutide discontinuation. This experimental protocol has been ethically reviewed by the Muen Biotech Laboratory Animal Care and Use Committee.

[0228] 3.1.1 Experimental methods

[0229] Experimental animals: C57BL / 6J mice were purchased from Guangdong Yaokang Biotechnology Co., Ltd.

[0230] Test strains: After thawing the glycerol cryopreserved tube of MNH19250 at 37°C, the strain was inoculated 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 liquid 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, and some of the MNH19250 live bacteria were incubated at 70°C for 30 minutes to obtain pasteurized MNH19250 bacteria (MNH19250-P).

[0231] Negative control: PBS containing 25% glycerol and 0.05% L-Cys HCl was used as a negative control.

[0232] Experimental Procedure: After the quarantine period, 8 C57BL / 6J male mice aged 5-6 weeks were randomly selected to be fed a basal diet. The remaining mice were fed a high-fat diet for 10 weeks. After 10 weeks, all mice fed the basal diet were transferred to the non-congested diet group. For the high-fat diet group, 32 mice weighing 36-44 g were randomly assigned to stratify according to body weight, with 8 mice per group. Five groups were divided: the non-congested diet group, the high-fat diet group, the high-fat diet group (HFD), the high-fat diet group (HFD+Sema), the MNH19250-L group, and the high-fat diet group. The NCD group continued to be fed a basal diet, while the other four groups continued to be fed a high-fat diet. Drug administration began after grouping (D1). The NCD and HFD groups received a negative control, while the remaining three groups received semaglutide (30 nmol / kg every three days). Semaglutide administration was discontinued after 4 weeks. The HFD+Sema group began to receive the negative control substance, the MNH19250-L group and the MNH19250-P group began to receive MNH19250-L and MNH19250-P, and the NCD group and the HFD group continued to receive the negative control substance. The drugs were administered once a day for a total of 28 days. During the administration period, the mice continued to receive high-fat feed. During the experiment, the mice had free access to water and food, and a 12h / 12h day and night cycle was adopted.

[0233] During the trial, general clinical observations were conducted once after each dosing period. The endpoint for this trial was the day after dosing (D56). Autopsies were performed according to the protocol, and data were summarized and analyzed for body weight, percentage change, autopsy data, and serum test results. Fasting blood glucose levels were measured, as well as blood glucose levels 15, 30, 60, 90, and 120 minutes after sugar administration. Each mouse was carefully timed, and blood glucose levels were accurately measured at six time points to obtain glycemic indicators.

[0234] 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.

[0235] 3.2 Experiment on the effect of MNH19250 on oral glucose tolerance in high-fat diet-induced type 2 diabetic mice:

[0236] Oral glucose tolerance test (OGTT): During the last week of dosing, perform an OGTT after a 12-hour fast (e.g., fasting from 8:30 PM to 8:30 AM the following day). Weigh the mice and administer glucose orally based on their fasting weight at a dose of 2 g / kg (g glucose / kg fasting weight). Measure fasting blood glucose and blood glucose levels 15, 30, 60, 90, and 120 minutes after glucose administration. Strictly time each mouse, and accurately measure blood glucose at these six time points.

[0237] 3.3 Experimental Results

[0238] The experimental results are shown in ​ 、 ​ as well as ​ All data are expressed as mean ± SD and analyzed using GraphPad Prism software. Pairwise comparisons were analyzed using the Student's t test. Significant differences are indicated with *, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0239] (1) MNH19250 can improve weight regain after cessation of semaglutide treatment. The results of the weight change experiment are as follows ​ shown. ​ 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 purpose of preventing weight regain.

[0240] (2) MNH19250 can improve the elevated blood glucose level after cessation of semaglutide treatment. ​ shown. ​ and ​ The results showed that MNH19250 can significantly reduce AUC-OGTT, 2-hour postprandial blood glucose and fasting blood glucose after discontinuation of semaglutide treatment, indicating that MNH19250 can improve the imbalance of blood glucose homeostasis after discontinuation of semaglutide treatment, improve blood glucose elevation, and has the purpose of preventing blood glucose elevation and maintaining blood glucose balance.

