Application of Akkermansia muciniphila in the preparation of products for the prevention, treatment and / or auxiliary treatment of metabolic diseases

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to the use of Akkermansia muciniphila in the preparation of products for preventing, treating and / or assisting in the treatment of metabolic diseases. The present invention provides an Akkermansia muciniphila, which has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 26, 2020, with a deposit number of CGMCC No. 20955. The Akkermansia muciniphila of the present invention can effectively lower blood sugar levels, lower TC, TG, and LDL-C levels, and increase HDL-C levels, especially having significant effects on increasing HDL-C levels, indicating that Akkermansia muciniphila has good blood sugar-lowering, blood lipid-lowering, and cholesterol-regulating effects on diabetic mice.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to application of Akkermansia muciniphila in preparing products for preventing, treating and / or assisting in treating metabolic diseases. Background Art

[0002] Metabolic diseases are diseases caused by metabolic problems, including metabolic disorders and hypermetabolism. When biochemical processes in the body are disturbed, certain metabolic substances such as sugar, fat, protein, purine, calcium and copper accumulate or are deficient, causing the disease. Symptoms vary in severity, and diagnosis relies on clinical manifestations and biochemical tests such as blood and urine. There is no effective cure, and the main treatment is to eliminate the cause and treat the symptoms. The prognosis depends on the cause, the severity of the symptoms and the effect of treatment. Common metabolic diseases with high incidence rates include obesity, diabetes, and lipid metabolism disorders.

[0003] Diabetes is a group of metabolic diseases characterized by chronic hyperglycemia caused by multiple causes. It is caused by defects in insulin secretion and (or) utilization. Long-term high blood sugar levels can cause damage to multiple systems, leading to chronic progressive lesions, functional impairment and failure of tissues and organs such as the eyes, kidneys, nerves, heart and blood vessels. When the condition is severe or stressful, it can cause acute and severe metabolic disorders, such as diabetic ketoacidosis and hyperosmolar hyperglycemic syndrome. At present, the drugs used to treat diabetes are mainly Western medicine preparations, including insulin preparations, sulfonylurea preparations, biguanide preparations, insulin resistance improvers, α-glucosidase inhibitors, etc. These drugs act on a certain link of blood sugar regulation to exert blood sugar control effects, with rapid onset of action, but large side effects, and a single efficacy, which is easy to produce resistance.

[0004] Studies have shown that chronic inflammation caused by endotoxins entering the blood due to dysbiosis is one of the important factors in the development of metabolic diseases such as obesity and diabetes. Imbalanced bacterial flora can trigger systemic chronic inflammatory reactions, leading to pancreatic beta cell damage and decreased insulin resistance. It also affects the absorption of sugar by other cells in the body for energy conversion, making it difficult for the human body to function normally, and ultimately causing a series of complications. Studies have shown that probiotics can effectively inhibit the development of prediabetes into type 2 diabetes. Probiotics help the human body lower blood sugar concentrations, thereby inhibiting the symptoms of prediabetes. [1] .

[0005] Patent CN113330109A relates to Akkermansia muciniphila SNUG61027 strain (preservation number KCTC13530BP) and its use. Specifically, a composition for appetite control or prevention, improvement, alleviation or treatment of metabolic diseases, a use for appetite control or prevention, improvement, alleviation and treatment of metabolic diseases, and a method for appetite control or prevention, improvement, alleviation and treatment of metabolic diseases using the composition are provided, the composition comprises the strain, or its culture fluid, etc. or B2UM07 protein separated therefrom as an active ingredient, and the metabolic disease is impaired glucose tolerance, diabetes, arteriosclerosis, hyperlipidemia, hypercholesterolemia, fatty liver, cardiovascular disease or obesity.

[0006] With the change of lifestyle, the prevalence of diabetes is increasing year by year, and more and more patients have other metabolic diseases, which seriously affects the quality of life and health of patients and also forms a huge clinical demand. Therefore, it is very important to provide patients with safe, effective, accessible and affordable innovative drugs.

[0007] [1] Zhu Lili et al. Meta-analysis of the clinical efficacy of probiotic preparations in the treatment of type 2 diabetes[J]. Chinese Journal of Microecology. 2019, 31(02).

