WO2023237038A1 - Akkermansia muciniphila, use thereof, and culturing method therefor - Google Patents

Akkermansia muciniphila, use thereof, and culturing method therefor Download PDF

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Publication number
WO2023237038A1
WO2023237038A1 PCT/CN2023/099055 CN2023099055W WO2023237038A1 WO 2023237038 A1 WO2023237038 A1 WO 2023237038A1 CN 2023099055 W CN2023099055 W CN 2023099055W WO 2023237038 A1 WO2023237038 A1 WO 2023237038A1
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akkermansia muciniphila
cgmcc
liver
group
application
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PCT/CN2023/099055
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French (fr)
Chinese (zh)
Inventor
郑丽君
刘洋洋
王薇
梁德宝
王晔
智发朝
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广州知易生物科技有限公司
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Publication of WO2023237038A1 publication Critical patent/WO2023237038A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D13/00—Finished or partly finished bakery products
    • A21D13/06—Products with modified nutritive value, e.g. with modified starch content
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
    • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the technical field of microorganisms, in particular to Akkermansia muciniphila and its application and culture methods.
  • Non-alcoholic fatty liver disease refers to a liver metabolic disease in which lipids, mainly triglycerides, accumulate in liver cells and become pathological changes caused by other clear liver-damaging factors in addition to long-term heavy drinking.
  • liver fat metabolism is impaired, causing a large amount of fatty substances to accumulate in liver cells (simple fatty liver, NAFL), which in turn leads to steatosis, liver cell damage, inflammatory reaction, and liver fibrosis (non-alcoholic fatty liver disease). hepatitis, NASH).
  • NAFLD neurodegenerative disease
  • Metformin, vitamin E, ursodeoxycholic acid, etc. are also used clinically for anti-insulin resistance, anti-oxidative stress and cell protection treatment.
  • Lipid-lowering drugs have also been reported to be used in the treatment of NAFLD, but they are still controversial. Generally, for patients who still have mixed hyperlipidemia after taking weight loss and hypoglycemic drugs or basic treatment for more than 3-6 months, the addition of statins and phenoxyaromatic acid lipid-lowering drugs should be considered, but liver function needs to be monitored.
  • Pioglitazone is an agonist of PPAR ⁇ .
  • pioglitazone can cause side effects such as water and sodium retention, osteoporosis, and weight gain. Its clinical application restricted. For obese patients with non-alcoholic fatty liver disease, if their weight cannot be reduced by more than 5% after 6-12 months of lifestyle changes, they should also use weight-loss drugs such as sibutramine and orlistat with caution to promote fat metabolism and improve fat metabolism. NAFLD symptoms.
  • PPARs are a type of nuclear hormone receptors. Distributed in liver, fat, skeletal muscle and other organs, it regulates lipid metabolism, transport and gluconeogenesis. PPAR ⁇ can promote the oxidative decomposition of fatty acids, and PPAR ⁇ also has anti-inflammatory effects. Elafibranor is a PPAR ⁇ / ⁇ agonist. Phase 2 clinical trials have proven that the drug can maintain blood sugar balance, improve lipid metabolism and reduce liver inflammation. It is a potential drug for the treatment of NAFLD.
  • GLP-1 is a glucagon-like polypeptide secreted by L cells in the small intestine. It can promote insulin secretion, increase the number of pancreatic beta cells, inhibit glucagon secretion, suppress appetite, and delay gastric emptying. Improve insulin sensitivity, etc.
  • Semaglutide is a GLP-1 analog that only needs to be administered once a week, and its clinical trials for the treatment of NASH are ongoing.
  • FXR is a multifunctional nuclear receptor that plays an important role in bile acid metabolism, glucose and lipid metabolism, liver protection, and regulation of intestinal bacterial growth.
  • Obeticholic acid is an FXR agonist that can not only reduce the degree of liver fat degeneration in NAFLD patients, but also improve insulin resistance and inhibit liver inflammation and fibrosis.
  • Obeticholic acid is currently in phase 3 clinical trials, and some subjects have experienced pruritus and increased low-density lipoprotein levels, so the safety of the drug needs to be further clarified.
  • Acetyl-CoA carboxylase ACC is a key enzyme in the de novo synthesis of fatty acids.
  • the ACC inhibitor PF-05221304 can suppress liver fat content in NAFLD patients, but the drug has the potential side effect of causing hypertriglyceridemia.
  • SCD-1 is stearoyl-CoA desaturase, which is the rate-limiting enzyme in the synthesis of unsaturated fatty acids.
  • Aramchol is an inhibitor of SCD-1. Clinical trials have found that the drug can reduce liver fat content in NAFLD patients.
  • Hepatocyte death is an important driving factor in promoting liver inflammation and fibrosis. Therefore, inhibiting liver cell death can help prevent and treat NASH.
  • Emricasan is a pancaspase inhibitor that can inhibit cell apoptosis, thereby alleviating liver inflammation and fibrosis. The drug is currently in Phase 2 clinical trials for the treatment of NASH.
  • Anti-inflammatory drugs Inflammatory cells and pro-inflammatory cytokines play an important role in the occurrence and development of NASH. Apoptosis signaling kinase ASK-1 can promote the activity of JNK, and JNK is an important kinase that promotes inflammation and cell death.
  • a short-term clinical trial found that an ASK-1 inhibitor (BGsertib) can reduce fibrosis in patients with NASH. BGsertib is currently in phase 3 clinical trials, and its efficacy awaits further evaluation.
  • surgical treatment can also be used to control non-alcoholic fatty liver disease.
  • bariatric surgery can help alleviate liver steatosis, fatty hepatitis and liver fibrosis.
  • liver transplantation is the only effective treatment.
  • surgical procedures are mainly targeted, expensive, and difficult to obtain and match liver sources.
  • Akkermansia muciniphila in the preparation of health foods or drugs for the prevention and treatment of non-alcoholic fatty liver disease.
  • the Akkermansia muciniphila is selected from CGMCC No. .22793 Akkermansia muciniphila and Akkermansia muciniphila with deposit number CGMCC No. 22794 or both.
  • the Akkermansia muciniphila deposited as CGMCC No. 22793 and the Akkermansia muciniphila deposited as CGMCC No. 22794 are each independently viable, sterilized One or more types of live bacteria.
  • Akkermansia muciniphila is provided with the deposit number CGMCC No. 22793, which was deposited at the General Microbiology Center of the Chinese Microbial Culture Collection Committee on June 28, 2021.
  • Akkermansia muciniphila is provided with the deposit number CGMCC No. 22794, which was deposited at the General Microbiology Center of the Chinese Microbial Culture Collection Committee on June 28, 2021.
  • a probiotic combination product which includes Akkermansia muciniphila with a deposit number of CGMCC No. 22793 and Akermansia muciniphila with a deposit number of CGMCC No. 22794.
  • Akkermansia species One or both Akkermansia species.
  • a health food comprising: one or both of the Akkermansia muciniphila described in the second aspect and the Akkermansia muciniphila described in the third aspect. , and food accessories.
  • the health food is pastry or drink.
  • a medicine comprising: one or both of the Akkermansia muciniphila described in the second aspect and the Akkermansia muciniphila described in the third aspect, and pharmaceutical excipients.
  • the dosage form of the drug is a pill, tablet, granule, capsule, solution, tube feeding preparation, suspension, cream, spray, ointment or patch.
  • the culture conditions include: using mucin as the sole carbon source; or/and, anaerobic environment; or/and, 36.5°C-37.5°C.
  • Figure 1 is a colony characteristic diagram of Akkermansia muciniphila AM02 cultured using the method of Example 2;
  • Figure 2 is a colony characteristic diagram of Akkermansia muciniphila AM06 cultured using the method of Example 2;
  • Figure 3 is a microscopic observation of Akkermansia muciniphila AM02 cultured using the method of Example 2 after Gram staining;
  • Figure 4 is a microscopic observation of Akkermansia muciniphila AM06 cultured using the method of Example 2 after Gram staining;
  • Figure 5 is a PCA analysis diagram of metabolites in the culture supernatant of Akkermansia muciniphila in Example 5;
  • Figure 6 is a fluorescence microscope picture of the effect of Akkermansia muciniphila on the reduction of tight junction protein ZO-1 expression in Caco2 cells induced by TNF- ⁇ and IFN- ⁇ in Example 6.
  • the Akkermansia muciniphila AM02 provided in this application is classified and named Akkermansia muciniphila. It has been deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 28, 2021. Address: Beichen West Road, Chaoyang District, Beijing No. 3, Hospital No. 1, the preservation number is CGMCC No. 22794; the strain was received and registered by the preservation center on June 28, 2021, and was detected as a surviving strain by the preservation center on June 28, 2021.
  • the Akkermansia muciniphila AM06 provided in this application is classified and named Akkermansia muciniphila. It has been deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 28, 2021. Address: Beichen West Road, Chaoyang District, Beijing No. 3, No. 1 Courtyard, the preservation number is CGMCC No. 22793; the strain was received and registered by the preservation center on June 28, 2021, and was detected as a surviving strain by the preservation center on June 28, 2021.
  • the technical solution of "A, and/or, B, and/or, C, and/or, D” includes any one of A, B, C, and D (that is, they are all connected with "logical OR” technical solution), also includes any and all combinations of A, B, C, and D, that is, including combinations of any two or any three of A, B, C, and D, and also includes A, B, C , four combinations of D (that is, technical solutions that are all connected by "logical AND").
  • first”, “second”, “third” and “fourth” etc. are for descriptive purposes only and shall not be understood as indicating or implying relative importance or quantity, nor shall they be understood as implicitly indicating the importance or quantity of indicated technical features.
  • first”, “second”, “third”, “fourth”, etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation of quantity.
  • the technical features described in open format include closed technical solutions composed of the listed features, and also include open technical solutions including the listed features.
  • the temperature parameters in this application are allowed to be treated at a constant temperature, or to vary within a certain temperature range. It should be understood that the thermostatic treatment described allows the temperature to fluctuate within the accuracy of the instrument control. It is allowed to fluctuate within the range of ⁇ 5°C, ⁇ 4°C, ⁇ 3°C, ⁇ 2°C and ⁇ 1°C.
  • % (w/w) and wt% both represent weight percentage
  • % (v/v) refers to volume percentage
  • % (w/v) refers to mass volume percentage
  • NAFLD non-alcoholic fatty liver disease
  • “Second hit” is a classic hypothesis for the onset of NAFLD proposed in 1998.
  • “First hit” refers to the accumulation of triglycerides in the liver caused by insulin resistance, and the liver's ability to tolerate endogenous damage factors, ischemia, hypoxia, etc. is reduced.
  • “Second hit” means that after triglycerides accumulate in liver cells, the liver cells will eventually be damaged under the action of inflammatory cytokines, oxidative stress, endoplasmic reticulum stress, etc., and pathological changes such as inflammation and fibrosis will occur in liver tissue. , the "second strike” doctrine gradually shifted to the “multiple strike” doctrine.
  • the lipotoxicity theory believes that the accumulation of triglycerides in the liver will not cause insulin resistance and liver cell damage.
  • the core mechanism causing NASH is endoplasmic reticulum stress and oxidation caused by free cholesterol, free fatty acids (FFA) and their metabolites. Stress and inflammatory response.
  • the traditional view is that triglycerides accumulated in hepatocytes promote lipid peroxidation, oxidative stress, inflammation and fibrosis, and are the driving factors for the development of NAFLD; however, this view is increasingly being challenged because triglycerides Esters may antagonize lipotoxicity.
  • palmitic acid C16:0
  • stearic acid C18:0
  • DAG diacylglycerol
  • ceramides ceramides
  • LPCs lysophosphatidic acid choline
  • fructose is mainly metabolized in the liver, which can promote large-scale lipid synthesis, inhibit mitochondrial ⁇ -oxidation, and cause hepatocyte steatosis. Due to its own instability (containing a five-membered furan ring), fructose will promote the generation of reactive oxygen species (ROS) and cause damage to liver cells.
  • ROS reactive oxygen species
  • ATP adenosine diphosphate and hypoxanthine nucleotides
  • uric acid adenosine diphosphate and hypoxanthine nucleotides
  • long-term intake of large amounts of fructose can lead to intestinal flora disorder and increased intestinal wall permeability.
  • Toxic products such as bacterial endotoxins enter the liver through the portal vein and promote liver inflammation.
  • Endotoxin then reaches the liver through the portal circulation, activates liver Kupffer cells, and promotes Kupffer cells to produce cytokines, thereby triggering an inflammatory cascade reaction, damaging liver cells, hindering their secretion, metabolism and other functions, and causing bile secretion disorders.
  • Abnormal bile secretion will affect the normal metabolism of fat, and abnormal accumulation of fat in the liver will lead to steatosis of liver cells and eventually the formation of NAFLD.
  • liver function After liver function is damaged, the liver's ability to process toxicants from the intestinal tract is reduced, and intestinal toxicants may accumulate and damage the intestinal mucosal barrier, leading to intestinal dysfunction; in addition, the reduction of related antibodies, lysozyme and secretions, coupled with The increase in endotoxin makes the environment conducive to the growth of Gram-negative bacteria, but restricts the growth of probiotics and reduces the local resistance of the intestinal wall. These factors can aggravate the intestinal flora imbalance in NAFLD patients.
  • the present application provides the application of Akkermansia muciniphila in the preparation of health foods or drugs for preventing and treating non-alcoholic fatty liver disease.
  • the Akkermansia muciniphila is selected from the group consisting of Akkermansia muciniphila deposited as CGMCC No. 22793.
  • Akkermansia and accession number CGMCC One or two species of Akkermansia muciniphila No. 22794.
  • the Akkermansia muciniphila described in this application can be a living cell, or it can be a biologically active Akkermansia muciniphila that has been inactivated, genetically recombined, transformed or modified, attenuated, chemically treated, or physically treated. , it may also be a bacterial lysate, a culture (such as a supernatant), or a component extracted from the supernatant.
  • the Akkermansia muciniphila deposited as CGMCC No. 22793 and the Akkermansia muciniphila deposited as CGMCC No. 22794 are each independently a live bacterium or an inactivated bacterium (which may be in the form One or more of the inactivated bacteria with complete structure or incomplete morphological structure).
  • Non-alcoholic fatty liver disease refers to a clinical syndrome characterized by hepatocellular steatosis, excluding excessive drinking and other clear liver damage factors, including simple fatty liver, non-alcoholic fatty liver disease and non-alcoholic fatty liver disease. Hepatitis and related cirrhosis and hepatocellular carcinoma.
  • prevention and treatment includes prevention, treatment, auxiliary treatment, etc.
  • preventing, treating,” or “treating” means alleviating, delaying progression, attenuating, preventing, or maintaining an existing disease or condition.
  • prevention and treatment also includes curing, preventing the development of, or alleviating to some extent one or more symptoms of a disease or condition.
  • the drugs described in this application may be human drugs or animal drugs.
  • drug includes any agent, compound, composition or mixture that provides a physiological and/or pharmacological effect in vivo or in vitro, often providing a beneficial effect.
  • the scope of physiological and/or pharmacological effects of a “drug” in the body is not particularly limited. It can have systemic effects or only local effects.
  • the activity of the "drug” is not particularly limited. It can be an active substance that can interact with other substances, or it can be an inert substance that does not interact.
  • This application provides Akkermansia muciniphila with the deposit number CGMCC No. 22793.
  • This strain is isolated from breast milk. Its colony culture characteristics include: round protrusions, neat edges, opaque, white, and uneven sizes. ; The 16S RNA sequence of this strain is shown in SEQ ID NO: 2; Compared with ATCC BAA-835, artificial gastric juice and artificial intestinal juice are better tolerated. The non-targeted metabolic difference analysis of the supernatant is shown in Figure 5 and Table 7. The effect of improving non-alcoholic fatty liver disease is more obvious, so BAA-835 can be identified as a new strain compared to ATCC.
  • This application provides Akkermansia muciniphila with the deposit number CGMCC No. 22794.
  • This strain is a strain isolated from feces. Its colony culture characteristics include: round protrusions, neat edges, opaque, white, and uneven sizes. ; The 16S RNA sequence of this strain is shown in SEQ ID NO: 1; Compared with ATCC BAA-835, artificial gastric juice and artificial intestinal juice are better tolerated. The non-targeted metabolic difference analysis of the supernatant is shown in Figure 5 and Table 7. The effect of improving non-alcoholic fatty liver disease is more obvious, so BAA-835 can be identified as a new strain compared to ATCC.
  • This application provides a probiotic combination product, which includes one of Akkermansia muciniphila deposited as CGMCC No. 22793 and Akkermansia muciniphila deposited as CGMCC No. 22794. Or both.
  • the present application provides a health food, comprising: one or both of the Akkermansia muciniphila described in the second aspect and the Akkermansia muciniphila described in the third aspect, and food accessories.
  • the health food is pastries or drinks.
  • “Nutraceutical” refers to an edible composition. It should be understood that in addition to the aforementioned Akkermansia muciniphila, the food composition is also allowed to contain any suitable other edible substances. In some embodiments, other edible substances can be selected from substances that are allowed to be added in the health care product management regulations, and further do not include substances that are prohibited from being added in the health care brand management regulations. Unless otherwise specified, health product management regulations refer to the current regulations at the time of production.
