Application of Akkermansia muciniphila in preparing products for preventing, treating and / or assisting in treating tumors

Abstract

The present invention relates to the field of microbial technology, and specifically to the use of Akkermansia muciniphila in the preparation of products for preventing, treating and / or assisting in the treatment of tumors. The deposit number of the Akkermansia muciniphila is CGMCC No. 20955. The present invention discloses for the first time the use of the above-mentioned Akkermansia muciniphila for anti-colorectal cancer, which can alleviate the increase in spleen and liver indexes and the decrease in body weight induced by AOM / DSS, effectively reduce the number of colorectal cancer tumors and the degree of rectal tissue lesions, upregulate the mRNA expression levels of caspase-9, Bid and Bim, reduce the degree of intestinal inflammation, and affect the occurrence and development of colorectal cancer.

Description

Technical Field

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

[0002] Akkermansia belongs to the phylum Verrucomycetes. It is a Gram-negative, strictly anaerobic, non-motile, non-spore-forming, oval bacterium that colonizes the surface of the gastrointestinal mucosa and uses gastric mucus protein as a carbon source. Akkermansia muciniphila (AKK) is found in fecal specimens of healthy people. It resides in the intestinal mucosa layer, has adhesion to epithelial cells, and can degrade mucin on the surface of the intestinal epithelium.

[0003] Colorectal cancer (CRC), also known as colorectal cancer, includes the ascending colon, transverse colon, descending colon, sigmoid colon and rectum. It is a common malignant tumor that seriously endangers human health. The clinical manifestations of early colorectal cancer are mostly asymptomatic, and a few only show abdominal pain, abdominal distension and diarrhea. In the middle and late stages, it manifests as changes in stool properties, abdominal pain, abdominal masses and intestinal obstruction. Currently, surgical resection is the main treatment for colorectal cancer, but surgical resection may increase the risk of tumor spread and metastasis. Radiotherapy and chemotherapy are commonly used treatments for patients with advanced colorectal cancer metastasis, but they are prone to drug resistance and side effects. Gene therapy, cell therapy and immunotherapy are expensive. At present, there is an urgent need for a method for treating colorectal cancer with high acceptance and good therapeutic effects.

[0004] Chinese patent CN108367031A discloses the use of Akkermansia muciniphila for treating inflammatory conditions, and specifically discloses that a composition comprising one or more strains of Akkermansia muciniphila has the effect of preventing and / or treating gastrointestinal inflammation associated with the following conditions: inflammatory bowel disease such as Crohn's disease or ulcerative colitis, irritable bowel syndrome, pouchitis, and post-infectious colitis.

[0005] Chinese patent CN116211896A discloses the use of Akkermansia muciniphila JF3 in improving tissue fibrosis and repairing mucosal damage in the intervention of radiation proctopathy, and the deposit number of Akkermansia muciniphila JF3 is CCTCC No: M20221499. The study in this patent found that Akkermansia muciniphila JF3 can effectively improve rectal fibrosis, rectal mucosal damage, anal damage and colorectal inflammation caused by radiation proctopathy. Akkermansia muciniphila JF3 can be used as a drug for treating rectal fibrosis, rectal mucosal damage, anal damage and colorectal inflammation caused by radiation proctopathy.

[0006] Currently, the prior art does not disclose that Akkermansia muciniphila with a deposit number of CGMCC No. 20955 has an anti-tumor effect.

[0007] The present invention is the downstream application technology development carried out by the inventor for the self-developed microbial resources: Akkermansia muciniphila with the deposit number of CGMCC No.20955 (other patents have been submitted on the same day).

[0008] After obtaining Akkermansia muciniphila, the inventors conducted application experiments in multiple fields including anti-tumor, cognitive disease, metabolic disease, inflammatory disease, bone and joint disease, cardiovascular disease, etc. and obtained positive experimental results in some fields.

[0009] Taking into account the relevant provisions of the Patent Law on unity, protection is requested separately for each indication that does not have unity.

[0010] The present invention is directed to the protection of anti-tumor application technology.