[0241] (3) MNH19250 can improve fat accumulation after cessation of semaglutide treatment. ​ shown. ​ The results showed that MNH19250 can 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 can improve fat accumulation after discontinuation of semaglutide treatment and has the purpose of preventing body fat accumulation and weight gain.

[0242] Example 4. Effect of strain MNH19250 on IFNβ expression

[0243] While GLP-1 agonists like liraglutide and semaglutide do have significant short-term weight loss effects, studies have found that they are associated with a higher incidence of four gastrointestinal adverse reactions compared to other weight loss medications, including biliary tract disease, pancreatitis, intestinal obstruction, and gastrospasm. Diarrhea and gastrointestinal inflammation are common side effects of GLP-1 agonists.

[0244] Type I interferon β (IFNβ) has been shown to have anti-inflammatory effects, promoting the reprogramming of macrophages to a pro-inflammatory phenotype, 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.

[0245] 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 established by Muen) to evaluate the effect of MNH19250 on IFNβ transcriptional activity.

[0246] 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 (for details, see the following literature: 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).

[0247] Prepare the culture supernatant of strain MNH19250: inoculate strain MNH19250 into 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 until use.

[0248] 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);

[0249] Strain MNH19250 group: DMEM complete medium containing 10% volume of strain MNH19250 culture supernatant.

[0250] THP-1-IFNβ-promoter reporter cells were seeded in 96-well plates at 1 × 10 5 Cells were treated according to the designated groups. After 24 hours of culture, the cells were centrifuged at 300 g for 5 minutes, the supernatant removed, and 50 μL of 1× Luminescence (Promega) was added for normalization to the control group (control) for evaluating the effect of strain MNH19250 on IFNβ transcriptional activity.

[0251] The experimental results are as follows ​ As shown, the metabolites of strain MNH19250 significantly promoted the transcriptional activity of IFNβ. Therefore, these results indicate that MNH19250 and its metabolites have anti-inflammatory and immune-regulating functions, thereby alleviating the side effects of GLP-1 agonists such as liraglutide and semaglutide.

[0252] Although the present invention has been disclosed with reference to certain embodiments, it is apparent that modifications and variations can 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 in the disclosure illustrate embodiments of the present invention, they are provided as non-limiting examples only and, therefore, should not be construed as limiting the various aspects of the invention thus described. The present invention is intended to have the full scope defined by the language of this disclosure, the following claims, and any equivalents thereof. Accordingly, the drawings and detailed description should be regarded as illustrative rather than restrictive.

Claims

1. Use of a composition in the preparation of a medicament for maintaining body weight and / or blood sugar balance after discontinuation of a GLP-1 receptor agonist, characterized in that: The composition comprises an effective amount of Akkermansia muciniphila ( 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) an inactivated fermentation broth of the Akkermansia muciniphila; C) the fermentation supernatant of the above A) or B) and the Akkermansia muciniphila; D) the concentrated or dried product of any one of A) to C) above; The inactivated bacteria are pasteurized bacteria; and the inactivated fermentation broth is a product obtained by pasteurizing the fermentation broth of Akkermansia muciniphila.

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

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

4. The use according to claim 3, characterized in that The other active agents are one or more of probiotics and prebiotics.

5. Use of a composition in the preparation of a medicament for preventing fat accumulation after discontinuation of a GLP-1 receptor agonist, characterized in that: The composition comprises an effective amount of Akkermansia muciniphila ( 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) an inactivated fermentation broth of the Akkermansia muciniphila; C) the fermentation supernatant of the above A) or B) and the Akkermansia muciniphila; D) the concentrated or dried product of any one of A) to C) above; The inactivated bacteria are pasteurized bacteria; and the inactivated fermentation broth is a product obtained by pasteurizing the fermentation broth of Akkermansia muciniphila.

6. Akkermansia muciniphila ( Akkermansia muciniphila ) or the culture of Akkermansia muciniphila in the preparation of a medicament for preventing weight regain after discontinuation of a GLP-1 receptor agonist, characterized in that, The Akkermansia muciniphila deposit number is GDMCC No: 63782, wherein the Akkermansia muciniphila includes live bacteria, freeze-dried 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) an inactivated fermentation broth of the Akkermansia muciniphila; C) the fermentation supernatant of the above A) or B) and the Akkermansia muciniphila; D) the concentrated or dried product of any one of A) to C) above; The inactivated bacteria are pasteurized bacteria; and the inactivated fermentation broth is a product obtained by pasteurizing the fermentation broth of Akkermansia muciniphila.