  • food additives also belong to edible excipients.
  • edible excipients include sugar, fructose, honey, glucose, starch, vitamins, beneficial trace elements and medium elements including calcium powder, soybean powder, mung bean powder, maltodextrin, milk powder, vegetable juice, fruit juice, spices or flavors , the edible excipients of this application can be used singly or in combination.
  • the present application provides a pharmaceutical composition, comprising: one or both of the Akkermansia muciniphila described in the second aspect and the Akkermansia muciniphila described in the third aspect, and pharmaceutical excipients. .
  • the dosage form of the medicament is pills, tablets, granules, capsules, solutions, tube feeding preparations, suspensions, creams, sprays, ointments or patches.
  • pharmaceutical composition refers to a composition that has preventive and therapeutic effects and can be used as a medicine.
  • excipients include but are not limited to mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium EDTA Sodium, monovalent alkali metal carbonates, acetates, phosphates or their aqueous solutions, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose , Fructooligosaccharides, dextran, glycine, starch, sucrose, maltodextrin, lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerol, Tween 80.
  • Agar Agar,
  • pharmaceutically acceptable refers to those ligands, materials, compositions and/or dosage forms that are, within the scope of reasonable medical judgment, suitable for administration to a patient and proportionate to a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
  • pharmaceutically acceptable carrier includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorbent agents that are compatible with the administration of the drug. Delay agents and the like. Each entity must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient.
  • Suitable examples include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch, potato starch and substituted or unsubstituted ⁇ -cyclodextrin; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) Powdered tragacanth; (5) Malt; (6) Gelatin; (7) Talc; (8) Fu excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols Alcohols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) Esters, such as ethyl oleate and ethyl laurate; (13) Agar; (14) Bu
  • the composition for preventing and treating alcoholic fatty liver disease is a pharmaceutical composition. Further, the composition for preventing and treating alcoholic fatty liver disease contains a therapeutically effective amount of Akkermansia muciniphila.
  • a "therapeutically effective amount” refers to an amount of a pharmaceutically active ingredient that will elicit a biological or medical response in an individual in response to a disease, disorder, and/or symptom, such as a physiological and/or pharmacological positive effect in the individual.
  • the amount of the compound of the present application, the physiological and/or pharmacological positive effects include but are not limited to reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, slowing down or delaying disease progression or preventing diseases, etc.
  • the present application provides a method for cultivating the Akkermansia muciniphila described in the second aspect or/and the Akkermansia muciniphila described in the third aspect.
  • the culture conditions include: using mucin as the sole carbon source. ; Or/and, anaerobic environment; or/and, 36.5°C-37.5°C.
  • the culture conditions are not particularly limited in this application, and a medium containing animal-derived components or a medium that does not contain animal-derived components can also be used, such as CN114350571A.
  • the temperature in the culture conditions is, for example, 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37°C, 37.1°C, 37.2°C, 37.3°C, 37.4°C, and 37.5°C.
  • the measurement parameters of raw material components are involved. Unless otherwise specified, there may be slight deviations within the range of weighing accuracy. Temperature and time parameters are involved, allowing for acceptable deviations due to instrument testing accuracy or operating accuracy.
  • the 16S sequence alignment result of SEQ ID NO:1 and ATCC BAA-835 shows that the Per.Ident value is 99.43%.
  • Freshly collected breast milk samples (from adult healthy women) were immediately injected into 5 mL anaerobic cillin bottles for storage, and then the samples were transferred to an anaerobic workstation at 37°C (85% N 2 , 10% H 2 , 5% CO 2 ), dilute the sample to 10 -6 according to the dilution method of 1:10, take 1 mL of each dilution solution and inoculate it into 9 mL of basal medium with mucin as the only carbon source, and culture it anaerobically for about 1 month.
  • the 16S sequence alignment result of SEQ ID NO:2 and ATCC BAA-835 shows that the Per.Ident value is 99.22%.
  • the first-level seed liquid undergoes Gram staining microscopy and should be G-bacillus, without spores and miscellaneous bacteria.
  • the first-level seed liquid undergoes Gram staining microscopy and should be G-bacillus, without spores and miscellaneous bacteria.
  • Survival rate number of viable bacteria at each time point/number of corresponding viable bacteria at 0 h ⁇ 100%
  • ATCC BAA-835, AM02, and AM06 strains are well tolerated by artificial intestinal fluid.
  • PCA is a data dimensionality reduction method, that is, reducing the dimensionality of multiple variables to a new set of comprehensive variables, and then selecting the first few principal components that reflect as much of the original variable information as possible to achieve the purpose of dimensionality reduction.
  • the PCA chart reflects the true distribution of samples and is mainly used to observe the separation trend between sample groups and whether there are abnormal points. It also reflects the variability between and within groups from the original data.
  • Example 6 Effect of Akkermansia muciniphila on the expression of tight junction protein ZO-1 in Caco2 cells induced by TNF- ⁇ and IFN- ⁇
  • Caco2 cells were seeded into a 96-well plate and cultured until the confluence was 80%-90%. Caco2 cells were induced with 100ng/mL TNF- ⁇ +100ng/mLIFN- ⁇ for 24 hours, and then AM02, AM06, and BAA-835 were added respectively to continue with The cells were incubated for 24 hours, and the experimental groups were as shown in Table 8. Five duplicate wells were made in each group. Immunofluorescence method was used to observe the effects of BAA-835, AM06 and AM02 on the expression of tight junction protein ZO-1 in Caco2 cells induced by TNF- ⁇ and IFN- ⁇ .
  • the livers of 8-week-old C57BL/6 mice were cut into 250 ⁇ m-thick slices using a microtome.
  • the liver slices were placed in a 6-well plate and cultured using slice culture medium with a culture volume of 3 ml/well.
  • the 6-well plate was cultured overnight at 95% oxygen, 5% carbon dioxide, 37 degrees Celsius and shaking at 70 rpm.
  • inflammation was induced by adding 2 ⁇ g/mL LPS to liver slices.
  • the experimental groups are as shown in Table 10, with 3 duplicate holes in each group.
  • an appropriate amount of culture medium, LPS, AM02, AM06 and BAA-835 were added to the 6-well plate according to the grouping, and the culture was continued for 48 hours, and the medium was changed every 24 hours.
  • liver sections were taken and QPCR method was used to detect the expression levels of IL-6 and IL-1 ⁇ genes in the liver sections.
  • test substance and inducer are added at the same time.
  • LPS could significantly induce the upregulation of IL-6 and IL-1 ⁇ gene expression levels in liver slices (P ⁇ 0.01).
  • the AM06 group, AM02 group, and BAA-835 group could significantly reduce the upregulation of IL-6 and IL-1 ⁇ gene expression levels in LPS-induced liver slices (P ⁇ 0.01).
  • the expression levels of IL-6 and IL-1 ⁇ genes in the AM06 group and AM02 group were significantly lower than those in the BAA-835 group. (P ⁇ 0.05).
  • ** indicates that compared with the inflammation model group, the difference is extremely significant at P ⁇ 0.01; a indicates that compared with the BAA-835 group, the difference is extremely significant at p ⁇ 0.05.
  • Experimental design 110 male C57BL/6 mice, 5 weeks old.
  • Experimental groups blank group, model group, positive drug group (obeticholic acid OCA, 3mg/mL), AM06 low dose (10 6 CFU/mL), AM06 medium dose (10 8 CFU/mL), AM06 high dose ( 10 10 CFU/mL) group, AM02 (10 8 CFU/mL) group, ATCC BAA-835 (10 8 CFU/mL) group, and the inactivated bacteria group of AM06, AM02 and BAA-835 (the dosage of each group Both are 10 8 CFU/mL). 10 animals per group. After a one-week adaptation period, the blank group was fed with ordinary feed, and the other groups except the blank group were fed with high-fat feed and given corresponding drugs at the same time.
  • mice After 10 weeks of administration, the mice were fasted for 6 hours and then euthanized with CO2 . Blood samples are collected by cardiac puncture. Separate serum to detect relevant indicators. Terminal body weight and liver weight were recorded. The liver tissue was removed and fixed in formalin for histopathological examination.
  • a means that compared with the BAA-835 medium-dose group, the difference is significant at p ⁇ 0.05, aa means that compared with the BAA-835 medium-dose group, the difference is extremely significant at p ⁇ 0.01;
  • b means that compared with the BAA-835 medium-dose group, the difference is significant at p ⁇ 0.05; bb means that compared with the BAA-835 medium-dose group, the difference is extremely significant at p ⁇ 0.01;
  • the weight of the mice in the model group increased significantly (p ⁇ 0.01), while the liver weight tended to increase, but there was no significant difference.
  • the AM06 medium-dose group and the AM06 inactivated bacteria group can significantly reduce body weight (p ⁇ 0.05).
  • the other administration groups have a tendency to reduce the body weight and liver weight of mice, but there is no significant difference.
  • HE staining of liver pathology was performed on mice in each group, and the scores were scored based on the degree of steatosis of the sections, the degree of ballooning degeneration of hepatocytes, and the degree of inflammation in the lobules, and the non-alcoholic fatty liver NAS score was calculated.
  • the results are shown in Table 7.
  • the NAS score of the model group increased significantly (p ⁇ 0.05), indicating that the animals developed non-alcoholic fatty liver disease.
  • each administration group can reduce NAS points to varying degrees
  • AM06 mid- and low-dose groups and inactivated strains can significantly reduce NAS points (p ⁇ 0.05).
  • the NAS scores of live bacteria of AM02 and AM06 are significantly lower than those of BAA-835 live bacteria (p ⁇ 0.05), and the NAS scores of inactivated bacteria of AM02 and AM06 tend to be lower than those of BAA-835 inactivated bacteria. , indicating that the live and inactivated bacteria of AM02 and AM06 are more effective than BAA-835 live and inactivated bacteria in preventing non-alcoholic fatty liver disease.
  • ALT and AST serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of mice were detected.
  • the results are shown in Table 7.
  • the ALT and AST of the mice in the model group were significantly increased (p ⁇ 0.01), indicating that the liver Functionality is compromised.
  • Each administration group can reduce the levels of ALT and AST in serum to varying degrees.
  • the positive drug group, AM06 low and high dose group, AM06 inactivated bacteria group and AM02 inactivated bacteria group can significantly reduce ALT (p ⁇ 0.05).
  • the positive drug group, all live bacteria group, AM06 inactivated bacteria group and AM02 inactivated bacteria group can significantly reduce AST (p ⁇ 0.05).
  • the AST content of live bacteria of AM02 and AM06 is slightly lower than that of BAA-835 live bacteria, and the AST content of inactivated bacteria of AM02 and AM06 is slightly lower than that of inactivated BAA-835 bacteria. This shows that AM02 and AM06 have slightly better protective effects on the liver than BAA-835.
  • mice The results of testing the serum LPS content of mice are shown in Table 7.
  • the LPS of mice in the model group increased significantly (p ⁇ 0.01), indicating that the model animals had mild endotoxemia.
  • the positive drug group failed to reduce the LPS in serum, while each Akkermansia muciniphila group could reduce the LPS content to varying degrees, among which the live bacteria medium-dose groups of AM02 and AM06, the live bacteria group of AM02 and AM06
  • the inactivated bacteria group can significantly reduce the LPS content (p ⁇ 0.05).
  • the medium-dose live bacteria group of AM02 and AM06 has a significantly better ability to reduce serum LPS than the BAA-835 live bacteria group (p ⁇ 0.05), while the inactivated bacteria group of AM02 and AM06 is slightly better than the BAA-835 inactivated bacteria group. , but there is no significant difference.
  • Akkermansia muciniphila can effectively prevent non-alcoholic fatty liver disease in mice, reduce liver damage and inhibit mild endotoxemia, and the efficacy of each strain is AM06 ⁇ AM02>BAA-835.
  • Example 10 Experiment on the efficacy of Akkermansia muciniphila in treating non-alcoholic steatohepatitis and liver fibrosis in mice
  • Experimental design 110 male C57BL/6 mice, 5 weeks old.
  • Experimental groups blank group, model group, positive drug group (obeticholic acid OCA, 3mg/mL), AM06 low dose (10 6 CFU/mL), AM06 medium dose (10 8 CFU/mL), AM06 high dose ( 10 10 CFU/mL) group, AM02 (10 8 CFU/mL) group, ATCC BAA-835 (10 8 CFU/mL) group, and the inactivated bacteria group of AM06, AM02 and BAA-835 (the dosage of each group Both are 10 8 CFU/mL). 10 animals per group.
  • the blank group was fed with ordinary feed.
  • the remaining groups except the blank group were fed high-fat feed until the 10th week, and then were fed modified high-fat feed (increased cholesterol content) from the 10th to 18th week of the experiment.
  • Each administration group started to receive administration from the 10th week to the 18th week.
  • mice After the administration, the mice were fasted for 6 hours and then euthanized with CO2 . Blood samples are collected by cardiac puncture. Separate serum to detect relevant indicators. Terminal body weight and liver weight were recorded. The liver tissue was removed and fixed in formalin for histopathological examination.
  • a means that compared with the BAA-835 medium-dose group, the difference is significant at p ⁇ 0.05, aa means that compared with the BAA-835 medium-dose group, the difference is extremely significant at p ⁇ 0.01;
  • b means that compared with the BAA-835 medium-dose group, the difference is significant at p ⁇ 0.05; bb means that compared with the BAA-835 medium-dose group, the difference is extremely significant at p ⁇ 0.01;
  • the weight of the mice in the model group increased significantly (p ⁇ 0.01), and the liver weight tended to increase, but there was no significant difference.
  • the AM06 low-dose group and the AM02 inactivated bacteria group could significantly reduce body weight (p ⁇ 0.05).
  • the other administration groups had a tendency to reduce the body weight and liver weight of mice, but there was no significant difference.
  • HE staining of liver pathology was performed on mice in each group, and the scores were scored based on the degree of steatosis of the sections, the degree of ballooning degeneration of hepatocytes, and the degree of inflammation in the lobules, and the non-alcoholic fatty liver NAS score was calculated.
  • the results are shown in Table 8.
  • the NAS score of the model group increased significantly (p ⁇ 0.05), indicating that the animals developed non-alcoholic fatty liver disease.
  • each administration group can reduce NAS points to varying degrees, and the positive drug group, AM06 mid-dose group, AM02 mid-dose group, AM02 and AM06 inactivated strains can significantly reduce NAS points (p ⁇ 0.05 ).
  • the NAS scores of live bacteria of AM02 and AM06 were significantly lower than those of BAA-835 live bacteria (p ⁇ 0.05), and the NAS scores of inactivated bacteria of AM02 and AM06 were significantly lower than those of BAA-835 inactivated bacteria, indicating that AM02 and AM06 are effective in treating Prevent non-alcoholic beverages
  • the efficacy of spermatozoa fatty liver disease is better than BAA-835.
  • mice serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of mice were detected.
  • the results are shown in Table 8.
  • the ALT and AST of the mice in the model group were significantly increased (p ⁇ 0.01), indicating that the liver Functionality is compromised.
  • the ALT levels of all administration groups were significantly reduced (p ⁇ 0.05), and the AST levels of the administration groups except the BAA-835 mid-dose group were significantly decreased (p ⁇ 0.05).
  • the ALT content of live or inactivated bacteria of AM02 and AM06 was significantly lower than that of BAA-835 live or inactivated bacteria (p ⁇ 0.05).
  • the AST content of AM02 live bacteria was significantly lower than that of BAA-835 live bacteria (p ⁇ 0.05), and the AST content of AM06 live bacteria was slightly lower than BAA-835 live bacteria, but there was no significant difference.
  • the AST content of AM02 and AM06 inactivated bacteria was significantly lower than that of BAA-835 inactivated bacteria (p ⁇ 0.05). This shows that AM02 and AM06 have better protective effects on the liver than BAA-835.
  • the liver tissue of mice was taken and QPCR method was used to detect the relative mRNA expression of fibrosis-related gene Col1a1.
  • the results are shown in Table 8.
  • the expression of Col1a1 in the model group mice was significantly increased (p ⁇ 0.01), indicating that liver fibrosis occurred in the liver of the model animals.
  • all administration groups except the BAA-835 mid-dose group could significantly reduce the expression of Col1a1 (p ⁇ 0.05).
  • the BAA-835 mid-dose group had a tendency to reduce the expression of Col1a1, but there was no significance. difference.
  • Akkermansia muciniphila can effectively treat non-alcoholic steatohepatitis in mice, reduce liver damage and inhibit liver fibrosis, and the efficacy of each strain is AM06 ⁇ AM02>BAA-835.
  • Akkermansia muciniphila especially Akkermansia muciniphila AM06 with deposit number CGMCC No. 22793 and Akkermansia muciniphila AM02 with deposit number CGMCC No. 22794 , in in vitro experiments, its tolerance to artificial gastric juice and artificial intestinal juice, its ability to inhibit inflammatory factors from destroying tight junction proteins in intestinal cells, and its ability to inhibit LPS-induced hepatitis in liver slices were all better than the standard strain BAA-835.
  • AM02 and AM06 bacteria can effectively prevent or treat non-alcoholic alcoholic diseases by reducing liver steatosis and hepatitis, improving liver function indicators (such as ALT and AST levels), reducing mild endotoxemia, and inhibiting liver fibrosis.
  • liver function indicators such as ALT and AST levels
  • reducing mild endotoxemia and inhibiting liver fibrosis.
  • the occurrence and development of fatty liver and its deterioration can improve the patient's quality of life.