[0011] 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.

[0012] Akkermansia muciniphila:

[0013] The conclusive information in this section is for the convenience of the review of 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.

[0014] 1. Akkermansia muciniphila was isolated and screened by the inventors;

[0015] 2. The deposit information of Akkermansia muciniphila: The deposit number is: CGMCC No.20955.

[0016] 3. 16srRNA of Akkermansia muciniphila: SEQ ID NO.1.

[0017] 4. Physiological and biochemical properties of Akkermansia muciniphila:

[0018] (1) Hydrophobicity: It increases with time and reaches more than 30% at 60 min.

[0019] (2) Self-aggregation: It increases with time and tends to be stable after 20 hours, maintaining at around 52%.

[0020] (3) Gastrointestinal fluid tolerance:

[0021] 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;

[0022] The survival rate in intestinal fluid showed a downward trend as a whole, and the survival rate was above 80% at 240 minutes.

[0023] (4) Biofilm formation ability: weak.

[0024] (5) Acute toxicity test:

[0025] The results of bacterial reverse mutation test showed that the strain had no mutagenicity;

[0026] Mice were acutely gavaged with high, medium, and low doses of Akkermansia muciniphila, and no mortality occurred;

[0027] There was no significant difference in the daily body weight changes between the test group mice and the control saline group;

[0028] 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;

[0029] 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;

[0030] There were no significant differences in the blood parameters between the acute gavage group and the control saline group;

[0031] In the liver, the triglyceride levels in the test mice were significantly lower than those in the control saline group mice;

[0032] There was no significant difference in serum triglyceride levels between the test mice and the control mice;

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

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

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

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

[0037] There was no significant difference in the activity of alanine aminotransferase in serum and liver between the test mice and the control mice.

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

[0039] There was no significant difference in serum creatinine content between the test mice and the control mice;

[0040] There was no significant difference in the urea nitrogen concentration in the serum between the test mice and the control mice;

[0041] 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;

[0042] There was no significant pathological damage in the liver and kidneys of the test mice and the control group mice.

[0043] (6) Subchronic toxicity test:

[0044] Mice were gavaged with high, medium, and low doses of AKK.pro for 90 consecutive days without mortality;

[0045] There was no significant difference in the weekly body weight change between the test group mice and the control saline group;

[0046] 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;

[0047] 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.

[0048] There were no significant differences in the blood parameters between the mice in the gavage group and the control saline group;

[0049] There was no significant difference in triglyceride levels in the liver and serum between the test mice and the control mice;

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

[0051] 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.

[0052] There was no significant difference in glucose levels in the liver and serum between the test mice and the control mice;

[0053] There was no significant difference in the total protein content in the liver and serum between the test mice and the control mice;

[0054] There was no significant difference in the activity of alanine aminotransferase in serum and liver between the test mice and the control mice.

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

[0056] There was no significant difference in serum creatinine content between the test mice and the control mice;

[0057] There was no significant difference in the urea nitrogen concentration in the serum between the test mice and the control mice;

[0058] 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;

[0059] There was no significant pathological damage in the liver and kidneys of the test mice and the control mice;

[0060] There was no significant difference in blood sugar regulation ability between the test mice and the control mice.

[0061] (7) Drug sensitivity analysis of Akkermansia muciniphila:

[0062] Akkermansia muciniphila was found to be sensitive to ampicillin, ceftriaxone, ceftriaxone, meropenem, tetracycline, moxifloxacin and chloramphenicol. Summary of the invention

[0063] The purpose of the present invention is to provide the preparation and application of Akkermansia muciniphila in anti-tumor. The Akkermansia muciniphila of the present invention alleviates the increase of spleen and liver indexes and the decrease of body weight induced by AOM / DSS, reduces the number of colorectal cancer tumors and the degree of damage to rectal tissues, and upregulates the mRNA expression levels of caspase-9, Bid and Bim in colon tissues, thus playing an important role in the treatment of colorectal cancer.

[0064] The present invention is directed to the downstream application technology of Akkermansia muciniphila for protection.