[0008] The present invention is the development of downstream application technology carried out by the inventor for the independently developed microbial resources: Akkermansia muciniphila with the deposit number of CGMCC No.20955 (other patents have been submitted on the same day).

[0009] After obtaining Akkermansia muciniphila, the inventors conducted application experiments in multiple fields including metabolic diseases, cardiovascular diseases, analgesia, anti-tumor, cognitive diseases, inflammatory diseases, bone and joint diseases, and obtained positive experimental results in some fields.

[0010] Taking into account the relevant provisions of the Patent Law on unity, protection is requested separately for each indication that does not have unity.

[0011] The present invention is for the protection of application technology for treating, assisting in the treatment or preventing metabolic diseases.

[0012] Since relevant evidence such as microbial identification and performance testing was published in the microbial patent submitted on the same day, in order to facilitate the review of the present invention and quickly understand the inventor's previous work, the relevant information of Akkermansiamuciniphila is briefly disclosed in the content of the present invention.

[0013] Akkermansia muciniphila:

[0014] The conclusive information in this section is for the convenience of reviewing the present invention, but is not intended to limit the scope of protection of the present invention. The specific experimental process and experimental results are recorded in the microbiological patent filed on the same day as the present invention.

[0015] 1. Akkermansia muciniphila was obtained by separation and screening by the present inventors.

[0016] 2. The deposit information of Akkermansia muciniphila: The deposit number is: CGMCC No.20955.

[0017] 3. 16srRNA of Akkermansia muciniphila: SEQ ID NO.1.

[0018] 4. Physiological and biochemical properties of Akkermansia muciniphila:

[0019] (1) Hydrophobicity: It increases with time and reaches more than 30% at 60 min.

[0020] (2) Self-aggregation: It increases with time and tends to be stable after 20 hours, maintaining at around 52%.

[0021] (3) Gastrointestinal fluid tolerance:

[0022] The survival rate in gastric juice showed a decreasing trend with the extension of time, and the survival rate was about 85% at 240 minutes;

[0023] The survival rate in intestinal fluid showed a downward trend as a whole, and the survival rate was above 80% at 240 minutes.

[0024] (4) Biofilm formation ability: weak.

[0025] (5) Acute toxicity test:

[0026] The results of bacterial reverse mutation test showed that the strain had no mutagenicity;

[0027] Mice were acutely gavaged with high, medium, and low doses of Akkermansia muciniphila, and no mortality occurred;

[0028] There was no significant difference in the daily body weight changes between the test group mice and the control saline group;

[0029] During the acute gavage period, there was no significant difference in the daily food intake of the test mice and the control saline group mice;

[0030] During the acute gavage period, there was no significant difference in the daily changes in blood glucose between the test mice and the control saline group mice;

[0031] There were no significant differences in the blood parameters between the acute gavage group and the control saline group;

[0032] In the liver, the triglyceride levels in the test mice were significantly lower than those in the control saline group mice;

[0033] There was no significant difference in serum triglyceride levels between the test mice and the control mice;

[0034] There was no significant difference in cholesterol levels in serum and liver between the test mice and the control mice;

[0035] There was no significant difference in the bile acid levels in serum and liver between the test mice and the control mice;

[0036] There was no significant difference in glucose levels in serum and liver between the test mice and the control mice;

[0037] There was no significant difference in the total protein levels in serum and liver between the test mice and the control mice;

[0038] There was no significant difference in the activity of alanine aminotransferase in serum and liver between the test mice and the control mice.

[0039] There was no significant difference in the activity of aspartate aminotransferase in serum and liver between the test mice and the control mice;

[0040] There was no significant difference in serum creatinine content between the test mice and the control mice;

[0041] There was no significant difference in the urea nitrogen concentration in the serum between the test mice and the control mice;

[0042] There was basically no significant difference in the weight of the heart, liver, spleen, kidney, thymus, brain, testis, lung, stomach and intestine of the test mice and the control mice;

[0043] There was no significant pathological damage in the liver and kidneys of the test mice and the control group mice.