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Abstract

Akkermansia muciniphila strains, which respectively have the preservation numbers CGMCC No. 22793 and CGMCC No. 22794, probiotic combination products comprising said strains, a health food or a pharmaceutical composition, a method for culturing the strains, and an application of the strains in preparation of a health food or a drug for preventing and treating non-alcoholic fatty liver disease. The strains obtained via separation may be used for preventing and treating the occurrence, development, and worsening of non-alcoholic fatty liver disease, can reduce liver fat and hepatitis, improve liver function indicators, reduce mild endotoxemia, and inhibit hepatic fibrosis.

Description

嗜粘蛋白阿克曼菌及其应用和培养方法Akkermansia muciniphila and its application and culture methods
相关申请Related applications
本申请要求2022年06月08日申请的，申请号为202210642440.1，名称为“嗜粘蛋白阿克曼菌及其应用和培养方法”的中国专利申请的优先权，在此将其全文引入作为参考。This application claims priority to the Chinese patent application filed on June 8, 2022, with application number 202210642440.1 and titled "Akermansia muciniphila and its application and culture methods", the full text of which is hereby incorporated by reference. .
技术领域Technical field
本发明涉及一种微生物技术领域，特别涉及嗜粘蛋白阿克曼菌及其应用和培养方法。The present invention relates to the technical field of microorganisms, in particular to Akkermansia muciniphila and its application and culture methods.
背景技术Background technique
非酒精性脂肪性肝病(NAFLD)是指除长期大量饮酒外，其他明确的损肝因素所引起的，以甘油三酯为主的脂质在肝细胞中蓄积为病理改变的肝脏代谢性疾病。NAFLD患者肝脏脂肪代谢功能出现障碍，使得大量脂肪类物质蓄积于肝细胞(单纯性脂肪肝，NAFL)，进而导致肝细胞发生脂肪变性、肝细胞损伤、炎症反应、肝脏纤维化(非酒精性脂肪性肝炎，NASH)。Non-alcoholic fatty liver disease (NAFLD) refers to a liver metabolic disease in which lipids, mainly triglycerides, accumulate in liver cells and become pathological changes caused by other clear liver-damaging factors in addition to long-term heavy drinking. In patients with NAFLD, liver fat metabolism is impaired, causing a large amount of fatty substances to accumulate in liver cells (simple fatty liver, NAFL), which in turn leads to steatosis, liver cell damage, inflammatory reaction, and liver fibrosis (non-alcoholic fatty liver disease). hepatitis, NASH).
目前还没有治疗NAFLD的针对性药物，NAFLD的主要治疗方法仍局限在改变饮食和生活方式上，但患者对这种治疗方法的依从性很低。临床上也配合应用二甲双胍、维生素E、熊去氧胆酸等进行抗胰岛素抵抗、抗氧化应激和细胞保护治疗。降脂类药物也有用于NAFLD治疗的报道，但仍然存在争议。一般针对用减肥降糖药或基础治疗3-6个月以上仍存在混合性高脂血症的患者，应考虑加用他汀类、苯氧芳酸类降脂药物，但是需要监测肝功能。匹格列酮是PPARγ的激动剂，它可以减轻患者脂肪肝、肝损伤、炎症和肝纤维化，但是匹格列酮因可导致水钠潴留、骨质疏松、体重增加等副作用，其临床应用受到限制。针对合并肥胖的非酒精性脂肪肝患者，如果改变生活方式6-12个月体重未能降低5％以上，还需谨慎使用***、奥利司他等减肥药物，促进脂肪代谢，改善NAFLD症状。There are currently no targeted drugs for the treatment of NAFLD. The main treatments for NAFLD are still limited to changes in diet and lifestyle, but patient compliance with this treatment is low. Metformin, vitamin E, ursodeoxycholic acid, etc. are also used clinically for anti-insulin resistance, anti-oxidative stress and cell protection treatment. Lipid-lowering drugs have also been reported to be used in the treatment of NAFLD, but they are still controversial. Generally, for patients who still have mixed hyperlipidemia after taking weight loss and hypoglycemic drugs or basic treatment for more than 3-6 months, the addition of statins and phenoxyaromatic acid lipid-lowering drugs should be considered, but liver function needs to be monitored. Pioglitazone is an agonist of PPARγ. It can reduce fatty liver, liver damage, inflammation and liver fibrosis in patients. However, pioglitazone can cause side effects such as water and sodium retention, osteoporosis, and weight gain. Its clinical application restricted. For obese patients with non-alcoholic fatty liver disease, if their weight cannot be reduced by more than 5% after 6-12 months of lifestyle changes, they should also use weight-loss drugs such as sibutramine and orlistat with caution to promote fat metabolism and improve fat metabolism. NAFLD symptoms.
有一些针对NASH的药物处于2期、3期临床试验阶段，药效还有待评估、确认，主要有如下几种类型的药物：(1)改善代谢类药物：PPARs是一类核激素受体，分布于肝脏、脂肪、骨骼肌等器官，调节脂质的代谢、转运以及糖异生等。PPARα可以促进脂肪酸的氧化分解，PPARδ还具有抗炎的作用。Elafibranor是一种PPARα/δ的激动剂，2期临床试验证实该药可以维持血糖平衡、改善脂质代谢及减轻肝脏炎症，是潜在的治疗NAFLD的药物。GLP-1是一种小肠L细胞分泌的胰高血糖素样的多肽，可以促进胰岛素的分泌、增加胰岛β细胞的数量、抑制胰高血糖素的分泌、抑制食欲、延缓胃内容物排空、提高胰岛素敏感性等。Semaglutide是GLP-1的类似物，只需要一周给药一次，其治疗NASH的临床试验正在进行中。FXR是一种多功能的核受体，在胆汁酸代谢、糖脂代谢、肝脏保护、调节肠道细菌生长等方面发挥重要作用。奥贝胆酸是一种FXR的激动剂，不仅可以降低NAFLD患者肝脏脂肪的变性程度，还可以改善患者胰岛素抵抗、抑制肝脏炎症和纤维化。目前奥贝胆酸正处于3期临床试验，一些受试者出现搔痒症和低密度脂蛋白水平升高的情况，因此该药物的安全性有待于进一步明确。乙酰辅酶A羧化酶ACC是脂肪酸从头合成的关键酶。ACC的抑制剂PF-05221304可以抑制NAFLD患者肝脏脂肪的含量，但是该药物有导致高甘油三酯血症的潜在副作用。SCD-1是硬脂酰辅酶A去饱和酶，是不饱和脂肪酸合成的限速酶。Aramchol是SCD-1的抑制剂，临床试验发现，该药物可以降低NAFLD患者肝脏脂肪的含量。(2)拮抗细胞死亡类药物：肝细胞的死亡是促进肝脏炎症、纤维化的重要驱动因素。因此，抑制肝细胞的死亡有助于防治NASH。Emricasan是泛天冬氨酸蛋白水解酶抑制剂(pancaspase inhibitor)，可以抑制细胞凋亡，从而缓解肝脏的炎症和纤维化。目前该药物处于NASH治疗的2期临床试验阶段。(3)拮抗炎症类药物：炎症细胞和促炎症的细胞因子在NASH发生发展过程中发挥重要的作用。凋亡信号激酶ASK-1可以促进JNK的活性，而JNK是促进炎症、细胞死亡的重要激酶。一项短期的临床试验发现，ASK-1的抑制剂(Selonsertib)可以减轻NASH患者的纤维化。目前Selonsertib正处于3期临床试验，其疗效有待于进一步的评估。There are some drugs targeting NASH in Phase 2 and Phase 3 clinical trials, and their efficacy has yet to be evaluated and confirmed. They mainly include the following types of drugs: (1) Metabolism-improving drugs: PPARs are a type of nuclear hormone receptors. Distributed in liver, fat, skeletal muscle and other organs, it regulates lipid metabolism, transport and gluconeogenesis. PPARα can promote the oxidative decomposition of fatty acids, and PPARδ also has anti-inflammatory effects. Elafibranor is a PPARα/δ agonist. Phase 2 clinical trials have proven that the drug can maintain blood sugar balance, improve lipid metabolism and reduce liver inflammation. It is a potential drug for the treatment of NAFLD. GLP-1 is a glucagon-like polypeptide secreted by L cells in the small intestine. It can promote insulin secretion, increase the number of pancreatic beta cells, inhibit glucagon secretion, suppress appetite, and delay gastric emptying. Improve insulin sensitivity, etc. Semaglutide is a GLP-1 analog that only needs to be administered once a week, and its clinical trials for the treatment of NASH are ongoing. FXR is a multifunctional nuclear receptor that plays an important role in bile acid metabolism, glucose and lipid metabolism, liver protection, and regulation of intestinal bacterial growth. Obeticholic acid is an FXR agonist that can not only reduce the degree of liver fat degeneration in NAFLD patients, but also improve insulin resistance and inhibit liver inflammation and fibrosis. Obeticholic acid is currently in phase 3 clinical trials, and some subjects have experienced pruritus and increased low-density lipoprotein levels, so the safety of the drug needs to be further clarified. Acetyl-CoA carboxylase ACC is a key enzyme in the de novo synthesis of fatty acids. The ACC inhibitor PF-05221304 can suppress liver fat content in NAFLD patients, but the drug has the potential side effect of causing hypertriglyceridemia. SCD-1 is stearoyl-CoA desaturase, which is the rate-limiting enzyme in the synthesis of unsaturated fatty acids. Aramchol is an inhibitor of SCD-1. Clinical trials have found that the drug can reduce liver fat content in NAFLD patients. (2) Drugs that antagonize cell death: Hepatocyte death is an important driving factor in promoting liver inflammation and fibrosis. Therefore, inhibiting liver cell death can help prevent and treat NASH. Emricasan is a pancaspase inhibitor that can inhibit cell apoptosis, thereby alleviating liver inflammation and fibrosis. The drug is currently in Phase 2 clinical trials for the treatment of NASH. (3) Anti-inflammatory drugs: Inflammatory cells and pro-inflammatory cytokines play an important role in the occurrence and development of NASH. Apoptosis signaling kinase ASK-1 can promote the activity of JNK, and JNK is an important kinase that promotes inflammation and cell death. A short-term clinical trial found that an ASK-1 inhibitor (Selonsertib) can reduce fibrosis in patients with NASH. Selonsertib is currently in phase 3 clinical trials, and its efficacy awaits further evaluation.
另外，外科手术治疗也可用于非酒精性脂肪性肝病的控制。重度肥胖患者合并睡眠呼吸障碍、心脏病等疾病时，可以考虑减重手术治疗，有助于缓解肝脏脂肪变性、脂肪肝性肝炎和肝纤维化。当NAFLD患者病程发展到肝硬化或肝癌等终末期肝病阶段时，肝移植是唯一有效的治疗手段。不过，外科手术主要的适用具有针对性，费用昂贵，并且肝源获得及配型不易。In addition, surgical treatment can also be used to control non-alcoholic fatty liver disease. When severely obese patients are complicated by sleep apnea, heart disease and other diseases, bariatric surgery can help alleviate liver steatosis, fatty hepatitis and liver fibrosis. When NAFLD patients progress to the stage of end-stage liver disease such as cirrhosis or liver cancer, liver transplantation is the only effective treatment. However, surgical procedures are mainly targeted, expensive, and difficult to obtain and match liver sources.
因此，有必要研发一种能有效改善非酒精性脂肪性肝病的药物。 Therefore, it is necessary to develop a drug that can effectively improve non-alcoholic fatty liver disease.
发明内容Contents of the invention
基于此，根据本申请的各种实施例，提供一种嗜粘蛋白阿克曼菌在制备保健食品或者防治非酒精性脂肪肝疾病的药物中的应用。技术方案为：Based on this, according to various embodiments of the present application, there is provided an application of Akkermansia muciniphila in the preparation of health foods or drugs for preventing and treating non-alcoholic fatty liver disease. The technical solution is:
在本申请的第一方面，提供嗜粘蛋白阿克曼菌在制备保健食品或者防治非酒精性脂肪肝疾病的药物中的应用，所述嗜粘蛋白阿克曼菌选自保藏编号为CGMCC No.22793的嗜粘蛋白阿克曼菌和保藏编号为CGMCC No.22794的嗜粘蛋白阿克曼菌中的一种或两种。In the first aspect of the present application, there is provided the application of Akkermansia muciniphila in the preparation of health foods or drugs for the prevention and treatment of non-alcoholic fatty liver disease. The Akkermansia muciniphila is selected from CGMCC No. .22793 Akkermansia muciniphila and Akkermansia muciniphila with deposit number CGMCC No. 22794 or both.
在本申请的一些实施方式中，所述保藏编号为CGMCC No.22793的嗜粘蛋白阿克曼菌和保藏编号为CGMCC No.22794的嗜粘蛋白阿克曼菌各自独立地为活菌、灭活菌中的一种或多种。In some embodiments of the present application, the Akkermansia muciniphila deposited as CGMCC No. 22793 and the Akkermansia muciniphila deposited as CGMCC No. 22794 are each independently viable, sterilized One or more types of live bacteria.
在本申请的第二方面，提供保藏编号为CGMCC No.22793的嗜粘蛋白阿克曼菌，2021年06月28日保藏于中国微生物菌种保藏管理委员会普通微生物中心。In the second aspect of this application, Akkermansia muciniphila is provided with the deposit number CGMCC No. 22793, which was deposited at the General Microbiology Center of the Chinese Microbial Culture Collection Committee on June 28, 2021.
在本申请的第三方面，提供保藏编号为CGMCC No.22794的嗜粘蛋白阿克曼菌，2021年06月28日保藏于中国微生物菌种保藏管理委员会普通微生物中心。In the third aspect of this application, Akkermansia muciniphila is provided with the deposit number CGMCC No. 22794, which was deposited at the General Microbiology Center of the Chinese Microbial Culture Collection Committee on June 28, 2021.
在本申请的第四方面，提供一种益生菌组合产品，所述益生菌组合产品包含保藏编号为CGMCC No.22793的嗜粘蛋白阿克曼菌和保藏编号为CGMCC No.22794的嗜粘蛋白阿克曼菌中的一种或两种。In the fourth aspect of the present application, a probiotic combination product is provided, which includes Akkermansia muciniphila with a deposit number of CGMCC No. 22793 and Akermansia muciniphila with a deposit number of CGMCC No. 22794. One or both Akkermansia species.
在本申请的第五方面，提供一种保健食品，包含：第二方面所述的嗜粘蛋白阿克曼菌和第三方面所述的嗜粘蛋白阿克曼菌中的一种或两种，以及食品辅料。In the fifth aspect of the present application, a health food is provided, comprising: one or both of the Akkermansia muciniphila described in the second aspect and the Akkermansia muciniphila described in the third aspect. , and food accessories.
在本申请的一些实施方式中，所述保健食品为糕点或者饮品。In some embodiments of the present application, the health food is pastry or drink.
在本申请的第六方面，提供一种药品，包含：第二方面所述的嗜粘蛋白阿克曼菌和第三方面所述的嗜粘蛋白阿克曼菌中的一种或两种，以及药用辅料。In the sixth aspect of the present application, a medicine is provided, comprising: one or both of the Akkermansia muciniphila described in the second aspect and the Akkermansia muciniphila described in the third aspect, and pharmaceutical excipients.
在本申请的一些实施方式中，所述药物的剂型为丸剂、片剂、颗粒剂、胶囊剂、溶液剂、管饲制剂、混悬剂、霜剂、喷雾剂、膏剂或贴剂。In some embodiments of the present application, the dosage form of the drug is a pill, tablet, granule, capsule, solution, tube feeding preparation, suspension, cream, spray, ointment or patch.
在本申请的第七方面，提供一种培养第二方面所述的嗜粘蛋白阿克曼菌或/和第三方面所述的嗜粘蛋白阿克曼菌的方法，In the seventh aspect of the present application, a method for cultivating Akkermansia muciniphila according to the second aspect or/and Akkermansia muciniphila according to the third aspect is provided,
培养的条件包括：以黏蛋白为唯一碳源；或/和，厌氧环境；或/和，36.5℃-37.5℃。The culture conditions include: using mucin as the sole carbon source; or/and, anaerobic environment; or/and, 36.5℃-37.5℃.
本申请的一个或多个实施例细节在下面的描述中提出，本申请的其他特征、目的和优点将从说明书及其权利要求书变得明显。The details of one or more embodiments of the present application are set forth in the description that follows, and other features, objects, and advantages of the present application will be apparent from the description and claims hereof.
附图说明Description of the drawings
为了更清楚地说明本申请实施例或传统技术中的技术方案，下面将对实施例或传统技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据公开的附图获得其他的附图。In order to more clearly explain the technical solutions in the embodiments of the present application or the traditional technology, the drawings needed to be used in the description of the embodiments or the traditional technology will be briefly introduced below. Obviously, the drawings in the following description are only for the purpose of explaining the embodiments or the technical solutions of the traditional technology. For the embodiments of the application, those of ordinary skill in the art can also obtain other drawings based on the disclosed drawings without exerting creative efforts.
图1为采用实施例2方法培养得到的嗜粘蛋白阿克曼菌AM02的菌落特征图；Figure 1 is a colony characteristic diagram of Akkermansia muciniphila AM02 cultured using the method of Example 2;
图2为采用实施例2方法培养得到的嗜粘蛋白阿克曼菌AM06的菌落特征图；Figure 2 is a colony characteristic diagram of Akkermansia muciniphila AM06 cultured using the method of Example 2;
图3为采用实施例2方法培养得到的嗜粘蛋白阿克曼菌AM02进行革兰氏染色后的显微镜观察图；Figure 3 is a microscopic observation of Akkermansia muciniphila AM02 cultured using the method of Example 2 after Gram staining;
图4为采用实施例2方法培养得到的嗜粘蛋白阿克曼菌AM06进行革兰氏染色后的显微镜观察图；Figure 4 is a microscopic observation of Akkermansia muciniphila AM06 cultured using the method of Example 2 after Gram staining;
图5为实施例5中嗜粘蛋白阿克曼菌培养上清代谢物PCA分析图；Figure 5 is a PCA analysis diagram of metabolites in the culture supernatant of Akkermansia muciniphila in Example 5;
图6为实施例6中嗜粘蛋白阿克曼菌对TNF-α和IFN-γ诱导Caco2细胞紧密连接蛋白ZO-1表达降低的影响的荧光显微镜拍摄图片。Figure 6 is a fluorescence microscope picture of the effect of Akkermansia muciniphila on the reduction of tight junction protein ZO-1 expression in Caco2 cells induced by TNF-α and IFN-γ in Example 6.
本申请提供的嗜粘蛋白阿克曼菌AM02，其分类命名为Akkermansia muciniphila，已于2021年06月28日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.22794；该菌株于2021年06月28日由保藏中心收到并登记入册，经保藏中心于2021年06月28日检测为存活菌株。The Akkermansia muciniphila AM02 provided in this application is classified and named Akkermansia muciniphila. It has been deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 28, 2021. Address: Beichen West Road, Chaoyang District, Beijing No. 3, Hospital No. 1, the preservation number is CGMCC No. 22794; the strain was received and registered by the preservation center on June 28, 2021, and was detected as a surviving strain by the preservation center on June 28, 2021.
本申请提供的嗜粘蛋白阿克曼菌AM06，其分类命名为Akkermansia muciniphila，已于2021年06月28日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.22793；该菌株于2021年06月28日由保藏中心收到并登记入册，经保藏中心于2021年06月28日检测为存活菌株。The Akkermansia muciniphila AM06 provided in this application is classified and named Akkermansia muciniphila. It has been deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on June 28, 2021. Address: Beichen West Road, Chaoyang District, Beijing No. 3, No. 1 Courtyard, the preservation number is CGMCC No. 22793; the strain was received and registered by the preservation center on June 28, 2021, and was detected as a surviving strain by the preservation center on June 28, 2021.