[0065] Specifically, it is the application of Akkermansia muciniphila in anti-tumor.

[0066] Specifically, in terms of application fields, the aforementioned applications include:

[0067] 1. Use Akkermansia muciniphila for the prevention, treatment, auxiliary prevention and auxiliary treatment of tumors in therapeutic and pharmaceutical applications;

[0068] 2. Use Akkermansia muciniphila in food preparation for the adjuvant prevention and adjuvant treatment of tumors;

[0069] 3. Use Akkermansia muciniphila in the preparation of health products for the auxiliary prevention and treatment of tumors;

[0070] 4. Use Akkermansia muciniphila for the prevention, treatment, auxiliary prevention and auxiliary treatment of tumors by using raw material preparation.

[0071] The present invention uses model mice as an example to verify the application of Akkermansia muciniphila in anti-tumor. The scope of application 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.

[0072] Specifically, in terms of tumor types, the aforementioned applications include, but are not limited to: musculoskeletal system tumors, digestive system tumors, respiratory system tumors, nervous system tumors, urinary system tumors, reproductive system tumors and / or endocrine system tumors.

[0073] Based on the fact that the occurrence of various tumors is due to the combined effects of multiple factors on the body, which lead to abnormal gene changes, including activation of oncogenes, inactivation of tumor suppressor genes, and accumulation of multiple gene abnormalities, ultimately forming tumors, those skilled in the art can reasonably conclude or infer that Akkermansia muciniphila can be equally applied to or at least has the potential for application to tumors of its system, organ, or tissue based on experiments on Akkermansia muciniphila in a specific tumor (colorectal cancer).

[0074] More specifically, the tumors can be divided into benign tumors and malignant tumors according to their nature.

[0075] The benign tumor is adenoma, fibroma, lipoma, leiomyoma, rhabdomyoma, hemangioma, lymphangioma, chondroma, glioma and / or ganglioneuroma.

[0076] The malignant tumor is breast cancer, nasopharyngeal cancer, lung cancer, colorectal cancer, rectal cancer, liver cancer, pancreatic cancer, malignant melanoma,

[0077] 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.

[0078] More specifically, the fermentation liquid refers to the liquid obtained by inoculating bacteria into a culture medium and culturing for a period of time.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] Specifically, from the food field, the food includes:

[0084] (1) Fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Akkermansia muciniphila;

[0085] (2) Food-friendly excipients.

[0086] More specifically, the food-acceptable auxiliary material is selected from one or a combination of two or more of an emulsifier, a suspension stabilizer, a flavor, a wetting agent, an excipient, a preservative, a lubricant, a sweetener and a diluent.

[0087] Preferably, the food-acceptable auxiliary material is at least one selected from calcium silicate, fine crystalline cellulose, sucrose, sorbitol, mineral oil, starch, propyl hydroxybenzoate, talc, magnesium stearate, gum arabic, calcium phosphate, alginate, lactose, glucose, gelatin, polyvinyl pyrrolidone, syrup, cellulose, water, methylcellulose, methyl hydroxybenzoate and mannose.

[0088] More specifically, the food includes but is not limited to: chocolate, solid beverages, liquid beverages, ice cream, hard candies, crisp candies, caramel candies, jelly candies, toffee candies, gum-based candies, aerated candies, compressed candies, jellies, gel particles and / or crystal balls.

[0089] Specifically, from the perspective of the pharmaceutical field, the drugs include:

[0090] (1) Fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Akkermansia muciniphila;

[0091] (2) Pharmaceutically acceptable excipients.

[0092] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of preservatives, antioxidants, buffers, excipients, diluents, wetting agents, emulsifiers, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, flavors, sweeteners and stabilizers.

[0093] Preferably, the pharmaceutically acceptable excipient is at least one selected from gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, lactose, mannose, starch, fine crystalline cellulose, water, syrup, methylcellulose, mineral oil, propyl hydroxybenzoate, talc, magnesium stearate, polyvinyl pyrrolidone, cellulose and methyl hydroxybenzoate.