[0044] (6) Subchronic toxicity test:

[0045] Mice were gavaged with high, medium, and low doses of AKK PROBIO for 90 consecutive days without mortality.

[0046] There was no significant difference in the weekly body weight change between the test group mice and the control saline group;

[0047] During the continuous gavage period, there was no significant difference in the weekly changes in food intake between the test mice and the control saline group mice;

[0048] During the continuous gavage period, the changes in blood glucose in the mice given a medium dose of oral administration were significantly different from those in the control saline group, but there were no significant differences between the other groups and the control group.

[0049] There were no significant differences in the blood parameters between the mice in the gavage group and the control saline group;

[0050] There was no significant difference in triglyceride levels in the liver and serum between the test mice and the control mice;

[0051] There was no significant difference in cholesterol levels in the liver and serum between the test mice and the control mice;

[0052] The bile acid levels in the liver of the mice given the medium dose and the control group were significantly different, while there were no significant differences in the serum and liver between the other groups and the control group.

[0053] There was no significant difference in glucose levels in the liver and serum between the test mice and the control mice;

[0054] There was no significant difference in the total protein content in the liver and serum between the test mice and the control mice;

[0055] There was no significant difference in the activity of alanine aminotransferase in serum and liver between the test mice and the control mice.

[0056] There was no significant difference in the activity of aspartate aminotransferase in serum and liver between the test mice and the control mice;

[0057] There was no significant difference in serum creatinine content between the test mice and the control mice;

[0058] There was no significant difference in the urea nitrogen concentration in the serum between the test mice and the control mice;

[0059] There was basically no significant difference in the weight of the heart, liver, spleen, kidney, thymus, brain, testis, lung, pancreas, stomach and intestine between the test mice and the control mice;

[0060] There was no significant pathological damage in the liver and kidneys of the test mice and the control mice;

[0061] There was no significant difference in blood sugar regulation ability between the test mice and the control group mice.

[0062] (7) Drug sensitivity analysis of Akkermansia muciniphila:

[0063] Akkermansia muciniphila was found to be sensitive to ampicillin, ceftriaxone, ceftriaxone, meropenem, tetracycline, moxifloxacin and chloramphenicol. Summary of the invention

[0064] In order to solve the above problems, the present invention provides an Akkermansia muciniphila, characterized in that it was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 26, 2020, and the deposit number is: CGMCC No. 20955. Akkermansia muciniphila can effectively lower blood sugar levels, lower TC, TG, and LDL-C levels, and increase HDL-C levels, especially having a significant effect on increasing HDL-C levels.

[0065] The present invention is directed to the downstream application technology of Akkermansia muciniphila for protection.

[0066] Specifically, the application of Akkermansia muciniphila in metabolic diseases.

[0067] Specifically, in terms of application fields, the aforementioned applications include:

[0068] 1. Use Akkermansia muciniphila for the prevention, treatment, auxiliary prevention and auxiliary treatment of metabolic diseases in therapeutic and pharmaceutical applications;

[0069] 2. Use Akkermansia muciniphila for the preparation of pharmaceutical raw materials for the prevention, treatment, auxiliary prevention and auxiliary treatment of metabolic diseases.

[0070] The present invention uses model mice as an example to verify the application of Akkermansia muciniphila in metabolic diseases. The scope of use of the present invention should not be limited to therapeutic methods or pharmaceutical uses based on the commonness of animal experiments in the medical field.

[0071] Specifically, in terms of the types of metabolic diseases, the aforementioned applications include, but are not limited to: abnormal glucose metabolism, abnormal lipid metabolism, abnormal calcium and phosphorus metabolism, abnormal bone metabolism and / or abnormal purine metabolism.

[0072] Based on the occurrence of various metabolic diseases, all of them are diseases caused by the disorders of substance anabolism and catabolism. Based on the experiments of Akkermansia muciniphila in a specific metabolic disease (diabetes), those skilled in the art can reasonably conclude or infer that it can be equally applied to or at least has the potential for application in other metabolic diseases.

[0073] More specifically, the abnormal glucose metabolism is diabetes and / or hypoglycemia.

[0074] Preferably, the abnormal glucose metabolism is diabetes.