具体实施方式 Detailed ways
下面结合附图、实施方式和实施例，对本申请作进一步详细的说明。应理解，这些实施方式和实施例仅用于说明本申请而不用于限制本申请的范围，提供这些实施方式和实施例的目的是使对本申请公开内容理解更加透彻全面。还应理解，本申请可以以许多不同的形式来实现，并不限于本文所描述的实施方式和实施例，本领域技术人员可以在不违背本申请内涵的情况下作各种改动或修改，得到的等价形式同样落于本申请的保护范围。此外，在下文的描述中，给出了大量具体的细节以便提供对本申请更为充分地理解，应理解，本申请可以无需一个或多个这些细节而得以实施。The present application will be described in further detail below in conjunction with the accompanying drawings, implementation modes and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the disclosure of the present application more thorough and comprehensive. It should also be understood that this application can be implemented in many different forms and is not limited to the implementation modes and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of this application, and obtain The equivalent forms also fall within the protection scope of this application. Additionally, in the following description, numerous specific details are given in order to provide a thorough understanding of the present application, and it is understood that the present application may be practiced without one or more of these details.
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述实施方式和实施例的目的，不是旨在于限制本申请。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing embodiments and examples only and is not intended to limit the application.
术语the term
除非另外说明或存在矛盾之处，本文中使用的术语或短语具有以下含义：Unless otherwise stated or contradictory, the terms or phrases used in this article have the following meanings:
本文所使用的术语“和/或”、“或/和”、“及/或”的选择范围包括两个或两个以上相关所列项目中任一个项目，也包括相关所列项目的任意的和所有的组合，所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。需要说明的是，当用至少两个选自“和/或”、“或/和”、“及/或”的连词组合连接至少三个项目时，应当理解，在本申请中，该技术方案毫无疑问地包括均用“逻辑与”连接的技术方案，还毫无疑问地包括均用“逻辑或”连接的技术方案。比如，“A及/或B”包括A、B和A+B三种并列方案。又比如，“A，及/或，B，及/或，C，及/或，D”的技术方案，包括A、B、C、D中任一项(也即均用“逻辑或”连接的技术方案)，也包括A、B、C、D的任意的和所有的组合，也即包括A、B、C、D中任两项或任三项的组合，还包括A、B、C、D的四项组合(也即均用“逻辑与”连接的技术方案)。The terms "and/or", "or/and" and "and/or" used in this article include any one of two or more related listed items, and also include any of the related listed items. and all combinations, including any two of the related listed items, any more of the related listed items, or a combination of all of the related listed items. It should be noted that when at least three items are connected in combination with at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that in this application, the technical solution It undoubtedly includes technical solutions that are all connected by "logical AND", and it also undoubtedly includes technical solutions that are all connected by "logical OR". For example, "A and/or B" includes three parallel solutions: A, B and A+B. For another example, the technical solution of "A, and/or, B, and/or, C, and/or, D" includes any one of A, B, C, and D (that is, they are all connected with "logical OR" technical solution), also includes any and all combinations of A, B, C, and D, that is, including combinations of any two or any three of A, B, C, and D, and also includes A, B, C , four combinations of D (that is, technical solutions that are all connected by "logical AND").
本申请中涉及“多个”、“多种”、“多次”、“多元”等，如无特别限定，指在数量上大于2或等于2。例如，“一种或多种”表示一种或大于等于两种。This application refers to "multiple", "multiple", "multiple", "multiple", etc., which means that the number is greater than 2 or equal to 2 unless otherwise specified. For example, "one or more" means one or more than two.
本文中所使用的“其组合”、“其任意组合”、“其任意组合方式”等中包括所列项目中任两个或任两个以上项目的所有合适的组合方式。As used herein, "combinations thereof", "any combinations thereof", "any combinations thereof", etc. include all suitable combinations of any two or more of the listed items.
本文中，“合适的组合方式”、“合适的方式”、“任意合适的方式”等中所述“合适”，以能够实施本申请的技术方案、解决本申请的技术问题、实现本申请预期的技术效果为准。In this article, "appropriate" mentioned in "appropriate combination", "appropriate way", "any suitable way", etc., can implement the technical solution of the present application, solve the technical problems of the present application, and realize the expectations of the present application. The technical effect shall prevail.
本文中，“优选”、“更好”、“更佳”、“为宜”仅为描述效果更好的实施方式或实施例，应当理解，并不构成对本申请保护范围的限制。In this article, "preferable", "better", "better" and "appropriate" are only used to describe implementations or examples with better effects. It should be understood that they do not limit the scope of protection of the present application.
本申请中，“进一步”、“更进一步”、“特别”等用于描述目的，表示内容上的差异，但并不应理解为对本申请保护范围的限制。In this application, "further", "further", "especially", etc. are used for descriptive purposes and indicate differences in content, but should not be understood as limiting the scope of protection of this application.
本申请中，“可选地”、“可选的”、“可选”，指可有可无，也即指选自“有”或“无”两种并列方案中的任一种。如果一个技术方案中出现多处“可选”，如无特别说明，且无矛盾之处或相互制约关系，则每项“可选”各自独立。In this application, "optional", "optional" and "optional" mean optional, that is, any one selected from the two parallel solutions of "with" or "without". If there are multiple "optionals" in a technical solution, each "optional" will be independent unless otherwise specified and there is no contradiction or mutual restriction.
本申请中，“第一方面”、“第二方面”、“第三方面”、“第四方面”等中，术语“第一”、“第二”、“第三”、“第四”等仅用于描述目的，不能理解为指示或暗示相对重要性或数量，也不能理解为隐含指明所指示的技术特征的重要性或数量。而且“第一”、“第二”、“第三”、“第四”等仅起到非穷举式的列举描述目的，应当理解并不构成对数量的封闭式限定。In this application, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third" and "fourth" etc. are for descriptive purposes only and shall not be understood as indicating or implying relative importance or quantity, nor shall they be understood as implicitly indicating the importance or quantity of indicated technical features. Furthermore, “first”, “second”, “third”, “fourth”, etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation of quantity.
本申请中，以开放式描述的技术特征中，包括所列举特征组成的封闭式技术方案，也包括包含所列举特征的开放式技术方案。In this application, the technical features described in open format include closed technical solutions composed of the listed features, and also include open technical solutions including the listed features.
本申请中，涉及到数值区间(也即数值范围)，如无特别说明，可选的数值分布在上述数值区间内视为连续，且包括该数值范围的两个数值端点(即最小值及最大值)，以及这两个数值端点之间的每一个数值。如无特别说明，当数值区间仅仅指向该数值区间内的整数时，包括该数值范围的两个端点整数，以及两个端点之间的每一个整数，在本文中，相当于直接列举了每一个整数，比如t为选自1～10的整数，表示t为选自由1、2、3、4、5、6、7、8、9和10构成的整数组的任一个整数。此外，当提供多个范围描述特征或特性时，可以合并这些范围。换言之，除非另有指明，否则本文中所公开之范围应理解为包括其中所归入的任何及所有的子范围。 In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the optional numerical distribution is considered to be continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value). value), and every value between the two numeric endpoints. Unless otherwise specified, when a numerical interval only points to integers within the numerical interval, including the two endpoint integers of the numerical range, and every integer between the two endpoints, in this article, it is equivalent to directly enumerating each Integer, for example, t is an integer selected from 1 to 10, indicating that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Additionally, when multiple scopes are provided to describe a feature or characteristic, these scopes can be combined. In other words, unless otherwise indicated, the ranges disclosed herein should be understood to include any and all subranges subsumed therein.
本申请中的温度参数，如无特别限定，既允许为恒温处理，也允许在一定温度区间内存在变动。应当理解的是，所述的恒温处理允许温度在仪器控制的精度范围内进行波动。允许在如±5℃、±4℃、±3℃、±2℃、±1℃的范围内波动。The temperature parameters in this application, unless otherwise specified, are allowed to be treated at a constant temperature, or to vary within a certain temperature range. It should be understood that the thermostatic treatment described allows the temperature to fluctuate within the accuracy of the instrument control. It is allowed to fluctuate within the range of ±5℃, ±4℃, ±3℃, ±2℃ and ±1℃.
本申请中，％(w/w)与wt％均表示重量百分比，％(v/v)指体积百分比，％(w/v)指质量体积百分数。In this application, % (w/w) and wt% both represent weight percentage, % (v/v) refers to volume percentage, and % (w/v) refers to mass volume percentage.
在本申请提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。除非和本申请的申请目的和/或技术方案相冲突，否则，本申请涉及的引用文献以全部内容、全部目的被引用。本申请中涉及引用文献时，相关技术特征、术语、名词、短语等在引用文献中的定义也一并被引用。本申请中涉及引用文献时，被引用的相关技术特征的举例、优选方式也可作为参考纳入本申请中，但以能够实施本申请为限。应当理解，当引用内容与本申请中的描述相冲突时，以本申请为准或者适应性地根据本申请的描述进行修正。All documents mentioned in this application are herein incorporated by reference to the same extent as if each individual document was individually incorporated by reference. Unless it conflicts with the application purpose and/or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When citing documents in this application, the examples and preferred modes of the cited relevant technical features may also be incorporated into this application as references, but only to the extent that they enable the implementation of this application. It should be understood that when the quoted content conflicts with the description in this application, this application shall prevail or be adapted to be modified according to the description in this application.
非酒精性脂肪肝病NAFLD的致病因素较为复杂，对其病因有多重假说：The causative factors of non-alcoholic fatty liver disease NAFLD are complex, and there are multiple hypotheses for its etiology:
“二次打击”是1998年提出的NAFLD发病的经典假说。“首次打击”指胰岛素抵抗造成的肝脏内甘油三酯堆积，肝脏对内外源性损害因子、缺血、缺氧等的耐受能力下降。“二次打击”是指甘油三酯堆积于肝细胞后，在炎性细胞因子、氧化应激和内质网应激等作用下肝细胞最终发生损伤，肝组织出现炎症、纤维化等病理改变，“二次打击”学说逐渐倾向“多重打击”学说转变。"Second hit" is a classic hypothesis for the onset of NAFLD proposed in 1998. "First hit" refers to the accumulation of triglycerides in the liver caused by insulin resistance, and the liver's ability to tolerate endogenous damage factors, ischemia, hypoxia, etc. is reduced. "Second hit" means that after triglycerides accumulate in liver cells, the liver cells will eventually be damaged under the action of inflammatory cytokines, oxidative stress, endoplasmic reticulum stress, etc., and pathological changes such as inflammation and fibrosis will occur in liver tissue. , the "second strike" doctrine gradually shifted to the "multiple strike" doctrine.
脂毒性学说认为，肝内甘油三酯堆积并不会引起胰岛素抵抗及肝细胞损伤，引起NASH的核心机制是游离胆固醇、游离脂肪酸(FFA)及其代谢产物所引起的内质网应激、氧化应激及炎性反应。传统观点认为，堆积在肝细胞中的甘油三酯促进了脂质过氧化、氧化应激、炎症和纤维化，是NAFLD发生发展的驱动因素；但是，这一观点正日益受到挑战，因为甘油三酯可能可以拮抗脂毒性。研究显示，动物脂肪和乳制品中含量丰富的棕榈酸(C16:0)和硬脂酸(C18:0)可以促进内质网应激、炎症小体的激活以及肝细胞的死亡。过多的棕榈酸、硬脂酸等游离脂肪酸堆积在肝脏可形成甘油二酯(DAG)、神经酰胺(ceramides)、溶血磷脂酸胆碱(LPCs)等代谢中间产物，发挥脂毒性的作用。The lipotoxicity theory believes that the accumulation of triglycerides in the liver will not cause insulin resistance and liver cell damage. The core mechanism causing NASH is endoplasmic reticulum stress and oxidation caused by free cholesterol, free fatty acids (FFA) and their metabolites. Stress and inflammatory response. The traditional view is that triglycerides accumulated in hepatocytes promote lipid peroxidation, oxidative stress, inflammation and fibrosis, and are the driving factors for the development of NAFLD; however, this view is increasingly being challenged because triglycerides Esters may antagonize lipotoxicity. Studies have shown that palmitic acid (C16:0) and stearic acid (C18:0), which are abundant in animal fats and dairy products, can promote endoplasmic reticulum stress, activation of inflammasomes, and liver cell death. Excessive free fatty acids such as palmitic acid and stearic acid accumulate in the liver and can form metabolic intermediates such as diacylglycerol (DAG), ceramides (ceramides), and lysophosphatidic acid choline (LPCs), which exert lipotoxic effects.
果糖的广泛使用也被认为是NAFLD的致病因素之一。果糖主要在肝脏代谢，可以促进脂质大量合成，抑制线粒体β氧化，引起肝细胞脂肪变性。果糖由于自身不稳定(含有五元呋喃环)，会促进活性氧(ROS)的生成，引起肝细胞的损伤。果糖经果糖激酶催化并快速磷酸化成1-磷酸果糖以及促进脂肪酸从头合成时，均需消耗肝脏中的ATP。ATP的大量消耗导致其代谢产物二磷酸腺苷和次黄嘌呤核苷酸产生增加，并转化为尿酸，促进尿酸产生增加，而尿酸可以加重代谢综合征。此外，长期大量果糖摄入，可以导致肠道菌群紊乱和肠壁通透性增加，细菌内毒素等毒性产物通过门静脉进入肝脏，促进肝脏炎症。The widespread use of fructose is also considered to be one of the causative factors of NAFLD. Fructose is mainly metabolized in the liver, which can promote large-scale lipid synthesis, inhibit mitochondrial β-oxidation, and cause hepatocyte steatosis. Due to its own instability (containing a five-membered furan ring), fructose will promote the generation of reactive oxygen species (ROS) and cause damage to liver cells. When fructose is catalyzed by fructokinase and rapidly phosphorylated into fructose 1-phosphate and promotes de novo synthesis of fatty acids, ATP in the liver is required. The large consumption of ATP leads to an increase in the production of its metabolites adenosine diphosphate and hypoxanthine nucleotides, which are converted into uric acid, promoting an increase in the production of uric acid, and uric acid can aggravate metabolic syndrome. In addition, long-term intake of large amounts of fructose can lead to intestinal flora disorder and increased intestinal wall permeability. Toxic products such as bacterial endotoxins enter the liver through the portal vein and promote liver inflammation.
随着肝-肠轴理论的提出，肠道菌群与NAFLD的关系和机制研究也落入了人们的视野。现有的研究显示，NAFLD与肠道菌群失调相互影响，彼此促进。肠道菌群失调导致NAFLD发生及发展的相关机制为：小肠中革兰阴性菌过度生长，增加内源性乙醇的生成。内源性乙醇联合其他因素促进致炎性细胞因子的分泌，诱导肠道巨噬细胞激活进而引起肠道屏障功能损坏。肠道通透性的改变可能便于细菌内毒素更多的进入到血液循环中，导致血液及门静脉中内毒素水平升高。内毒素进而通过门静脉循环到达肝脏，激活肝脏Kupffer细胞，并促进Kupffer细胞生成细胞因子，进而引发炎症瀑布反应，损伤肝细胞，阻碍其分泌、代谢等功能，引起胆汁分泌障碍。胆汁分泌异常会影响脂肪正常的新陈代谢，而脂肪异常堆积在肝脏导致肝细胞脂肪变性，并最终形成NAFLD。肝功能受损后，肝脏处理肠道来源毒物的能力降低，肠道毒物可能蓄积且损害肠黏膜屏障，从而导致肠道功能失调；此外，相关抗体、溶菌酶及分泌液的减少，再加上内毒素增加，促使环境有利于革兰阴性菌的生长，但是限制益生菌的生长，使得肠壁局部抵抗力下降，这些因素均可加剧NAFLD患者肠道菌群失调。With the proposal of the liver-gut axis theory, research on the relationship and mechanism between intestinal flora and NAFLD has also fallen into people's field of vision. Existing research shows that NAFLD and intestinal flora imbalance interact and promote each other. The relevant mechanism that intestinal flora imbalance leads to the occurrence and development of NAFLD is: the overgrowth of Gram-negative bacteria in the small intestine increases the production of endogenous ethanol. Endogenous ethanol combined with other factors promotes the secretion of pro-inflammatory cytokines, induces activation of intestinal macrophages, and causes damage to intestinal barrier function. Changes in intestinal permeability may allow more bacterial endotoxins to enter the blood circulation, leading to increased endotoxin levels in the blood and portal vein. Endotoxin then reaches the liver through the portal circulation, activates liver Kupffer cells, and promotes Kupffer cells to produce cytokines, thereby triggering an inflammatory cascade reaction, damaging liver cells, hindering their secretion, metabolism and other functions, and causing bile secretion disorders. Abnormal bile secretion will affect the normal metabolism of fat, and abnormal accumulation of fat in the liver will lead to steatosis of liver cells and eventually the formation of NAFLD. After liver function is damaged, the liver's ability to process toxicants from the intestinal tract is reduced, and intestinal toxicants may accumulate and damage the intestinal mucosal barrier, leading to intestinal dysfunction; in addition, the reduction of related antibodies, lysozyme and secretions, coupled with The increase in endotoxin makes the environment conducive to the growth of Gram-negative bacteria, but restricts the growth of probiotics and reduces the local resistance of the intestinal wall. These factors can aggravate the intestinal flora imbalance in NAFLD patients.
基于上述理论，调节肠道菌群以治疗NAFLD的治疗方法被开发出来。目前已有益生菌/益生元/合生元调节法；抗生素调节法；黏附分子调节法和粪菌移植(FMT)等方法。但是，这些治疗方法所采用的菌种以一代益生菌为主，效果更好、安全性更佳、耐受力更强的二代益生菌有必要被开发出来。Based on the above theory, therapeutic methods to modulate intestinal flora to treat NAFLD have been developed. At present, there are probiotic/prebiotic/synbiotic regulation methods; antibiotic regulation methods; adhesion molecule regulation methods and fecal bacterial transplantation (FMT) and other methods. However, the strains used in these treatments are mainly first-generation probiotics, and it is necessary to develop second-generation probiotics that are more effective, safer, and more tolerant.
本申请的第一方面First aspect of this application