[0094] More specifically, the dosage form of the drug can be an oral preparation or an injection preparation.

[0095] Preferably, the oral preparation can be a tablet, powder, capsule, granule, pill, powder, paste, solid beverage or oral liquid.

[0096] More specifically, the drug comprises other drugs for treating tumors.

[0097] Specifically, in terms of health care products, the health care products include:

[0098] (1) Fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Akkermansia muciniphila;

[0099] (2) Acceptable excipients in health products.

[0100] More specifically, the auxiliary material is selected from one or a combination of two or more of a sweetener, a filler, a stabilizer, a pigment, a solvent, a flavoring agent, and a capsule shell material.

[0101] Preferably, the sweetener is selected from at least one of natural sweeteners and artificial sweeteners;

[0102] The filler is selected from at least one of starch, corn flour and grapes;

[0103] The stabilizer is selected from at least one of an antioxidant and a preservative;

[0104] The pigment is selected from at least one of natural pigment and artificial pigment;

[0105] The solvent is selected from at least one of water, alcohol, glycerol and ethanol;

[0106] The flavoring agent is selected from at least one of natural flavors and artificial flavors;

[0107] The capsule shell material is selected from at least one of gelatin, hydroxypropyl methylcellulose and polyvinyl alcohol.

[0108] 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.

[0109] 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.

[0110] More preferably, the viable count of Akkermansia muciniphila is 1×10 9 -1×10 12 CFU / g or 1×10 9 -1×10 12 CFU / 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 9CFU / 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.

[0111] The present invention also provides a method for treating tumors, which comprises administering Akkermansiamuciniphila to tumor patients.

[0112] Specifically, the tumor patients are patients clinically diagnosed with musculoskeletal tumors, digestive system tumors, respiratory system tumors, nervous system tumors, urinary system tumors, reproductive system tumors and / or endocrine system tumors.

[0113] Specifically, the method of administration is selected from oral administration, intravenous injection, topical administration, intradermal administration and / or subcutaneous administration.

[0114] Specifically, the drugs administered to tumor patients include:

[0115] (1) Fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Akkermansia muciniphila;

[0116] (2) Pharmaceutically acceptable excipients.

[0117] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of preservatives, antioxidants, buffers, excipients, diluents, wetting agents, emulsifiers, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, flavors, sweeteners and stabilizers.

[0118] Preferably, the pharmaceutically acceptable excipient is at least one selected from gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, lactose, mannose, starch, fine crystalline cellulose, water, syrup, methylcellulose, mineral oil, propyl hydroxybenzoate, talc, magnesium stearate, polyvinyl pyrrolidone, cellulose and methyl hydroxybenzoate.

[0119] More specifically, the dosage form of the drug can be an oral preparation or an injection preparation.

[0120] Preferably, the oral preparation can be a tablet, powder, capsule, granule, pill, powder, paste, solid beverage or oral liquid.

[0121] The present invention also provides a method for treating tumors using Akkermansia muciniphila produced, derived or derived from the above application.

[0122] Specifically, based on the use of Akkermansia muciniphila in treating or preventing tumors, the strain can also be improved to produce, derive, or derive Akkermansia muciniphila with better treatment or prevention effects on tumors, and the improvement methods include traditional methods and genetic engineering methods.

[0123] More specifically, the conventional method may be a physical method, a chemical method and / or a selection method.

[0124] More specifically, the physical method refers to the use of radiation or high pressure to induce mutations, thereby achieving strain improvement.

[0125] Preferably, the radiation method comprises ultraviolet radiation and / or X-ray radiation;

[0126] The high pressure method may be to expose the microorganisms to a high pressure environment to cause them to undergo adaptive changes.

[0127] More specifically, the chemical method refers to the use of chemical agents to induce mutation of bacterial strains.

[0128] Preferably, the chemical agent may be nitrite, acetylcholine and / or nitrogen mustard.

[0129] More specifically, the selection method refers to the process of selecting microorganisms with excellent traits for reproduction, thereby achieving strain improvement.