[0075] More specifically, the abnormal lipid metabolism may be obesity, fatty liver, hyperlipidemia and / or anorexia.

[0076] More specifically, the abnormal calcium-phosphorus metabolism may be hypercalcemia, hypocalcemia, hyperparathyroidism, hypoparathyroidism and / or parathyroid adenoma.

[0077] More specifically, the abnormal bone metabolism may be osteoporosis, osteomalacia and / or osteodystrophy.

[0078] More specifically, the abnormal purine metabolism may be hyperuricemia, acute gouty arthritis and / or chronic gouty arthritis.

[0079] Specifically, in terms of raw material form, the aforementioned application includes the use of fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, dead bacteria and / or any ingredients, components, compositions, metabolites directly or indirectly derived from Akkermansia muciniphila; and may or may not contain current or future excipients allowed in a specific application field.

[0080] More specifically, the fermentation liquid refers to the liquid obtained by inoculating bacteria into a culture medium and culturing for a period of time.

[0081] More specifically, the fermentation broth supernatant refers to the clarified liquid on the upper layer of the fermentation broth after centrifugation; it contains abundant metabolites of bacterial growth and reproduction and some bacterial fragments. The acidic substances and bacteriocins secreted by bacteria have antagonistic and killing effects on harmful bacteria; the amino acids and synthesized vitamins after bacteria decompose food are all in the culture medium, and also include enzymes secreted by bacteria that are useful to the human body; and some bacterial components also have an immune-promoting effect on the human body.

[0082] More specifically, the fermentation liquid precipitate refers to the liquid precipitate obtained by centrifugation, including free protein, residual bacteria, broken cells, and residues of the culture matrix, mainly protein and intracellular matrix.

[0083] More specifically, the live bacteria, also called active flora, can colonize and multiply in the intestine, which is beneficial to increase the number of beneficial bacteria.

[0084] More specifically, the dead bacteria are microorganisms that have lost their vitality and are unable to grow and reproduce. The probiotics lose their vitality due to the production process, such as high temperature treatment or excessive drying.

[0085] Specifically, in the field of medicine, the medicine includes:

[0086] (1) Fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Akkermansia muciniphila;

[0087] (2) Pharmaceutically acceptable excipients.

[0088] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0089] Preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0090] More specifically, the dosage form of the drug is tablets, capsules, granules or injections.

[0091] More specifically, the medicine comprises other medicines for treating metabolic diseases.

[0092] Specifically, in terms of the active dose in the above fields, the viable count of Akkermansia muciniphila is not less than 1×10 8 CFU / g or 1×10 8 CFU / mL.

[0093] Preferably, the viable count of Akkermansia muciniphila is 1×10 8 -1×10 12 CFU / g or 1×10 8 -1×10 12 CFU / mL.

[0094] More preferably, the viable count of Akkermansia muciniphila is 1×10 9 -1×10 12 CFU / g or 1×10 9 -1×10 12CFU / mL, such as 1×10 9 CFU / g (CFU / mL), 2×10 9 CFU / g (CFU / mL), 3×10 9 CFU / g (CFU / mL), 4×10 9 CFU / g (CFU / mL), 5×10 9 CFU / g (CFU / mL), 6×10 9 CFU / g (CFU / mL), 7×10 9 CFU / g (CFU / mL), 8×10 9 CFU / g (CFU / mL), 9×10 9 CFU / g (CFU / mL), 10×10 9 CFU / g, etc. Other point values ​​within this numerical range can be selected.

[0095] In another aspect, the present invention also provides a method for treating a metabolic disease, which comprises administering Akkermansia muciniphila to a patient with the metabolic disease.

[0096] Specifically, the metabolic disease patients are patients clinically diagnosed with abnormal glucose metabolism, abnormal lipid metabolism, abnormal calcium and phosphorus metabolism, abnormal bone metabolism and / or abnormal purine metabolism.

[0097] Specifically, the method of administration is selected from oral administration, intravenous injection, topical administration, intradermal administration and / or subcutaneous administration.