本申请提供嗜粘蛋白阿克曼菌在制备保健食品或者防治非酒精性脂肪肝疾病的药物中的应用，所述嗜粘蛋白阿克曼菌选自保藏编号为CGMCC No.22793的嗜粘蛋白阿克曼菌和保藏编号为CGMCC  No.22794的嗜粘蛋白阿克曼菌中的一种或两种。The present application provides the application of Akkermansia muciniphila in the preparation of health foods or drugs for preventing and treating non-alcoholic fatty liver disease. The Akkermansia muciniphila is selected from the group consisting of Akkermansia muciniphila deposited as CGMCC No. 22793. Akkermansia and accession number CGMCC One or two species of Akkermansia muciniphila No. 22794.
本申请所述嗜粘蛋白阿克曼菌可以是活菌体，也可以经过灭活、基因重组、改造或修饰、减毒、化学处理、物理处理的保留生物活性的嗜黏蛋白阿克曼菌，还可以是菌体的裂解物、培养物(例如上清液)或者从上培养物中提取到的成分。优选地，所述保藏编号为CGMCC No.22793的嗜粘蛋白阿克曼菌和保藏编号为CGMCC No.22794的嗜粘蛋白阿克曼菌各自独立地为活菌、灭活菌(可以是形态结构完整的灭活菌、也可以是形态结构不完整的)中的一种或多种。The Akkermansia muciniphila described in this application can be a living cell, or it can be a biologically active Akkermansia muciniphila that has been inactivated, genetically recombined, transformed or modified, attenuated, chemically treated, or physically treated. , it may also be a bacterial lysate, a culture (such as a supernatant), or a component extracted from the supernatant. Preferably, the Akkermansia muciniphila deposited as CGMCC No. 22793 and the Akkermansia muciniphila deposited as CGMCC No. 22794 are each independently a live bacterium or an inactivated bacterium (which may be in the form One or more of the inactivated bacteria with complete structure or incomplete morphological structure).
本申请所述的非酒精性脂肪肝病，是指除外过量饮酒和其他明确的肝损伤因素所导致的、以肝细胞脂肪变为特征的临床综合征，包括单纯性脂肪肝、非酒精性脂肪性肝炎以及相关的肝硬化和肝细胞肝癌。Non-alcoholic fatty liver disease as described in this application refers to a clinical syndrome characterized by hepatocellular steatosis, excluding excessive drinking and other clear liver damage factors, including simple fatty liver, non-alcoholic fatty liver disease and non-alcoholic fatty liver disease. Hepatitis and related cirrhosis and hepatocellular carcinoma.
在本申请中，“防治”包括预防、治疗、辅助治疗等方面。如本文所用，如本文所用，“防治”是指减轻、延缓进展、衰减、预防，或维持现有疾病或病症。“防治”还包括将疾病或病症的一个或多个症状治愈、预防其发展或减轻到某种程度。In this application, "prevention and treatment" includes prevention, treatment, auxiliary treatment, etc. As used herein, "preventing, treating," or "treating" means alleviating, delaying progression, attenuating, preventing, or maintaining an existing disease or condition. "Prevention and treatment" also includes curing, preventing the development of, or alleviating to some extent one or more symptoms of a disease or condition.
本申请所述的药物，可以是人药，也可以是动物用药。The drugs described in this application may be human drugs or animal drugs.
在本申请中，“药物”包括在体内或体外提供生理和/或药理作用的任何药剂、化合物、组合物或混合物，且往往提供的是有益效果。“药物”在体内产生生理和/或药理作用的范围没有特别限制，可以为全身效果，也可以只在局部产生效果。所述“药物”的活性没有特别限制，可以为能与其它物质发生相互作用的活性物质，也可以为不发生相互作用的惰性物质。As used herein, "drug" includes any agent, compound, composition or mixture that provides a physiological and/or pharmacological effect in vivo or in vitro, often providing a beneficial effect. The scope of physiological and/or pharmacological effects of a "drug" in the body is not particularly limited. It can have systemic effects or only local effects. The activity of the "drug" is not particularly limited. It can be an active substance that can interact with other substances, or it can be an inert substance that does not interact.
在本申请中，“保健食品”指可食用的物质。In this application, "nutraceutical" refers to edible substances.
本申请的第二方面Second aspect of this application
本申请提供保藏编号为CGMCC No.22793的嗜粘蛋白阿克曼菌，该菌株是从母乳分离得到的菌株，其菌落培养特征包括：圆形凸起、边缘整齐、不透明、白色、大小不均一；该菌株的16S RNA序列如SEQ ID NO:2所示；相对于ATCC BAA-835，人工胃液和人工肠液的耐受性更好，上清液非靶向代谢学差异分析见图5和表7，改善非酒精性脂肪肝病的效果更明显，因此相对于ATCC BAA-835可以鉴定为新菌株。This application provides Akkermansia muciniphila with the deposit number CGMCC No. 22793. This strain is isolated from breast milk. Its colony culture characteristics include: round protrusions, neat edges, opaque, white, and uneven sizes. ; The 16S RNA sequence of this strain is shown in SEQ ID NO: 2; Compared with ATCC BAA-835, artificial gastric juice and artificial intestinal juice are better tolerated. The non-targeted metabolic difference analysis of the supernatant is shown in Figure 5 and Table 7. The effect of improving non-alcoholic fatty liver disease is more obvious, so BAA-835 can be identified as a new strain compared to ATCC.
本申请的第三方面Third aspect of this application
本申请提供保藏编号为CGMCC No.22794的嗜粘蛋白阿克曼菌，该菌株是粪便中分离得到的菌株，其菌落培养特征包括：圆形凸起、边缘整齐、不透明、白色、大小不均一；该菌株的16S RNA序列如SEQ ID NO:1所示；相对于ATCC BAA-835，人工胃液和人工肠液的耐受性更好，上清液非靶向代谢学差异分析见图5和表7，改善非酒精性脂肪肝病的效果更明显，因此相对于ATCC BAA-835可以鉴定为新菌株。This application provides Akkermansia muciniphila with the deposit number CGMCC No. 22794. This strain is a strain isolated from feces. Its colony culture characteristics include: round protrusions, neat edges, opaque, white, and uneven sizes. ; The 16S RNA sequence of this strain is shown in SEQ ID NO: 1; Compared with ATCC BAA-835, artificial gastric juice and artificial intestinal juice are better tolerated. The non-targeted metabolic difference analysis of the supernatant is shown in Figure 5 and Table 7. The effect of improving non-alcoholic fatty liver disease is more obvious, so BAA-835 can be identified as a new strain compared to ATCC.
本申请的第四方面The fourth aspect of this application
本申请提供益生菌组合产品，所述益生菌组合产品包含保藏编号为CGMCC No.22793的嗜粘蛋白阿克曼菌和保藏编号为CGMCC No.22794的嗜粘蛋白阿克曼菌中的一种或两种。This application provides a probiotic combination product, which includes one of Akkermansia muciniphila deposited as CGMCC No. 22793 and Akkermansia muciniphila deposited as CGMCC No. 22794. Or both.
本申请的第五方面The fifth aspect of this application
本申请提供一种保健食品，包含：第二方面所述的嗜粘蛋白阿克曼菌和第三方面所述的嗜粘蛋白阿克曼菌中的一种或两种，以及食品辅料。The present application provides a health food, comprising: one or both of the Akkermansia muciniphila described in the second aspect and the Akkermansia muciniphila described in the third aspect, and food accessories.
在其中一些实施例中，所述保健食品为糕点或者饮品。In some embodiments, the health food is pastries or drinks.
“保健食品”指可食用的组合物。应当理解，的食品组合物除包含前述的嗜粘蛋白阿克曼菌外，还允许包含任意合适的其他可食用物质。在一些实施例中，其他可食用物质可选自保健品管理规范中允许添加的物质，进一步地，不包含保健品牌管理规范中禁止添加的物质。如无特别限定，保健品管理规范指生产时的现行规范。"Nutraceutical" refers to an edible composition. It should be understood that in addition to the aforementioned Akkermansia muciniphila, the food composition is also allowed to contain any suitable other edible substances. In some embodiments, other edible substances can be selected from substances that are allowed to be added in the health care product management regulations, and further do not include substances that are prohibited from being added in the health care brand management regulations. Unless otherwise specified, health product management regulations refer to the current regulations at the time of production.
在本申请中，食品添加剂也属于可食用辅料。可食用辅料的举例如食糖，果糖，蜂蜜，葡萄糖，淀粉，维生素，有益微量元素和中量元素包括钙粉，大豆粉，绿豆粉，麦芽糊精，奶粉，蔬菜汁，水果汁，香料或香精，本申请的可食用辅料可以单一或复合的使用。In this application, food additives also belong to edible excipients. Examples of edible excipients include sugar, fructose, honey, glucose, starch, vitamins, beneficial trace elements and medium elements including calcium powder, soybean powder, mung bean powder, maltodextrin, milk powder, vegetable juice, fruit juice, spices or flavors , the edible excipients of this application can be used singly or in combination.
本申请的第六方面The sixth aspect of this application
本申请提供一种药物组合物，包含：第二方面所述的嗜粘蛋白阿克曼菌和第三方面所述的嗜粘蛋白阿克曼菌中的一种或两种，以及药用辅料。 The present application provides a pharmaceutical composition, comprising: one or both of the Akkermansia muciniphila described in the second aspect and the Akkermansia muciniphila described in the third aspect, and pharmaceutical excipients. .
在其中一些实施例中，所述药物的剂型为丸剂、片剂、颗粒剂、胶囊剂、溶液剂、管饲制剂、、混悬剂、霜剂、喷雾剂、膏剂或贴剂。In some embodiments, the dosage form of the medicament is pills, tablets, granules, capsules, solutions, tube feeding preparations, suspensions, creams, sprays, ointments or patches.
在本申请中，“药物组合物”指具有防治作用、可作为药物使用的组合物。In this application, "pharmaceutical composition" refers to a composition that has preventive and therapeutic effects and can be used as a medicine.
本申请中，“辅料”包括但不限于甘露醇、山梨醇、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、维生素C、EDTA二钠、EDTA钙钠，一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、氯化钠、氯化钾、乳酸钠、木糖醇、麦芽糖、葡萄糖、果糖、低聚果糖、右旋糖苷、甘氨酸、淀粉、蔗糖、麦芽糊精、乳糖、甘露糖醇、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、吐温80、琼脂、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、磷脂类材料、高岭土、滑石粉、硬脂酸钙、硬脂酸镁。In this application, "excipients" include but are not limited to mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium EDTA Sodium, monovalent alkali metal carbonates, acetates, phosphates or their aqueous solutions, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose , Fructooligosaccharides, dextran, glycine, starch, sucrose, maltodextrin, lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerol, Tween 80. Agar, calcium carbonate, calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate.
在本申请中，“药学上可接受的”指在合理医学判断范围内适于施用患者且与合理益处/风险比相称的那些配体、材料、组合物和/或剂型。As used herein, "pharmaceutically acceptable" refers to those ligands, materials, compositions and/or dosage forms that are, within the scope of reasonable medical judgment, suitable for administration to a patient and proportionate to a reasonable benefit/risk ratio.
在本申请中，“药学上可接受的载体”指药学上可接受的材料、组合物或媒剂，例如液体或固体填充剂、稀释剂、赋形剂、溶剂或囊封材料。如本文所用，语言“药学上可接受的载体”包括与药物施用相容的缓冲剂、注射用无菌水、溶剂、分散介质、包衣、抗细菌剂及抗真菌剂、等渗剂及吸收延迟剂及诸如此类。在与配制物中其他成分兼容且对患者无害的意义上，每种体必须为“药学上可接受的”。合适的实例包括但不限于：(1)糖，例如乳糖、葡萄糖及蔗糖；(2)淀粉，例如玉米淀粉、马铃薯淀粉及经取代或未经取代的β-环糊精；(3)纤维素及其衍生物，例如羧甲基纤维素钠、乙基纤维素及乙酸纤维素；(4)粉状黄蓍胶；(5)麦芽；(6)明胶；(7)滑石；(8)赋形剂，例如可可脂及栓剂蜡；(9)油类，例如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油及大豆油；(10)二醇，例如丙二醇；(11)多元醇，例如甘油、山梨醇、甘露醇及聚乙二醇；(12)酯类，例如油酸乙酯及月桂酸乙酯；(13)琼脂；(14)缓冲剂，例如氢氧化镁及氢氧化铝；(15)海藻酸；(16)无热原水；(17)等渗盐水；(18)林格氏溶液；(19)乙醇；(20)磷酸盐缓冲液；及(21)药物配制物中所采用的其他无毒兼容物质。As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. As used herein, the language "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorbent agents that are compatible with the administration of the drug. Delay agents and the like. Each entity must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient. Suitable examples include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch, potato starch and substituted or unsubstituted β-cyclodextrin; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) Powdered tragacanth; (5) Malt; (6) Gelatin; (7) Talc; (8) Fu excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols Alcohols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) Esters, such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, such as magnesium hydroxide and hydrogen Aluminum oxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) pharmaceutical preparation Other non-toxic compatible substances used in products.
在一些实施方式中，防治酒精性脂肪肝的组合物为药物组合物。进一步地，防治酒精性脂肪肝的组合物中含有治疗有效量的所述嗜粘蛋白阿克曼菌。In some embodiments, the composition for preventing and treating alcoholic fatty liver disease is a pharmaceutical composition. Further, the composition for preventing and treating alcoholic fatty liver disease contains a therapeutically effective amount of Akkermansia muciniphila.
在本申请中，“治疗有效量”是指针对疾病、病症和/或症状，将引起个体的生物学或医学响应的药物活性成分的量，例如为个体带来生理和/或药理上积极效果的本申请化合物的量，所述生理和/或药理上积极效果包括但不限于降低或抑制酶或蛋白质活性或改善症状、缓解病症、减缓或延迟疾病进程或预防疾病等。As used herein, a "therapeutically effective amount" refers to an amount of a pharmaceutically active ingredient that will elicit a biological or medical response in an individual in response to a disease, disorder, and/or symptom, such as a physiological and/or pharmacological positive effect in the individual. The amount of the compound of the present application, the physiological and/or pharmacological positive effects include but are not limited to reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, slowing down or delaying disease progression or preventing diseases, etc.
本申请的第七方面The seventh aspect of this application
本申请提供一种培养第二方面所述的嗜粘蛋白阿克曼菌或/和第三方面所述的嗜粘蛋白阿克曼菌的方法，培养的条件包括：以黏蛋白为唯一碳源；或/和，厌氧环境；或/和，36.5℃-37.5℃。The present application provides a method for cultivating the Akkermansia muciniphila described in the second aspect or/and the Akkermansia muciniphila described in the third aspect. The culture conditions include: using mucin as the sole carbon source. ; Or/and, anaerobic environment; or/and, 36.5℃-37.5℃.
可以理解的是，本申请对培养的条件(包括采用的培养基)不做特别限定，也可以采用含有动物源成分的培养基，也可以采用不含有动物源成分的培养基，例如CN114350571A。It can be understood that the culture conditions (including the medium used) are not particularly limited in this application, and a medium containing animal-derived components or a medium that does not contain animal-derived components can also be used, such as CN114350571A.
培养的条件中的温度例如36.5℃、36.6℃、36.7℃、36.8℃、36.9℃、37℃、37.1℃、37.2℃、37.3℃、37.4℃、37.5℃。The temperature in the culture conditions is, for example, 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37°C, 37.1°C, 37.2°C, 37.3°C, 37.4°C, and 37.5°C.
具体实施例Specific embodiments
下面将结合实施例对本申请的实施方案进行详细描述。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。下列实施例中未注明具体条件的实验方法，优先参考本申请中给出的指引，还可以按照本领域的实验手册或常规条件，还可以按照制造厂商所建议的条件，或者参考本领域已知的实验方法。The embodiments of the present application will be described in detail below with reference to examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. For experimental methods that do not indicate specific conditions in the following examples, priority is given to the guidelines given in this application. You can also follow the experimental manuals or conventional conditions in this field. You can also follow the conditions recommended by the manufacturer, or refer to the experimental methods in this field. Known experimental methods.
下述的具体实施例中，涉及原料组分的量度参数，如无特别说明，可能存在称量精度范围内的细微偏差。涉及温度和时间参数，允许仪器测试精度或操作精度导致的可接受的偏差。In the following specific examples, the measurement parameters of raw material components are involved. Unless otherwise specified, there may be slight deviations within the range of weighing accuracy. Temperature and time parameters are involved, allowing for acceptable deviations due to instrument testing accuracy or operating accuracy.
实施例1、嗜粘蛋白阿克曼菌的分离及鉴定Example 1. Isolation and identification of Akkermansia muciniphila
(1)AM02菌株的分离及鉴定(1) Isolation and identification of AM02 strain
用无菌取样勺，取黄豆粒大小粪便(样本来源于成年健康男性)于10mL离心管中，取样完成后将样本立即转移至37℃厌氧工作站中(85％N2、10％H2、5％CO2)，按1：10的稀释方式将样本稀释至10-9， 取各稀释度溶液各1mL接种至9mL以黏蛋白为唯一碳源的基础培养基中，厌氧培养7天。取10-4稀释度接种的培养液1mL，按1：10的稀释方式将培养液稀释至10-6，分别取各稀释度100μL分别于黏蛋白琼脂培养基上涂布，厌氧培养7天，挑取单菌落接种至2mL BHI肉汤(含N-乙酰-D-氨基葡萄糖培养基)。对培养后的菌液进行16S RNA测序，结果鉴定为嗜粘蛋白阿克曼氏菌，16S RNA测序结果如下SEQ ID NO:1所示：
Use a sterile sampling spoon to take soybean-sized feces (the sample comes from a healthy adult male) in a 10mL centrifuge tube. After sampling is completed, transfer the sample immediately to a 37°C anaerobic workstation (85% N 2 , 10% H 2 , 5% CO 2 ), dilute the sample to 10 -9 according to the dilution method of 1:10, Take 1 mL of each dilution solution and inoculate it into 9 mL of basal medium with mucin as the only carbon source, and culture it anaerobically for 7 days. Take 1 mL of the culture medium inoculated at a dilution of 10 -4 , dilute the culture medium to 10 -6 according to the dilution method of 1:10, take 100 μL of each dilution and spread it on the mucin agar medium, and culture it anaerobically for 7 days. , pick a single colony and inoculate it into 2mL BHI broth (containing N-acetyl-D-glucosamine medium). The cultured bacterial liquid was subjected to 16S RNA sequencing, and the result was identified as Akkermansia muciniphila. The 16S RNA sequencing result is as follows: SEQ ID NO: 1:
SEQ ID NO:1与ATCC BAA-835的16S序列比对结果显示Per.Ident值为99.43％。The 16S sequence alignment result of SEQ ID NO:1 and ATCC BAA-835 shows that the Per.Ident value is 99.43%.
(2)AM06菌株的分离及鉴定(2) Isolation and identification of AM06 strain
将新鲜采集的母乳样本(来源于成年健康女性)立即注射于5mL厌氧西林瓶中保存，然后将样本转移至37℃厌氧工作站中(85％N2、10％H2、5％CO2)，按1：10的稀释方式将样本稀释至10-6，取各稀释度溶液各1mL接种至9mL以黏蛋白为唯一碳源的基础培养基中，厌氧培养约1个月。取10-1-10-4稀释度接种的培养液1mL，按1：10的稀释方式将培养液稀释至10-6，分别取各稀释度100μL分别于黏蛋白琼脂培养基上涂布，厌氧培养7天，挑取单菌落接种至2mL BHI肉汤(含N-乙酰-D-氨基葡萄糖培养基)。对培养后的菌液进行16S RNA测序鉴定，将16S RNA序列于NCBI上进行序列比对，结果鉴定为嗜粘蛋白阿克曼氏菌，16S RNA测序结果如下SEQ ID NO:2所示：