7. Akkermansia muciniphila ( Akkermansia muciniphila ) or the culture of Akkermansia muciniphila in the preparation of a medicament for maintaining weight loss after discontinuation of a GLP-1 receptor agonist, characterized in that, The Akkermansia muciniphila deposit number is GDMCC No: 63782, wherein the Akkermansia muciniphila includes live bacteria, freeze-dried 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) an inactivated fermentation broth of the Akkermansia muciniphila; C) the fermentation supernatant of the above A) or B) and the Akkermansia muciniphila; D) the concentrated or dried product of any one of A) to C) above; The inactivated bacteria are pasteurized bacteria; and the inactivated fermentation broth is a product obtained by pasteurizing the fermentation broth of Akkermansia muciniphila.

8. Akkermansia muciniphila ( Akkermansia muciniphila ) or the culture of Akkermansia muciniphila in the preparation of a medicament for preventing blood sugar elevation after discontinuation of a GLP-1 receptor agonist, characterized in that, The Akkermansia muciniphila deposit number is GDMCC No: 63782, wherein the Akkermansia muciniphila includes live bacteria, freeze-dried 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) an inactivated fermentation broth of the Akkermansia muciniphila; C) the fermentation supernatant of the above A) or B) and the Akkermansia muciniphila; D) the concentrated or dried product of any one of A) to C) above; The inactivated bacteria are pasteurized bacteria; and the inactivated fermentation broth is a product obtained by pasteurizing the fermentation broth of Akkermansia muciniphila.

9. Akkermansia muciniphila ( Akkermansia muciniphila ) or the culture of Akkermansia muciniphila in the preparation of a medicament for maintaining blood sugar balance after discontinuation of a GLP-1 receptor agonist, characterized in that, The Akkermansia muciniphila deposit number is GDMCC No: 63782, wherein the Akkermansia muciniphila includes live bacteria, freeze-dried 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) an inactivated fermentation broth of the Akkermansia muciniphila; C) the fermentation supernatant of the above A) or B) and the Akkermansia muciniphila; D) the concentrated or dried product of any one of A) to C) above; The inactivated bacteria are pasteurized bacteria; and the inactivated fermentation broth is a product obtained by pasteurizing the fermentation broth of Akkermansia muciniphila.

10. Akkermansia muciniphila ( Akkermansia muciniphila ) or a culture of Akkermansia muciniphila in the preparation of a medicament for preventing fat accumulation after discontinuation of a GLP-1 receptor agonist, characterized in that, The Akkermansia muciniphila deposit number is GDMCC No: 63782, wherein the Akkermansia muciniphila includes live bacteria, freeze-dried 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) an inactivated fermentation broth of the Akkermansia muciniphila; C) the fermentation supernatant of the above A) or B) and the Akkermansia muciniphila; D) the concentrated or dried product of any one of A) to C) above; The inactivated bacteria are pasteurized bacteria; and the inactivated fermentation broth is a product obtained by pasteurizing the fermentation broth of Akkermansia muciniphila.

11. Akkermansia muciniphila ( Akkermansia muciniphila ) or the culture of Akkermansia muciniphila in the preparation of a health product for helping to control body fat and / or maintain healthy blood sugar levels after discontinuation of a GLP-1 receptor agonist, characterized in that, The Akkermansia muciniphila deposit number is GDMCC No: 63782, wherein the Akkermansia muciniphila includes live bacteria, freeze-dried 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) an inactivated fermentation broth of the Akkermansia muciniphila; C) the fermentation supernatant of the above A) or B) and the Akkermansia muciniphila; D) the concentrated or dried product of any one of A) to C) above; The inactivated bacteria are pasteurized bacteria; and the inactivated fermentation broth is a product obtained by pasteurizing the fermentation broth of Akkermansia muciniphila.

12. The use according to any one of claims 1 to 11, characterized in that The GLP-1 receptor agonist includes liraglutide, semaglutide or tilportide.

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