[0130] Preferably, the selection method is used to improve the metabolic pathway and / or growth conditions of the microorganism to obtain Akkermansia muciniphila with better tumor treatment or prevention effect.

[0131] 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 tumors.

[0132] 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 tumors.

[0133] 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 tumors.

[0134] More specifically, the gene editing refers to the use of technical means to accurately modify the target gene sequence to achieve the regulation of microbial traits in order to obtain strains with better effects in treating or preventing tumors.

[0135] More specifically, the synthetic biology refers to the use of chemical synthesis machine technology to construct new gene sequences and introduce them into microorganisms to achieve regulation of microbial traits in order to obtain strains with better effects in treating or preventing tumors.

[0136] The present invention also provides products containing Akkermansia muciniphila produced, derived or derived based on the above application.

[0137] Specifically, a drug for treating or preventing tumors, the drug comprising:

[0138] (1) The fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Akkermansia muciniphila produced, derived or derived;

[0139] (2) Pharmaceutically acceptable excipients.

[0140] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of preservatives, antioxidants, buffers, excipients, diluents, wetting agents, emulsifiers, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, flavors, sweeteners and stabilizers.

[0141] Preferably, the pharmaceutically acceptable excipient is at least one selected from gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, lactose, mannose, starch, fine crystalline cellulose, water, syrup, methylcellulose, mineral oil, propyl hydroxybenzoate, talc, magnesium stearate, polyvinyl pyrrolidone, cellulose and methyl hydroxybenzoate.

[0142] More specifically, the dosage form of the drug can be an oral preparation or an injection preparation.

[0143] Preferably, the oral preparation can be a tablet, powder, capsule, granule, pill, powder, paste, solid beverage or oral liquid.

[0144] More specifically, the drug comprises other drugs for treating tumors.

[0145] Specifically, a food for treating or preventing tumors, the food comprising:

[0146] (1) The fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Akkermansia muciniphila produced, derived or derived;

[0147] (2) Food-friendly excipients.

[0148] More specifically, the food-acceptable auxiliary material is selected from one or a combination of two or more of an emulsifier, a suspension stabilizer, a flavor, a wetting agent, an excipient, a preservative, a lubricant, a sweetener and a diluent.

[0149] Preferably, the food-acceptable auxiliary material is at least one selected from calcium silicate, fine crystalline cellulose, sucrose, sorbitol, mineral oil, starch, propyl hydroxybenzoate, talc, magnesium stearate, gum arabic, calcium phosphate, alginate, lactose, glucose, gelatin, polyvinyl pyrrolidone, syrup, cellulose, water, methylcellulose, methyl hydroxybenzoate and mannose.

[0150] More specifically, the food includes but is not limited to: chocolate, solid beverages, liquid beverages, ice cream, hard candies, crisp candies, caramel candies, jelly candies, toffee candies, gum-based candies, aerated candies, compressed candies, jellies, gel particles and / or crystal balls.

[0151] More specifically, the food includes but is not limited to: chocolate, solid beverages, liquid beverages, ice cream, hard candies, crisp candies, caramel candies, jelly candies, toffee candies, gum-based candies, aerated candies, compressed candies, jellies, gel particles and / or crystal balls.

[0152] Specifically, a health product for treating or preventing tumors, the health product comprising:

[0153] (1) The fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria of Akkermansia muciniphila produced, derived or derived;

[0154] (2) Acceptable excipients in health products.

[0155] More specifically, the auxiliary material is selected from one or a combination of two or more of a sweetener, a filler, a stabilizer, a pigment, a solvent, a flavoring agent, and a capsule shell material.

[0156] Preferably, the sweetener is selected from at least one of natural sweeteners and artificial sweeteners;

[0157] The filler is selected from at least one of starch, corn flour and grapes;

[0158] The stabilizer is selected from at least one of an antioxidant and a preservative;

[0159] The pigment is selected from at least one of natural pigment and artificial pigment;

[0160] The solvent is selected from at least one of water, alcohol, glycerol and ethanol;

[0161] The flavoring agent is selected from at least one of natural flavors and artificial flavors;

[0162] The capsule shell material is selected from at least one of gelatin, hydroxypropyl methylcellulose and polyvinyl alcohol.