Freshly collected breast milk samples (from adult healthy women) were immediately injected into 5 mL anaerobic cillin bottles for storage, and then the samples were transferred to an anaerobic workstation at 37°C (85% N 2 , 10% H 2 , 5% CO 2 ), dilute the sample to 10 -6 according to the dilution method of 1:10, take 1 mL of each dilution solution and inoculate it into 9 mL of basal medium with mucin as the only carbon source, and culture it anaerobically for about 1 month. Take 1 mL of the culture medium inoculated at dilutions of 10 -1 -10 -4 , dilute the culture medium to 10 -6 according to the dilution method of 1:10, take 100 μL of each dilution and spread it on the mucin agar medium. After 7 days of oxygen culture, a single colony was picked and inoculated into 2 mL BHI broth (containing N-acetyl-D-glucosamine medium). The cultured bacterial liquid was identified by 16S RNA sequencing, and the 16S RNA sequence was compared on NCBI. The result was identified as Akkermansia muciniphila. The 16S RNA sequencing result is as follows: SEQ ID NO: 2:

SEQ ID NO:2与ATCC BAA-835的16S序列比对结果显示Per.Ident值为99.22％。The 16S sequence alignment result of SEQ ID NO:2 and ATCC BAA-835 shows that the Per.Ident value is 99.22%.
实施例2、嗜粘蛋白阿克曼菌的培养Example 2. Culture of Akkermansia muciniphila
将嗜粘蛋白阿克曼菌种划线接种于BHA平板，厌氧培养3天。观察菌落形态特征、染色特性、大小、球杆状和分布情况等。Akkermansia muciniphila was streaked onto BHA plates and cultured anaerobically for 3 days. Observe the morphological characteristics, staining characteristics, size, club shape and distribution of the colonies.
菌落特征：嗜粘蛋白阿克曼菌在上述培养基上培养3天后，呈现圆形凸起、边缘整齐、不透明、白色、大小不均一的菌落，参见图1(嗜粘蛋白阿克曼菌AM02)和图2(嗜粘蛋白阿克曼菌AM06)。Colony characteristics: After Akkermansia muciniphila was cultured on the above-mentioned medium for 3 days, colonies with round convex shapes, neat edges, opaque, white, and uneven sizes appeared, see Figure 1 (Akkermansia muciniphila AM02 ) and Figure 2 (Akermansia muciniphila AM06).
显微镜下形态：嗜粘蛋白阿克曼菌进行革兰氏染色镜检，为革兰阴性细菌，椭圆形、单个或成链状排列，参见图3(嗜粘蛋白阿克曼菌AM02)和图4(嗜粘蛋白阿克曼菌AM06)。Morphology under the microscope: Gram staining of Akkermansia muciniphila shows that it is a Gram-negative bacterium, oval, single or chain-like, see Figure 3 (Akermansia muciniphila AM02) and Figure 3 4 (Akermansia muciniphila AM06).
选取单个菌落接种于BHI肉汤中培养48小时(温度为37℃)，所得菌液离心沉淀，转速16000×g，离心30min，去上清，收集沉淀物，即得嗜粘蛋白阿克曼菌菌泥。分别培养得到AM02、AM06、ATCC BAA-835嗜粘蛋白阿克曼菌。Select a single bacterial colony and inoculate it in BHI broth and culture it for 48 hours (temperature is 37°C). The resulting bacterial liquid is centrifuged and precipitated at a speed of 16000×g for 30 minutes. The supernatant is removed and the precipitate is collected to obtain Akkermansia muciniphila. Bacteria slime. AM02, AM06, and ATCC BAA-835 Akkermansia muciniphila were cultured respectively.
实施例3、嗜粘蛋白阿克曼菌对于人工胃液的耐受性Example 3. Tolerance of Akkermansia muciniphila to artificial gastric juice
(1)实验方法及分组(1) Experimental methods and grouping
表1、实验分组
Table 1. Experimental grouping
表2、实验方法
Table 2. Experimental methods
“+”表示需要检测"+" means detection is required
1)取嗜粘蛋白阿克曼菌菌种1支，去除标签，75％(v/v)酒精对甘油冻存管外表面进行擦拭消毒，涡旋振荡混匀，开启。吸取100-500μL菌液接种至10mL/管的BHI肉汤中，摇匀，共制备3管，同时不接菌作阴性对照，置于37℃厌氧培养2-4天，得一级种子液。1) Take 1 Akkermansia muciniphila strain, remove the label, wipe and disinfect the outer surface of the glycerol cryopreservation tube with 75% (v/v) alcohol, vortex and mix, and open. Inoculate 100-500 μL of bacterial solution into 10 mL/tube of BHI broth, shake well, and prepare a total of 3 tubes. At the same time, do not inoculate the bacteria as a negative control, and place it at 37°C for anaerobic culture for 2-4 days to obtain the first-level seed solution. .
一级种子液进行革兰氏染色镜检，应为G-杆菌，无芽孢，无杂菌。 The first-level seed liquid undergoes Gram staining microscopy and should be G-bacillus, without spores and miscellaneous bacteria.
2)取上述一级种子液10mL，12000×g、4℃离心10min，弃上清，加入1mL0.9wt％NaCl溶液重悬，分别制得菌液，备用。2) Take 10 mL of the above-mentioned first-level seed liquid, centrifuge at 12000×g and 4°C for 10 min, discard the supernatant, add 1 mL of 0.9wt% NaCl solution and resuspend, respectively, to prepare bacterial liquid for later use.
3)按表2将AM06、AM02及标准菌株的菌液分别加入至0.9wt％NaCl、pH3.0以及pH2.0的人工胃液中，混匀，分装成5mL/管，置于厌氧手套箱37℃孵育0h、1.5h以及3h后取出检测各样品的菌浓度。每个实验组做3个平行。3) According to Table 2, add the bacterial liquid of AM06, AM02 and standard strains to 0.9wt% NaCl, pH3.0 and pH2.0 artificial gastric juice respectively, mix well, divide into 5mL/tube, and place in anaerobic gloves After incubation at 37°C for 0 h, 1.5 h and 3 h, the bacterial concentration of each sample was taken out and tested. Each experimental group did 3 parallel runs.
4)活菌数测定：4) Determination of viable bacterial count:
取实验样品，10倍系列稀释后取100μL稀释液接种至BHA平板上，涂布均匀，每个稀释度共做2个平皿，一般做2-3个稀释度，同时取稀释液100μL于BHA平板上，作为阴性对照，所有涂布平皿正置厌氧条件下培养约3-5天，观察平皿上菌落生长情况，并计数。Take the experimental sample, and after 10-fold serial dilution, take 100 μL of the dilution and inoculate it on the BHA plate. Spread evenly. Make a total of 2 plates for each dilution, usually 2-3 dilutions. At the same time, take 100 μL of the dilution on the BHA plate. As a negative control, all coated plates were cultured upright under anaerobic conditions for about 3-5 days. The growth of colonies on the plates was observed and counted.
根据2个平皿菌落数之和按下列公式计算活菌数：
活菌数(CFU/mL)＝2个平皿菌落数之和/2×10×最终稀释度Calculate the number of viable bacteria based on the sum of the number of colonies on 2 plates according to the following formula:
Number of viable bacteria (CFU/mL) = sum of the number of colonies on 2 plates/2×10×final dilution
存活率计算：
Survival rate calculation:
(2)实验结果(2)Experimental results
表3、嗜粘蛋白阿克曼菌人工胃液耐受性存活率结果统计表
Table 3. Statistical table of survival rate results of Akkermansia muciniphila artificial gastric juice tolerance
由表可见，不同嗜粘蛋白阿克曼菌株的人工胃液耐受性依次为AM02＞AM06＞标准株。It can be seen from the table that the artificial gastric juice tolerance of different mucinophilic Akkermann strains is AM02>AM06>standard strain.
实施例4、嗜粘蛋白阿克曼菌对于人工肠液的耐受性Example 4. Tolerance of Akkermansia muciniphila to artificial intestinal fluid
(1)实验方法及分组(1) Experimental methods and grouping
表4、实验分组
Table 4. Experimental grouping
1)一级种子液制备1) Preparation of first-level seed liquid
取嗜粘蛋白阿克曼菌菌种1支去除标签，75％(v/v)酒精对甘油冻存管外表面进行擦拭消毒，涡旋振荡混匀，开启。吸取100μL菌液接种至10mL/管的BHI肉汤中，摇匀，共制备3管，同时不接菌作阴性对照，置于37℃厌氧培养2-4天，得一级种子液。Take 1 Akkermansia muciniphila strain and remove the label, wipe and disinfect the outer surface of the glycerol cryovial with 75% (v/v) alcohol, vortex and mix, and open. Pipette 100 μL of bacterial solution and inoculate it into 10 mL/tube of BHI broth, shake well, and prepare a total of 3 tubes. At the same time, do not inoculate the bacteria as a negative control, and place it at 37°C for anaerobic culture for 2-4 days to obtain the first-level seed solution.
一级种子液进行革兰氏染色镜检，应为G-杆菌，无芽孢，无杂菌。The first-level seed liquid undergoes Gram staining microscopy and should be G-bacillus, without spores and miscellaneous bacteria.
2)菌泥制备2) Preparation of bacteria puree
分别上述一级种子液分装成1.5mL/管，12000rpm、离心10min，弃上清，制得菌泥，AM06、AM02及标准菌株各制备3管菌泥。Divide the above-mentioned first-level seed liquid into 1.5 mL/tube, centrifuge at 12000 rpm for 10 min, discard the supernatant, and prepare bacterial mud. Prepare 3 tubes of bacterial mud for each of AM06, AM02 and standard strains.
3)菌株人工肠液耐受性评价3) Evaluation of bacterial strain tolerance to artificial intestinal fluid
如表5所示，向2)中制得的菌泥中每管分别加入1.5mL人工肠液，混匀，然后每管溶液按照0.5mL/支进行分装，分装3管，分别于37℃厌氧孵育0、4h和8h，取样检测活菌数。每组各做3个平行。As shown in Table 5, add 1.5 mL of artificial intestinal fluid to each tube of the bacterial slurry prepared in 2), mix well, and then divide the solution into 3 tubes at 0.5 mL/tube and incubate at 37°C. Anaerobically incubate for 0, 4 and 8 hours, and take samples to detect the number of viable bacteria. Do 3 parallel reps in each group.
表5、实验方法
Table 5. Experimental methods
“+”表示需要检测"+" means detection is required
4)活菌数测定4) Determination of viable bacterial count
分别取孵育后的样品10倍系列稀释后取100μL稀释液接种至BHA平板上，涂布均匀，每个稀释度共做2个平皿，一般做2-3个稀释度，同时取稀释液100μL于BHA平板上，作为阴性对照，所有涂布平皿正置厌氧条件下培养约3-5天，观察平皿上菌落生长情况，并计数。
活菌数(CFU/mL)＝2个平皿菌落数之和/2×10×最终稀释度Take 10-fold serial dilutions of the incubated samples and inoculate 100 μL of the dilution onto the BHA plate. Spread evenly on the BHA plate. Make a total of 2 plates for each dilution, usually 2-3 dilutions. At the same time, take 100 μL of the dilution on the BHA plate. On the BHA plate, as a negative control, all coated plates were cultured upright under anaerobic conditions for about 3-5 days. The growth of colonies on the plates was observed and counted.
Number of viable bacteria (CFU/mL) = sum of the number of colonies on 2 plates/2×10×final dilution
存活率计算：
存活率＝各时间点活菌数/对应0h活菌数×100％Survival rate calculation:
Survival rate = number of viable bacteria at each time point/number of corresponding viable bacteria at 0 h × 100%
(2)实验结果(2)Experimental results
表6、存活率统计表
Table 6. Survival rate statistics table
如表所示，ATCC BAA-835、AM02、AM06菌株人工肠液耐受性良好。As shown in the table, ATCC BAA-835, AM02, and AM06 strains are well tolerated by artificial intestinal fluid.
实施例5、嗜粘蛋白阿克曼菌培养上清非靶向代谢学差异分析Example 5. Analysis of non-targeted metabolic differences in culture supernatants of Akkermansia muciniphila
(1)样本制备(1)Sample preparation
取实施例2中培养结束后的各个嗜粘蛋白阿克曼菌(AM02、AM06、ATCC BAA-835)的培养上清，各取1mL菌液12000rpm离心5min后，上清过0.22μm滤膜后，取滤液作为待测样本，进行非靶向代谢组学分析。每个菌株各制备5份平行的待测样本。Take the culture supernatant of each Akkermansia muciniphila (AM02, AM06, ATCC BAA-835) after the culture in Example 2, take 1 mL of each bacterial solution and centrifuge it at 12000 rpm for 5 minutes, and then pass the supernatant through a 0.22 μm filter. , take the filtrate as a sample to be tested for non-targeted metabolomic analysis. Prepare 5 parallel samples for each strain.
(2)实验结果(2)Experimental results
PCA是一种数据降维方法，即把多个变量降维到一组新的综合变量，再从中选取尽可能多地反映原有变量信息的前几个主成分，从而达到降维的目的。PCA图反应样本的真实分布情况，主要用于观察样本组间分离趋势，以及是否有异常点出现，同时从原始数据上反映组间和组内的变异度。PCA is a data dimensionality reduction method, that is, reducing the dimensionality of multiple variables to a new set of comprehensive variables, and then selecting the first few principal components that reflect as much of the original variable information as possible to achieve the purpose of dimensionality reduction. The PCA chart reflects the true distribution of samples and is mainly used to observe the separation trend between sample groups and whether there are abnormal points. It also reflects the variability between and within groups from the original data.
实验结果如图5所示，包含了QC样本和所有样本的PCA分析。其中各个QC样本在两个主成分分析图中均聚集在一起，说明检测期间仪器稳定，采集数据的重复性好。同时结果还显示，AM06培养上清中的代谢物与BAA-835的较接近，AM02与BAA-835的培养上清代谢物差异较大。The experimental results are shown in Figure 5, including PCA analysis of QC samples and all samples. Each QC sample is clustered together in the two principal component analysis diagrams, indicating that the instrument is stable during the detection and the repeatability of the collected data is good. At the same time, the results also showed that the metabolites in the culture supernatant of AM06 were close to those of BAA-835, while the metabolites in the culture supernatants of AM02 and BAA-835 were quite different.
各菌株之间差异代谢物的数量比较结果如表7所示。可见AM02与标准株BAA-835相比，正离子(pos)模式检测的差异代谢物和负离子(neg)模式检测的差异代谢物分别为205和135个，AM06与标准株BAA-835相比，正离子(pos)模式检测的差异代谢物和负离子(neg)模式检测的差异代谢物分别为111和62个。因此，保藏编号为CGMCC No.22794的AM02和保藏编号为CGMCC No.22793的AM06鉴定为不同于ATCC BAA-835的新菌种。The results of quantitative comparison of differential metabolites between strains are shown in Table 7. It can be seen that compared with the standard strain BAA-835, AM02 has 205 differential metabolites detected in the positive ion (pos) mode and 135 differential metabolites detected in the negative ion (neg) mode. Compared with the standard strain BAA-835, AM06 has The number of differential metabolites detected in the positive ion (pos) mode and the negative ion (neg) mode were 111 and 62, respectively. Therefore, AM02 with the deposit number CGMCC No. 22794 and AM06 with the deposit number CGMCC No. 22793 were identified as new strains different from ATCC BAA-835.
表7、差异代谢物统计表