[0098] Specifically, a medicament comprising the following (1) and (2) is administered to a patient with a metabolic disease:

[0099] (1) Fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Akkermansia muciniphila;

[0100] (2) Pharmaceutically acceptable excipients.

[0101] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0102] Preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0103] More specifically, the medicine comprises other medicines for treating metabolic diseases.

[0104] In another aspect, the present invention also provides a method for preventing and treating metabolic diseases based on Akkermansia muciniphila produced, derived and derived from the above application.

[0105] Specifically, based on the use of Akkermansia muciniphila in treating or preventing metabolic diseases, the strain can also be improved to produce, derive, or derive Akkermansia muciniphila with better treatment or prevention effects on metabolic diseases, and the improvement methods include traditional methods and genetic engineering methods.

[0106] More specifically, the conventional method may be a physical method, a chemical method and / or a selection method.

[0107] More specifically, the physical method refers to the use of radiation or high pressure to induce mutations, thereby achieving strain improvement.

[0108] Preferably, the radiation method comprises ultraviolet radiation and / or X-ray radiation;

[0109] The high pressure method may be to expose the microorganisms to a high pressure environment to cause them to undergo adaptive changes.

[0110] More specifically, the chemical method refers to the use of chemical agents to induce mutation of bacterial strains.

[0111] Preferably, the chemical agent may be nitrite, acetylcholine and / or nitrogen mustard.

[0112] More specifically, the selection method refers to the process of selecting microorganisms with excellent traits for reproduction, thereby achieving strain improvement.

[0113] Preferably, the selection method is used to improve the metabolic pathway and / or growth conditions of the microorganism to obtain Akkermansia muciniphila with better effect in treating or preventing metabolic diseases.

[0114] More specifically, the genetic engineering method may be gene cloning, gene knockout, gene editing and / or synthetic biology, so as to obtain strains with better effects in treating or preventing metabolic diseases.

[0115] More specifically, the gene cloning refers to cutting out the target gene from one cell and inserting it into another cell to achieve the expression of the target gene in the new host, so as to enhance the effect of the original strain in treating or preventing metabolic diseases.

[0116] More specifically, the gene knockout refers to deleting the target gene through technical means to achieve the regulation of microbial traits in order to obtain strains with better effects in treating or preventing metabolic diseases.

[0117] More specifically, the gene editing refers to the precise modification of the target gene sequence through technical means to achieve the regulation of microbial traits in order to obtain strains that are better for treating or preventing metabolic diseases.

[0118] More specifically, the synthetic biology refers to the use of chemical synthesizer technology to construct new gene sequences and introduce them into microorganisms to achieve regulation of microbial traits in order to obtain strains that are better at treating or preventing metabolic diseases.

[0119] Specifically, a drug for treating or preventing metabolic diseases, the drug comprising:

[0120] (1) The fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Akkermansia muciniphila produced, derived or derived;

[0121] (2) Pharmaceutically acceptable excipients.

[0122] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0123] Preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0124] More specifically, the dosage form of the drug is tablets, capsules, granules or injections.

[0125] More specifically, the medicine comprises other medicines for treating metabolic diseases.

[0126] Specifically, the pharmaceutical raw material for treating or preventing metabolic diseases is the produced, derived, or derived Akkermansia muciniphila fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria.

[0127] The technical effects achieved by the present invention are:

[0128] (1) Akkermansia muciniphila can effectively lower blood sugar levels and has good blood sugar lowering function.

[0129] (2) Akkermansia muciniphila can reduce TC, TG, and LDL-C levels and increase HDL-C levels, especially in increasing HDL-C levels.

[0130] Storage Instructions:

[0131] Strain name: AKK PROBIO;

[0132] Deposit number: CGMCC No.20955;

[0133] Taxonomic nomenclature: Akkermansia muciniphila;

[0134] Deposit date: October 26, 2020;

[0135] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0136] Address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Figure 1 is the level of high-density lipoprotein cholesterol (HDL-C) in mouse serum. DETAILED DESCRIPTION

[0138] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0139] Nouns and terms:

[0140] Triglyceride (TG): Also known as neutral fat, it is a lipid formed by the reaction of glycerol molecules and fatty acids. It is one of the components of blood lipids and has the function of providing energy for cell metabolism. Triglyceride determination is an important part of blood lipid examination. It is the sum of triglycerides contained in each lipoprotein in plasma.