Table 7. Statistical table of differential metabolites

实施例6、嗜粘蛋白阿克曼菌对TNF-α和IFN-γ诱导Caco2细胞紧密连接蛋白ZO-1表达的影响Example 6. Effect of Akkermansia muciniphila on the expression of tight junction protein ZO-1 in Caco2 cells induced by TNF-α and IFN-γ
(1)实验方法和分组(1) Experimental methods and grouping
将Caco2细胞接种至96孔板培养至汇合度为80％-90％，使用100ng/mL TNF-α+100ng/mLIFN-γ诱导Caco2细胞24h后，再分别加入AM02、AM06、BAA-835继续与细胞孵育24h，实验分组如表8所示，每组做5个复孔。使用免疫荧光法观察BAA-835、AM06和AM02对TNF-α和IFN-γ诱导Caco2细胞紧密连接蛋白ZO-1表达的影响。Caco2 cells were seeded into a 96-well plate and cultured until the confluence was 80%-90%. Caco2 cells were induced with 100ng/mL TNF-α+100ng/mLIFN-γ for 24 hours, and then AM02, AM06, and BAA-835 were added respectively to continue with The cells were incubated for 24 hours, and the experimental groups were as shown in Table 8. Five duplicate wells were made in each group. Immunofluorescence method was used to observe the effects of BAA-835, AM06 and AM02 on the expression of tight junction protein ZO-1 in Caco2 cells induced by TNF-α and IFN-γ.
表8、实验分组
Table 8. Experimental grouping
(2)实验结果(2)Experimental results
对拍摄的图片进行荧光强度统计，结果如图6和表9所示。与空白对照组相比，经过TNF-α和IFN-γ诱导48h后炎症模型组细胞间隙的荧光强度显著减弱，说明ZO-1蛋白表达减少，细胞间的紧密连接被破坏。与炎症模型组相比，给予AM02、AM06、BAA-835干预后细胞的处理组荧光强度显著增加(P＜0.05)，且AM02和AM06抑制炎症因子诱导Caco2细胞ZO-1蛋白减少的能力显著优于BAA-825(P＜0.05)。The fluorescence intensity statistics of the captured pictures were performed, and the results are shown in Figure 6 and Table 9. Compared with the blank control group, the fluorescence intensity of the intercellular spaces in the inflammation model group was significantly weakened after 48 hours of TNF-α and IFN-γ induction, indicating that the expression of ZO-1 protein was reduced and the tight junctions between cells were destroyed. Compared with the inflammation model group, the fluorescence intensity of the cells in the treatment group after intervention with AM02, AM06, and BAA-835 increased significantly (P<0.05), and AM02 and AM06 were significantly better at inhibiting the reduction of ZO-1 protein in Caco2 cells induced by inflammatory factors. in BAA-825 (P<0.05).
表9、荧光强度统计
Table 9. Fluorescence intensity statistics
注：**表示与模型组比较，差异极显著p＜0.01；aa表示与BAA-835组相比，差异极显著p＜0.01。Note: ** means that compared with the model group, the difference is extremely significant at p<0.01; aa means that compared with the BAA-835 group, the difference is extremely significant at p<0.01.
实施例7、嗜粘蛋白阿克曼菌对LPS诱导的肝脏切片炎症的影响Example 7. Effect of Akkermansia muciniphila on LPS-induced inflammation in liver slices
(1)实验方法(1) Experimental methods
使用切片机将8周大C57BL/6小鼠的肝脏切成250μm厚的切片，肝切片置于6孔板中，使用切片培养基进行培养，培养体积为3ml/孔。将6孔板置于95％氧气，5％二氧化碳，37摄氏度和70转摇晃的条件下培养过夜。The livers of 8-week-old C57BL/6 mice were cut into 250 μm-thick slices using a microtome. The liver slices were placed in a 6-well plate and cultured using slice culture medium with a culture volume of 3 ml/well. The 6-well plate was cultured overnight at 95% oxygen, 5% carbon dioxide, 37 degrees Celsius and shaking at 70 rpm.
本实验通过在肝切片中加入2μg/mL LPS来诱导炎症。实验分组如见表10所示，每组作3个复孔。肝切片过夜培养后根据分组在6孔板中加入适量的培养基、LPS、AM02、AM06和BAA-835，继续培养48小时，每24小时换液一次。培养结束后取肝切片用QPCR法检测肝切片的IL-6和IL-1β基因的表达水平。In this experiment, inflammation was induced by adding 2 μg/mL LPS to liver slices. The experimental groups are as shown in Table 10, with 3 duplicate holes in each group. After the liver slices were cultured overnight, an appropriate amount of culture medium, LPS, AM02, AM06 and BAA-835 were added to the 6-well plate according to the grouping, and the culture was continued for 48 hours, and the medium was changed every 24 hours. After the culture, liver sections were taken and QPCR method was used to detect the expression levels of IL-6 and IL-1β genes in the liver sections.
表10、实验分组

Table 10. Experimental grouping

注：受试物和诱导物同时加入。Note: The test substance and inducer are added at the same time.
(2)实验结果(2)Experimental results
与空白对照组相比，LPS可显著诱导肝脏切片中的IL-6和IL-1β基因表达水平的上调(P＜0.01)。与炎症模型组相比，AM06组、AM02组，BAA-835组均能显著降低LPS诱导肝切片的IL-6和IL-1β基因基因表达水平的上调(P＜0.01)。且AM06组、AM02组的IL-6和IL-1β基因表达水平显著低于BAA-835组。(P＜0.05)。Compared with the blank control group, LPS could significantly induce the upregulation of IL-6 and IL-1β gene expression levels in liver slices (P<0.01). Compared with the inflammation model group, the AM06 group, AM02 group, and BAA-835 group could significantly reduce the upregulation of IL-6 and IL-1β gene expression levels in LPS-induced liver slices (P<0.01). Moreover, the expression levels of IL-6 and IL-1β genes in the AM06 group and AM02 group were significantly lower than those in the BAA-835 group. (P<0.05).
表11、IL-6和IL-1β的相对表达
Table 11. Relative expression of IL-6 and IL-1β
注：**表示与炎症模型组相比，差异极显著P＜0.01；a表示与BAA-835组相比，差异显著p＜0.05。Note: ** indicates that compared with the inflammation model group, the difference is extremely significant at P < 0.01; a indicates that compared with the BAA-835 group, the difference is extremely significant at p < 0.05.
实施例8、嗜粘蛋白阿克曼菌预防小鼠非酒精性脂肪肝的药效实验Example 8. Experiment on the efficacy of Akkermansia muciniphila in preventing non-alcoholic fatty liver disease in mice
(1)实验设计及方法(1) Experimental design and methods
实验设计：110只雄性C57BL/6小鼠，5周龄。实验分组：空白组、模型组、阳性药组(奥贝胆酸OCA，3mg/mL)、AM06低剂量(106CFU/mL)、AM06中剂量(108CFU/mL)、AM06高剂量(1010CFU/mL)组、AM02(108CFU/mL)组、ATCC BAA-835(108CFU/mL)组，以及AM06、AM02和BAA-835的灭活菌组(各组给药剂量均为108CFU/mL)。每组10只动物。一周适应期后，空白组饲喂普通饲料，空白组外的其余各组饲喂高脂饲料，并同时给予相应药物。Experimental design: 110 male C57BL/6 mice, 5 weeks old. Experimental groups: blank group, model group, positive drug group (obeticholic acid OCA, 3mg/mL), AM06 low dose (10 6 CFU/mL), AM06 medium dose (10 8 CFU/mL), AM06 high dose ( 10 10 CFU/mL) group, AM02 (10 8 CFU/mL) group, ATCC BAA-835 (10 8 CFU/mL) group, and the inactivated bacteria group of AM06, AM02 and BAA-835 (the dosage of each group Both are 10 8 CFU/mL). 10 animals per group. After a one-week adaptation period, the blank group was fed with ordinary feed, and the other groups except the blank group were fed with high-fat feed and given corresponding drugs at the same time.
给药10周后，小鼠禁食6小时后用CO2对动物实施安乐死。心脏穿刺采集血样。分离血清检测相关指标。记录终末体重和肝脏重量。并取肝脏组织固定在***中进行组织病理学检查。After 10 weeks of administration, the mice were fasted for 6 hours and then euthanized with CO2 . Blood samples are collected by cardiac puncture. Separate serum to detect relevant indicators. Terminal body weight and liver weight were recorded. The liver tissue was removed and fixed in formalin for histopathological examination.
(2)实验结果(2)Experimental results
第十周时。各检测指标结果如下表所示。On the tenth week. The results of each test indicator are shown in the table below.
表12、各项检测指标统计(mean±SD，n＝10)
Table 12. Statistics of various detection indicators (mean ± SD, n = 10)
表13、各项检测指标统计(mean±SD，n＝10)

Table 13. Statistics of various detection indicators (mean ± SD, n = 10)

注1：*表示与模型组比较，差异显著p＜0.05,**表示与模型组比较，差异极显著p＜0.01；Note 1: * indicates that compared with the model group, the difference is significant at p < 0.05, ** indicates that compared with the model group, the difference is extremely significant at p < 0.01;
注2：a表示与BAA-835中剂量组比较，差异显著p＜0.05，aa表示与BAA-835中剂量组比较，差异极显著p＜0.01；Note 2: a means that compared with the BAA-835 medium-dose group, the difference is significant at p<0.05, aa means that compared with the BAA-835 medium-dose group, the difference is extremely significant at p<0.01;
注3：b表示与BAA-835中剂量组比较，差异显著p＜0.05，bb表示与BAA-835中剂量组比较，差异极显著p＜0.01；Note 3: b means that compared with the BAA-835 medium-dose group, the difference is significant at p<0.05; bb means that compared with the BAA-835 medium-dose group, the difference is extremely significant at p<0.01;
1)小鼠体重和肝脏重量1) Mouse body weight and liver weight
如表7所示，与空白组相比，模型组小鼠的体重显著增加(p＜0.01)，而肝脏重量有增加趋势，但无显著性差异。与模型组相比，AM06中剂量组和AM06灭活菌组可显著减少体重(p＜0.05)，其余各个给药组有减少小鼠体重和肝脏重量的趋势，但无显著性差异As shown in Table 7, compared with the blank group, the weight of the mice in the model group increased significantly (p<0.01), while the liver weight tended to increase, but there was no significant difference. Compared with the model group, the AM06 medium-dose group and the AM06 inactivated bacteria group can significantly reduce body weight (p<0.05). The other administration groups have a tendency to reduce the body weight and liver weight of mice, but there is no significant difference.
2)小鼠肝脏病理评分2) Mouse liver pathology score
对各组小鼠进行肝脏病理HE染色，根据切片的脂肪变性程度、肝细胞气球样变性程度以及小叶内炎症程度进行评分，计算非酒精性脂肪肝NAS积分，结果如表7所示。与空白对照组相比，模型组的NAS积分显著增加(p＜0.05)，说明动物出现非酒精性脂肪肝。与模型组相比，各个给药组均可不同程度地降低NAS积分，且AM06中低剂量组和灭活菌株可显著降低NAS积分(p＜0.05)。在同等剂量水平下，AM02和AM06的活菌的NAS积分显著低于BAA-835活菌(p＜0.05)，AM02和AM06的灭活菌的NAS积分有低于BAA-835灭活菌的趋势，说明AM02和AM06的活菌和灭活菌在预防非酒精性脂肪肝的疗效要优于BAA-835活菌和灭活菌。HE staining of liver pathology was performed on mice in each group, and the scores were scored based on the degree of steatosis of the sections, the degree of ballooning degeneration of hepatocytes, and the degree of inflammation in the lobules, and the non-alcoholic fatty liver NAS score was calculated. The results are shown in Table 7. Compared with the blank control group, the NAS score of the model group increased significantly (p<0.05), indicating that the animals developed non-alcoholic fatty liver disease. Compared with the model group, each administration group can reduce NAS points to varying degrees, and AM06 mid- and low-dose groups and inactivated strains can significantly reduce NAS points (p<0.05). At the same dose level, the NAS scores of live bacteria of AM02 and AM06 are significantly lower than those of BAA-835 live bacteria (p<0.05), and the NAS scores of inactivated bacteria of AM02 and AM06 tend to be lower than those of BAA-835 inactivated bacteria. , indicating that the live and inactivated bacteria of AM02 and AM06 are more effective than BAA-835 live and inactivated bacteria in preventing non-alcoholic fatty liver disease.
3)小鼠血清肝功能检测3) Mouse serum liver function test
对小鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)进行检测，结果如表7所示，与空白组相比，模型组小鼠的ALT和AST显著增加(p＜0.01)，说明肝功能受到了损害。各个给药组均可不同程度地减少血清中的ALT和AST含量。其中阳性药组，AM06低高剂量组，AM06灭活菌组和AM02灭活菌组可显著降低ALT(p＜0.05)。而阳性药组，所有活菌组，AM06灭活菌组和AM02灭活菌组可显著降低AST(p＜0.05)。且同等剂量水平下，AM02和AM06的活菌的AST含量要略低于BAA-835活菌，AM02和AM06的灭活菌的AST含量略要低于BAA-835灭活菌。说明AM02和AM06对肝脏的保护作用要略优于BAA-835。The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of mice were detected. The results are shown in Table 7. Compared with the blank group, the ALT and AST of the mice in the model group were significantly increased (p<0.01), indicating that the liver Functionality is compromised. Each administration group can reduce the levels of ALT and AST in serum to varying degrees. Among them, the positive drug group, AM06 low and high dose group, AM06 inactivated bacteria group and AM02 inactivated bacteria group can significantly reduce ALT (p<0.05). The positive drug group, all live bacteria group, AM06 inactivated bacteria group and AM02 inactivated bacteria group can significantly reduce AST (p<0.05). And at the same dosage level, the AST content of live bacteria of AM02 and AM06 is slightly lower than that of BAA-835 live bacteria, and the AST content of inactivated bacteria of AM02 and AM06 is slightly lower than that of inactivated BAA-835 bacteria. This shows that AM02 and AM06 have slightly better protective effects on the liver than BAA-835.
4)血清LPS4)Serum LPS
小鼠的血清LPS含量进行检测结果如表7所示，与空白组相比，模型组小鼠的LPS显著增加(p＜0.01)，说明模型动物存在轻微的内毒素血症。与模型组相比，阳性药组未能降低血清中的LPS，而各个嗜粘蛋白阿克曼菌组可不同程度地减少LPS含量，其中AM02和AM06的活菌中剂量组，AM02和AM06的灭活菌组可显著降低LPS含量(p＜0.05)。且AM02和AM06的活菌中剂量组减少血清LPS的能力要显著优于BAA-835活菌组(p＜0.05)，而AM02和AM06的灭活菌组要略优于BAA-835灭活菌组，但无显著性差异。The results of testing the serum LPS content of mice are shown in Table 7. Compared with the blank group, the LPS of mice in the model group increased significantly (p<0.01), indicating that the model animals had mild endotoxemia. Compared with the model group, the positive drug group failed to reduce the LPS in serum, while each Akkermansia muciniphila group could reduce the LPS content to varying degrees, among which the live bacteria medium-dose groups of AM02 and AM06, the live bacteria group of AM02 and AM06 The inactivated bacteria group can significantly reduce the LPS content (p<0.05). Moreover, the medium-dose live bacteria group of AM02 and AM06 has a significantly better ability to reduce serum LPS than the BAA-835 live bacteria group (p<0.05), while the inactivated bacteria group of AM02 and AM06 is slightly better than the BAA-835 inactivated bacteria group. , but there is no significant difference.
综上所述，嗜粘蛋白阿克曼菌能够有效预防小鼠非酒精性脂肪肝，减少肝脏损伤并抑制轻微地内毒素血症，且各菌株的疗效AM06≈AM02＞BAA-835。In summary, Akkermansia muciniphila can effectively prevent non-alcoholic fatty liver disease in mice, reduce liver damage and inhibit mild endotoxemia, and the efficacy of each strain is AM06≈AM02>BAA-835.
实施例10、嗜粘蛋白阿克曼菌治疗小鼠非酒精性脂肪肝炎和肝纤维化的药效实验Example 10. Experiment on the efficacy of Akkermansia muciniphila in treating non-alcoholic steatohepatitis and liver fibrosis in mice
(1)实验设计及方法(1) Experimental design and methods
实验设计：110只雄性C57BL/6小鼠，5周龄。实验分组：空白组、模型组、阳性药组(奥贝胆酸OCA，3mg/mL)、AM06低剂量(106CFU/mL)、AM06中剂量(108CFU/mL)、AM06高剂量(1010CFU/mL)组、AM02(108CFU/mL)组、ATCC BAA-835(108CFU/mL)组，以及AM06、AM02和BAA-835的灭活菌组(各组给药剂量均为108CFU/mL)。每组10只动物。实验期间，空白组饲一直喂普通饲料。 空白组外的其余各组饲喂高脂饲料至第10周，然后在实验第10周至第18周喂食改良的高脂饲料(增加胆固醇含量)。各个给药组由第10周开始给药只第18周。Experimental design: 110 male C57BL/6 mice, 5 weeks old. Experimental groups: blank group, model group, positive drug group (obeticholic acid OCA, 3mg/mL), AM06 low dose (10 6 CFU/mL), AM06 medium dose (10 8 CFU/mL), AM06 high dose ( 10 10 CFU/mL) group, AM02 (10 8 CFU/mL) group, ATCC BAA-835 (10 8 CFU/mL) group, and the inactivated bacteria group of AM06, AM02 and BAA-835 (the dosage of each group Both are 10 8 CFU/mL). 10 animals per group. During the experiment, the blank group was fed with ordinary feed. The remaining groups except the blank group were fed high-fat feed until the 10th week, and then were fed modified high-fat feed (increased cholesterol content) from the 10th to 18th week of the experiment. Each administration group started to receive administration from the 10th week to the 18th week.
给药结束后，小鼠禁食6小时后用CO2对动物实施安乐死。心脏穿刺采集血样。分离血清检测相关指标。记录终末体重和肝脏重量。并取肝脏组织固定在***中进行组织病理学检查。After the administration, the mice were fasted for 6 hours and then euthanized with CO2 . Blood samples are collected by cardiac puncture. Separate serum to detect relevant indicators. Terminal body weight and liver weight were recorded. The liver tissue was removed and fixed in formalin for histopathological examination.
(2)实验结果(2)Experimental results
第18周时。各检测指标结果如下表所示。At week 18. The results of each test indicator are shown in the table below.
表14、各项检测指标统计(mean±SD，n＝10)
Table 14. Statistics of various detection indicators (mean ± SD, n = 10)
表15、各项检测指标统计(mean±SD，n＝10)
Table 15. Statistics of various detection indicators (mean ± SD, n = 10)
注1：*表示与模型组比较，差异显著p＜0.05，**表示与模型组比较，差异极显著p＜0.01；Note 1: * indicates that compared with the model group, the difference is significant at p < 0.05, ** indicates that compared with the model group, the difference is extremely significant at p < 0.01;
注2：a表示与BAA-835中剂量组比较，差异显著p＜0.05，aa表示与BAA-835中剂量组比较，差异极显著p＜0.01；Note 2: a means that compared with the BAA-835 medium-dose group, the difference is significant at p<0.05, aa means that compared with the BAA-835 medium-dose group, the difference is extremely significant at p<0.01;
注3：b表示与BAA-835中剂量组比较，差异显著p＜0.05，bb表示与BAA-835中剂量组比较，差异极显著p＜0.01；Note 3: b means that compared with the BAA-835 medium-dose group, the difference is significant at p<0.05; bb means that compared with the BAA-835 medium-dose group, the difference is extremely significant at p<0.01;
1)小鼠体重和肝脏重量、1) Mouse body weight and liver weight,
如表8所示，与空白组相比，模型组小鼠的体重显著增加(p＜0.01)，而肝脏重量有增加趋势，但无显著性差异。与模型组相比，AM06低剂量组和AM02灭活菌组可显著减少体重(p＜0.05)，其余各个给药组有减少小鼠体重和肝脏重量的趋势，但无显著性差异。As shown in Table 8, compared with the blank group, the weight of the mice in the model group increased significantly (p<0.01), and the liver weight tended to increase, but there was no significant difference. Compared with the model group, the AM06 low-dose group and the AM02 inactivated bacteria group could significantly reduce body weight (p<0.05). The other administration groups had a tendency to reduce the body weight and liver weight of mice, but there was no significant difference.
2)小鼠肝脏病理评分2) Mouse liver pathology score
对各组小鼠进行肝脏病理HE染色，根据切片的脂肪变性程度、肝细胞气球样变性程度以及小叶内炎症程度进行评分，计算非酒精性脂肪肝NAS积分，结果如表8所示。与空白对照组相比，模型组的NAS积分显著增加(p＜0.05)，说明动物出现非酒精性脂肪肝。与模型组相比，各个给药组均可不同程度地降低NAS积分，且阳性药组，AM06中剂量组，AM02中剂量组，AM02和AM06的灭活菌株可显著降低NAS积分(p＜0.05)。且AM02和AM06的活菌的NAS积分显著低于BAA-835活菌(p＜0.05)，AM02和AM06的灭活菌的NAS积分显著低于BAA-835灭活菌，说明AM02和AM06在治疗防非酒 精性脂肪肝的疗效要优于BAA-835。HE staining of liver pathology was performed on mice in each group, and the scores were scored based on the degree of steatosis of the sections, the degree of ballooning degeneration of hepatocytes, and the degree of inflammation in the lobules, and the non-alcoholic fatty liver NAS score was calculated. The results are shown in Table 8. Compared with the blank control group, the NAS score of the model group increased significantly (p<0.05), indicating that the animals developed non-alcoholic fatty liver disease. Compared with the model group, each administration group can reduce NAS points to varying degrees, and the positive drug group, AM06 mid-dose group, AM02 mid-dose group, AM02 and AM06 inactivated strains can significantly reduce NAS points (p<0.05 ). Moreover, the NAS scores of live bacteria of AM02 and AM06 were significantly lower than those of BAA-835 live bacteria (p<0.05), and the NAS scores of inactivated bacteria of AM02 and AM06 were significantly lower than those of BAA-835 inactivated bacteria, indicating that AM02 and AM06 are effective in treating Prevent non-alcoholic beverages The efficacy of spermatozoa fatty liver disease is better than BAA-835.
3)小鼠血清肝功能检测3) Mouse serum liver function test
对小鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)进行检测，结果如表8所示，与空白组相比，模型组小鼠的ALT和AST显著增加(p＜0.01)，说明肝功能受到了损害。与模型组相比，所有给药组的ALT水平均显著降低(p＜0.05)，除BAA-835中剂量组以外的给药组的AST水平均显著降低(p＜0.05)。The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of mice were detected. The results are shown in Table 8. Compared with the blank group, the ALT and AST of the mice in the model group were significantly increased (p<0.01), indicating that the liver Functionality is compromised. Compared with the model group, the ALT levels of all administration groups were significantly reduced (p<0.05), and the AST levels of the administration groups except the BAA-835 mid-dose group were significantly decreased (p<0.05).
且同等剂量水平下，AM02和AM06的活菌或灭活菌的ALT含量显著低于BAA-835活菌或灭活菌(p＜0.05)。AM02的活菌的AST含量显著低于BAA-835活菌(p＜0.05),AM06的活菌的AST含量略低于BAA-835活菌，但无显著性差异。而AM02和AM06的灭活菌的AST含量均显著低于BAA-835灭活菌(p＜0.05)。说明AM02和AM06对肝脏的保护作用要优于BAA-835。And at the same dose level, the ALT content of live or inactivated bacteria of AM02 and AM06 was significantly lower than that of BAA-835 live or inactivated bacteria (p<0.05). The AST content of AM02 live bacteria was significantly lower than that of BAA-835 live bacteria (p<0.05), and the AST content of AM06 live bacteria was slightly lower than BAA-835 live bacteria, but there was no significant difference. The AST content of AM02 and AM06 inactivated bacteria was significantly lower than that of BAA-835 inactivated bacteria (p<0.05). This shows that AM02 and AM06 have better protective effects on the liver than BAA-835.
4)纤维化相关基因Col1a1的相对mRNA表达4) Relative mRNA expression of fibrosis-related gene Col1a1
取小鼠的肝组织用QPCR法检测纤维化相关基因Col1a1的相对mRNA表达，结果如表8所示。与空白组相比，模型组小鼠的Col1a1的表达显著增加(p＜0.01)，说明模型动物肝脏出现肝纤维化。与模型组相比，除了BAA-835中剂量组外的各个给药组均能显著降低Col1a1的表达(p＜0.05)，BAA-835中剂量组有降低Col1a1的表达的趋势，但无显著性差异。在同等剂量水平下，AM06和AM02的活菌或灭活菌的Col1a1表达均显著低于BAA-835活菌或灭活菌(p＜0.05)，说明AM02和AM06对抑制肝纤维化的能力要优于BAA-835The liver tissue of mice was taken and QPCR method was used to detect the relative mRNA expression of fibrosis-related gene Col1a1. The results are shown in Table 8. Compared with the blank group, the expression of Col1a1 in the model group mice was significantly increased (p<0.01), indicating that liver fibrosis occurred in the liver of the model animals. Compared with the model group, all administration groups except the BAA-835 mid-dose group could significantly reduce the expression of Col1a1 (p<0.05). The BAA-835 mid-dose group had a tendency to reduce the expression of Col1a1, but there was no significance. difference. At the same dose level, the Col1a1 expression of live or inactivated bacteria of AM06 and AM02 was significantly lower than that of BAA-835 live or inactivated bacteria (p<0.05), indicating that AM02 and AM06 have greater ability to inhibit liver fibrosis. Better than BAA-835
综上所述，嗜粘蛋白阿克曼菌能够有效治疗小鼠非酒精性脂肪肝炎，减少肝脏损伤并抑制肝纤维化，且各菌株的疗效AM06≈AM02＞BAA-835。In summary, Akkermansia muciniphila can effectively treat non-alcoholic steatohepatitis in mice, reduce liver damage and inhibit liver fibrosis, and the efficacy of each strain is AM06≈AM02>BAA-835.
整体上，通过大量实验证明，嗜粘蛋白阿克曼菌特别是保藏编号为CGMCC No.22793的嗜粘蛋白阿克曼菌AM06和保藏编号为CGMCC No.22794的嗜粘蛋白阿克曼菌AM02，在体外实验中对人工胃液和人工肠液的耐受性，抑制炎症因子破坏肠细胞的紧密连接蛋白的能力，抑制LPS诱导肝切片肝炎的作用均优于标准株BAA-835。动物实验证明，AM02和AM06菌可通过减少肝脏脂肪变和肝炎，改善肝功能指标(如ALT和AST水平)，减少轻微的内毒素血症，抑制肝纤维化，从而有效预防或治疗非酒精性脂肪肝的发生发展及其恶化，提高患者的生活质量。On the whole, a large number of experiments have proved that Akkermansia muciniphila, especially Akkermansia muciniphila AM06 with deposit number CGMCC No. 22793 and Akkermansia muciniphila AM02 with deposit number CGMCC No. 22794 , in in vitro experiments, its tolerance to artificial gastric juice and artificial intestinal juice, its ability to inhibit inflammatory factors from destroying tight junction proteins in intestinal cells, and its ability to inhibit LPS-induced hepatitis in liver slices were all better than the standard strain BAA-835. Animal experiments have proven that AM02 and AM06 bacteria can effectively prevent or treat non-alcoholic alcoholic diseases by reducing liver steatosis and hepatitis, improving liver function indicators (such as ALT and AST levels), reducing mild endotoxemia, and inhibiting liver fibrosis. The occurrence and development of fatty liver and its deterioration can improve the patient's quality of life.
以上所述实施方式和实施例的各技术特征可以进行任意合适方式的组合，为使描述简洁，未对上述实施方式和实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为在本说明书记载的范围中。The technical features of the above-described embodiments and examples can be combined in any suitable manner. To simplify the description, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as these There is no contradiction in the combination of technical features, and they should be considered to be within the scope of this specification.
以上所述实施例仅表达了本申请的几种实施方式，便于具体和详细地理解本申请的技术方案，但并不能因此而理解为对申请专利保护范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。此外应理解，在阅读了本申请的上述讲授内容之后，本领域技术人员可以对本申请作各种改动或修改，得到的等价形式同样落于本申请的保护范围。还应当理解，本领域技术人员在本申请提供的技术方案的基础上，通过合乎逻辑的分析、推理或者有限的试验得到的技术方案，均在本申请所附权利要求的保护范围内。因此，本申请专利的保护范围应以所附权利要求的内容为准，说明书及附图可以用于解释权利要求的内容。 The above-mentioned embodiments only express several implementation modes of the present application to facilitate a specific and detailed understanding of the technical solutions of the present application, but should not be construed as limiting the scope of patent protection. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all fall within the protection scope of the present application. In addition, it should be understood that after reading the above teaching content of this application, those skilled in the art can make various changes or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited testing based on the technical solutions provided in this application are within the protection scope of the claims appended to this application. Therefore, the protection scope of the patent of this application shall be subject to the contents of the appended claims, and the description and drawings may be used to interpret the contents of the claims.

Claims (10)

  1. 嗜粘蛋白阿克曼菌在制备保健食品或者防治非酒精性脂肪肝疾病的药物中的应用，其特征在于，所述嗜粘蛋白阿克曼菌选自保藏编号为CGMCC No.22793的嗜粘蛋白阿克曼菌和保藏编号为CGMCC No.22794的嗜粘蛋白阿克曼菌中的一种或两种。The application of Akkermansia muciniphila in the preparation of health foods or drugs for the prevention and treatment of non-alcoholic fatty liver disease is characterized in that the Akkermansia muciniphila is selected from the group consisting of Akkermansia muciniphila with the deposit number CGMCC No. 22793. One or both of Akkermansia albuminosa and Akkermansia muciniphila with deposit number CGMCC No. 22794.
  2. 根据权利要求1所述的应用，其特征在于，所述保藏编号为CGMCC No.22793的嗜粘蛋白阿克曼菌和保藏编号为CGMCC No.22794的嗜粘蛋白阿克曼菌各自独立地为活菌、灭活菌中的一种或多种。The application according to claim 1, wherein the Akkermansia muciniphila deposited as CGMCC No. 22793 and the Akkermansia muciniphila deposited as CGMCC No. 22794 are each independently One or more of live bacteria and inactivated bacteria.
  3. 保藏编号为CGMCC No.22793的嗜粘蛋白阿克曼菌，2021年06月28日保藏于中国微生物菌种保藏管理委员会普通微生物中心。The deposit number of Akkermansia muciniphila is CGMCC No. 22793, which was deposited at the General Microbiology Center of the Chinese Microbial Culture Collection Committee on June 28, 2021.
  4. 保藏编号为CGMCC No.22794的嗜粘蛋白阿克曼菌，2021年06月28日保藏于中国微生物菌种保藏管理委员会普通微生物中心。Akkermansia muciniphila, with the deposit number CGMCC No. 22794, was deposited at the General Microbiology Center of the Chinese Microbial Culture Collection Committee on June 28, 2021.
  5. 益生菌组合产品，其特征在于，所述益生菌组合产品包含保藏编号为CGMCC No.22793的嗜粘蛋白阿克曼菌和保藏编号为CGMCC No.22794的嗜粘蛋白阿克曼菌中的一种或两种。A probiotic combination product, characterized in that the probiotic combination product contains one of Akkermansia muciniphila with a deposit number of CGMCC No. 22793 and Akkermansia muciniphila with a deposit number of CGMCC No. 22794. One or two species.
  6. 保健食品，其特征在于，包含：权利要求3所述的嗜粘蛋白阿克曼菌和权利要求4所述的嗜粘蛋白阿克曼菌中的一种或两种，以及食品辅料。A health food, characterized in that it contains: one or both of the Akkermansia muciniphila described in claim 3 and the Akkermansia muciniphila described in claim 4, and food supplements.
  7. 根据权利要求6所述保健食品，其特征在于，所述保健食品为糕点或者饮品。The health food according to claim 6, characterized in that the health food is pastries or drinks.
  8. 药物组合物，其特征在于，包含：权利要求3所述的嗜粘蛋白阿克曼菌和权利要求4所述的嗜粘蛋白阿克曼菌中的一种或两种，以及药用辅料。A pharmaceutical composition is characterized by comprising: one or both of the Akkermansia muciniphila described in claim 3 and the Akkermansia muciniphila described in claim 4, and pharmaceutical excipients.
  9. 根据权利要求8所述的药物组合物，其特征在于，所述药物的剂型为丸剂、片剂、颗粒剂、胶囊剂、溶液剂、管饲制剂、混悬剂、霜剂、喷雾剂、膏剂或贴剂。The pharmaceutical composition according to claim 8, characterized in that the dosage form of the drug is pills, tablets, granules, capsules, solutions, tube feeding preparations, suspensions, creams, sprays, ointments or patches.
  10. 培养权利要求3和权利要求4所述的嗜粘蛋白阿克曼菌中的一种或者多种的方法，其特征在于，培养满足如下条件中的一个或者几个：The method for cultivating one or more Akkermansia muciniphila described in claim 3 and claim 4 is characterized in that the cultivation satisfies one or more of the following conditions:
    (1)以黏蛋白为唯一碳源；(1) Use mucin as the only carbon source;
    (2)厌氧环境；和，(2) Anaerobic environment; and,
    (3)约36.5℃-37.5℃。 (3) About 36.5℃-37.5℃.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104918626A (en) * 2012-11-19 2015-09-16 卢万天主教大学 Use of akkermansia for treating metabolic disorders
CN108289918A (en) * 2015-09-10 2018-07-17 卢万天主教大学 Ai Keman bacterium through pasteurization are used to treat the purposes of metabolic disorder
CN113330109A (en) * 2018-10-11 2021-08-31 Ko生物技术有限公司 Akkermansia muciniphila strain and application thereof
CN113906129A (en) * 2019-08-23 2022-01-07 安泰微生物科技有限公司 Ackermanella muciniphila EB-AMDK19 strain and application thereof
CN114514028A (en) * 2020-08-26 2022-05-17 安泰微生物科技有限公司 Pharmaceutical composition for preventing or treating atopic diseases comprising akkermansia muciniphila strain
CN114949003A (en) * 2022-06-08 2022-08-30 广州知易生物科技有限公司 Ackermanella muciniphila and application and culture method thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104918626A (en) * 2012-11-19 2015-09-16 卢万天主教大学 Use of akkermansia for treating metabolic disorders
CN108289918A (en) * 2015-09-10 2018-07-17 卢万天主教大学 Ai Keman bacterium through pasteurization are used to treat the purposes of metabolic disorder
CN113330109A (en) * 2018-10-11 2021-08-31 Ko生物技术有限公司 Akkermansia muciniphila strain and application thereof
CN113906129A (en) * 2019-08-23 2022-01-07 安泰微生物科技有限公司 Ackermanella muciniphila EB-AMDK19 strain and application thereof
CN114514028A (en) * 2020-08-26 2022-05-17 安泰微生物科技有限公司 Pharmaceutical composition for preventing or treating atopic diseases comprising akkermansia muciniphila strain
CN114949003A (en) * 2022-06-08 2022-08-30 广州知易生物科技有限公司 Ackermanella muciniphila and application and culture method thereof

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