[0141] Total cholesterol (TC): The sum of cholesterol contained in various lipoproteins in the blood. Cholesterol is the main component of cell membranes and an important raw material for synthesizing physiologically active substances such as adrenal cortex hormones, sex hormones, bile acids and vitamin D. The total cholesterol concentration in human blood can be used as an indicator of lipid metabolism to assess the risk of atherosclerosis and ischemic cardiovascular and cerebrovascular diseases.

[0142] High-density lipoprotein cholesterol (HDL-C) is an anti-atherosclerotic lipoprotein that can transport cholesterol from extrahepatic tissues to the liver for metabolism and excrete it from the body in bile. Its plasma level is negatively correlated with the risk of metabolic diseases.

[0143] Low density lipoprotein cholesterol (LDL-C): It is the main lipoprotein in fasting plasma, accounting for about 2 / 3 of plasma lipoproteins. It is the main carrier for transporting cholesterol to extrahepatic tissues. Its concentration is significantly positively correlated with the incidence of metabolic diseases.

[0144] The Akkermansia muciniphila in the present invention is AKK PROBIO.

[0145] Example 1 Cultivation of AKK PROBIO

[0146] (1) Culture medium

[0147] Solid plate culture: BHI (Brain Heart Infusion, OXOID, Catalog No.: CM1032B)) + 5% sheep blood

[0148] Weigh a certain volume of BHI powder according to the finished product requirements, add agar powder at a ratio of 20g / L, dilute to the corresponding volume, and sterilize at 121℃ for 20min. After sterilization, add 5% sterile defibrinated sheep blood when it cools to 53℃ (not hot to hold), shake well and pour 15-20mL into a plate for later use.

[0149] (2) Culture conditions

[0150] Culture temperature: 37°C;

[0151] Culture conditions: completely anaerobic;

[0152] Liquid fermentation broth culture: BHI;

[0153] Culture temperature: 37°C;

[0154] Culture conditions: Completely anaerobic culture on 8 layers of gauze.

[0155] (3) Expansion culture of bacterial strains

[0156] Level 1: Take a 2 ml glycerol tube and inoculate it into a test tube containing 9 mL BHI at a 10% inoculum volume, and culture anaerobically at 37°C for 24-48 hours.

[0157] Secondary: Take the primary fermentation test tube and inoculate it into a Erlenmeyer flask containing 90 mL of BHI at a 5% inoculation rate, and culture anaerobically at 37°C for 24 h.

[0158] Level 3: Take the fermentation liquid from the second-stage Erlenmeyer flask and inoculate it into a Erlenmeyer flask containing 300 mL of BHI at a 5% inoculation rate. Incubate anaerobically at 37°C for 13 h and store in a 20% glycerol tube at -80°C.

[0159] Example 2 Animal Experiment

[0160] 2.1 Animals

[0161] A total of 40 8-week-old female C57BL / 6 mice (Shanghai Model Organisms Technology Co., Ltd.) weighing 18-22 g and free of specific pathogens were used. They were adaptively fed for one week before the experiment, with free access to food and water, and the breeding environment met the SPF level requirements.

[0162] 2.2 Reagents and instruments

[0163] Blood glucose meter: Aikelai animal blood glucose meter BS-7110.

[0164] Positive drug: Metformin hydrochloride tablets, Shanghai Bristol-Myers Squibb Pharmaceuticals Co., Ltd., approval number: H20023371.

[0165] Streptozotocin (STZ), Shanghai Beinuo Biotechnology Co., Ltd., catalog number: Sigma 100mg.

[0166] High-density lipoprotein cholesterol (HDL-C) detection kit, Shanghai Yanzun Biotechnology Co., Ltd., catalog number: YZ-P63995.

[0167] Low-density lipoprotein cholesterol (LDL-C) detection kit, Shanghai Yanqi Biotechnology Co., Ltd., catalog number: YQ-2710.

[0168] Triglyceride (TG) detection kit, Shanghai Xuanya Biotechnology Co., Ltd., catalog number: XY-SJH-XS1465.

[0169] Total cholesterol (TC) detection kit, Shanghai Qiyuan Biotechnology Co., Ltd., catalog number: QYS-234007.

[0170] 2.3 Animal modeling and drug intervention

[0171] Low-fat diet: Research Diets D12450B, Shanghai Huaya Sichuang Biotechnology Co., Ltd., item number: D12450J; high-fat diet: Research Diets D12451, Shanghai Huaya Sichuang Biotechnology Co., Ltd., item number: D12451. Streptozotocin (STZ), dissolved in 0.1 mol / L, pH 4.4 citric acid solution, 0.22 μm microfiltration, kept in an ice bath; stored at 4 ° C, prepared before use.

[0172] The STZ-induced diabetic mouse model was established. The control group (10 mice) was fed with a low-fat diet, and the diabetic model group (50 mice) was fed with a high-fat diet. After 4 weeks of feeding, the mice were fasted for 1 hour before STZ injection. The diabetic model group was intraperitoneally injected with a small dose of 70 mg / kg STZ for 5 consecutive days, and the control group was injected with an equal volume of citric acid buffer. After injection, the control group was fed a low-fat diet; the diabetic model group continued to be given a high-fat diet. After fasting for 8 hours, blood was collected from the tail vein on the 7th day to measure fasting blood sugar. The tail was cut with sterile small scissors to collect blood sugar, and blood sugar was measured using a blood glucose meter. Blood sugar ≥11.1mmol / L was determined to be a successful diabetic model.

[0173] The diabetic model mice were randomly divided into 3 groups: model control, positive drug group, and AKK PROBIO group, with 10 mice in each group. The positive drug group was given metformin hydrochloride tablets at an intragastric dose of 150 mg / kg; the AKK PROBIO group was given metformin hydrochloride tablets at an intragastric dose of 1×10 9 CFU / mouse. All drugs were administered orally twice a day (once in the morning and once in the evening) according to the corresponding dose, and the control group and the model group were administered with the same amount of citric acid solution for 5 weeks.

[0174] 2.4 Index detection

[0175] 2.4.1 Blood glucose in mice

[0176] The fasting blood glucose levels of mice in each group were measured before administration and 5 weeks after administration.

[0177] 2.4.2 Serum index detection

[0178] After administration in the fifth week, mice were fasted for 8 hours, and fresh blood was collected from the heart. The blood of each group of mice was placed at room temperature for 1 hour, and then centrifuged at 1000×g for 20 minutes to obtain serum samples. Serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured according to the kit instructions.

[0179] 2.5 Experimental Results

[0180] 2.5.1 Blood glucose and body weight of mice

[0181] The blood glucose of mice is shown in Table 1. The blood glucose level of the model group was significantly higher than that of the control group, indicating that the mouse hyperglycemia model was successfully established. The blood glucose levels of the positive drug group and the AKK PROBIO group were significantly reduced compared with the model group, and the AKK PROBIO group almost reached the same level as the positive drug group, indicating that AKK PROBIO has a good blood glucose-lowering effect.

[0182] Table 1

[0183]

[0184] 2.5.2 Serum detection indicators

[0185] The serum test indicators are shown in Table 2. Compared with the control group, the serum TC, TG, and LDL-C levels of the model group mice were significantly increased, and the HDL-C level was significantly decreased. Compared with the model group, the positive drug group and the AKK PROBIO group effectively reduced the TC, TG, and LDL-C levels, increased the HDL-C level, and the HDL-C level in the AKK PROBIO group was higher than that in the positive drug group. This shows that AKKPROBIO has the effect of regulating blood lipids and total cholesterol in diabetic mice, and effectively reduces low-density lipoprotein and increases high-density lipoprotein.

[0186] Table 2

[0187]

[0188]

[0189] Comparative Example

[0190] Referring to the culture method of Example 1, the serum detection index experiment in Example 2 was performed by replacing the AKKPROBIO of the present invention with the ATCC standard strain of Akkermansia (ATCC BAA 835). The specific results are shown in Table 3.

[0191] Table 3

[0192]

[0193] The above results show that the ATCC standard strain of Akkermansia can also increase the HDL-C level in the serum of diabetic mice, but the effect is far less than that of AKK PROBIO of the present application.

[0194] Application Example 1: Preparation of AKK PROBIO live bacteria

[0195] AKK PROBIO live bacteria culture method:

[0196] (1) Culture medium

[0197] Solid plate culture: BHI + 5% sheep blood

[0198] Weigh a certain volume of BHI powder according to the finished product requirements, add agar powder at a ratio of 20g / L, dilute to the corresponding volume, and sterilize at 121℃ for 20min. After sterilization, add 5% sterile defibrinated sheep blood when it cools to 53℃ (not hot to hold), shake well and pour 15-20ml into a plate for later use.

[0199] (2) Culture conditions

[0200] Culture temperature: 37°C;

[0201] Culture conditions: completely anaerobic;

[0202] Liquid fermentation broth culture: BHI;

[0203] Culture temperature: 37°C;

[0204] Culture conditions: Completely anaerobic culture on 8 layers of gauze.

[0205] (3) Expansion culture of bacterial strains

[0206] Level 1: Take a 2 ml glycerol tube and inoculate it into a test tube containing 9 mL BHI at a 10% inoculum volume, and culture anaerobically at 37°C for 24-48 hours.

[0207] Secondary: Take the primary fermentation test tube and inoculate it into a Erlenmeyer flask containing 90 mL of BHI at a 5% inoculation rate, and culture anaerobically at 37°C for 24 h.

[0208] Level 3: Take the fermentation liquid from the second-stage Erlenmeyer flask and inoculate it into a Erlenmeyer flask containing 300 mL of BHI at a 5% inoculation rate. Incubate anaerobically at 37°C for 13 h and store in a 20% glycerol tube at -80°C.

[0209] (4) Fermentation tank culture

[0210] The activated bacteria were placed in a large fermentation tank and cultured under the same conditions as above. After the culture was completed, the wet bacteria were obtained by centrifugation.

[0211] Application Example 2: Preparation of AKK PROBIO dead bacteria

[0212] The wet bacteria prepared in Example 1 are killed by chemical or physical methods to obtain dead bacteria;

[0213] The chemical method may be gas sterilization and / or liquid sterilization;

[0214] Specifically, the gas sterilization may be ozone, ethylene oxide, formaldehyde, propylene glycol, glycerin or peracetic acid vapor;

[0215] The liquid sterilization agent is a chemical reagent, such as ethanol;

[0216] The physical method may be heat sterilization, light sterilization and / or microwave sterilization;

[0217] Specifically, the thermal sterilization method may be a burning method, a dry baking method, a boiling method and / or a pressure steam sterilization method;

[0218] The light sterilization can be sunlight exposure, ultraviolet irradiation and / or ionizing radiation.

[0219] Application Example 3: An oral medicine containing AKK PROBIO

[0220] The wet bacteria prepared in Example 1 are mixed with auxiliary materials in a certain ratio, and the mixture is made into live bacteria tablets, capsules, granules, etc. by spray drying, tabletting, etc.;

[0221] Alternatively, the wet bacteria prepared in Example 1 are subjected to cell disruption, and then the beneficial components are extracted by appropriate techniques; the extraction methods include centrifugation, distillation, leaching, extraction, etc., and the specific selection depends on the components contained in the probiotics; the extracted components can be prepared into different dosage forms such as capsules, oral liquids, granules, injections, etc. according to clinical use needs.

Claims

1. Akkermansia muciniphila The use of the method in preparing a drug for reducing blood sugar is characterized in that: The Akkermansia muciniphila The deposit number is: CGMCC No. 20955; Akkermansia muciniphila The number of viable bacteria is not less than 1×10 8 CFU / g or 1×10 8 CFU / mL.

2. The use according to claim 1, characterized in that: The dosage form of the medicine is tablet, capsule or granule.

3. The use according to claim 2, characterized in that: The medicines described include other blood sugar lowering medicines.

4. The use according to any one of claims 1 to 3, characterized in that: The medicine also includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients are selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

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