[0163] Specifically, in terms of active dose, the viable count of Akkermansia muciniphila produced, derived, or derived is not less than 1×10 8 CFU / g or 1×10 8 CFU / mL.

[0164] 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.

[0165] More preferably, the viable count of Akkermansia muciniphila is 1×10 9 -1×10 12 CFU / g or 1×10 9 -1×10 12 CFU / 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.

[0166] Specifically, the food raw material for treating or preventing tumors is the produced, derived, or derived Akkermansia muciniphila fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria.

[0167] Specifically, the drug raw material for treating or preventing tumors is the produced, derived, or derived Akkermansia muciniphila fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria.

[0168] Specifically, the industrial raw materials used for treating or preventing tumors are produced, derived, and derived Akkermansia muciniphila fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria and / or dead bacteria.

[0169] The beneficial effects of the present invention are:

[0170] The present invention discloses for the first time the application of Akkermansia muciniphila (AKK PROBIO, with a deposit number of CGMCC No. 20955) in preventing colorectal cancer, and verifies through experiments that it has an obvious regulatory effect on colorectal cancer, can alleviate the increase of spleen and liver indexes and the decrease of body weight induced by AOM / DSS, and effectively reduce the number of colorectal cancer tumors and the degree of rectal tissue lesions; can upregulate the mRNA expression levels of caspase-9, Bid and Bim in colon tissue, reduce the degree of intestinal inflammation, and plays an important role in the occurrence and development of colorectal cancer.

[0171] Collection Instructions

[0172] Strain name: AKK PROBIO;

[0173] Deposit number: CGMCC No.20955;

[0174] Taxonomic nomenclature: Akkermansia muciniphila;

[0175] Deposit date: October 26, 2020;

[0176] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0177] Abbreviation of depository unit: CGMCC;

[0178] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0179] Figure 1The figure is a statistical chart of the changes in body weight of mice in each group.

[0180] Figure 2 The figure is a statistical chart of the colon length of each group of mice.

[0181] Figure 3 Statistical graph of the ratio of colon weight to length (colon weight / length) of each group of mice.

[0182] Figure 4 The figure is a statistical chart of the tumor number of mice in each group.

[0183] Figure 5 is the spleen index of mice in each group.

[0184] Figure 6 is the liver index (Liver index) of mice in each group.

[0185] Figure 7 is the kidney index (Kidney index) of mice in each group.

[0186] Figure 8 The rectal tissue pathological images of normal group mice.

[0187] Fig. 9 These are the rectal tissue pathology images of mice in the model group.

[0188] Fig.10 These are the rectal tissue pathology images of mice in the aspirin-treated group.

[0189] Fig.11 These are the rectal tissue pathology images of mice in the low-dose AKK PROBIO treatment group.

[0190] Fig.12 These are the rectal tissue pathology images of mice in the high-dose AKK PROBIO treatment group.

[0191] Fig.13 is the content of interleukin 1β (IL-1β) in the serum of mice in each group.

[0192] Fig.14 is the content of interleukin 6 (IL-6) in the serum of mice in each group.

[0193] Fig.15 is the content of interferon-γ (IFN-γ) in the serum of mice in each group.

[0194] Fig.16 is the content of inducible NO synthase (iNOS) in the serum of mice in each group.

[0195] Fig.17 is the content of nuclear factor κB (NF-κB) in the serum of mice in each group.

[0196] Fig.18 is the relative expression level of p50 mRNA, a gene related to the inflammatory pathway in the colon tissue of mice in each group.

[0197] Fig.19 is the relative expression level of p52 mRNA, a gene related to the inflammatory pathway in the colon tissue of each group of mice.

[0198] Fig. 20 is the relative expression level of p65 mRNA, a gene related to the inflammatory pathway in the colon tissue of each group of mice.

[0199] Fig.21 The relative expression levels of IκBβ mRNA, a gene related to the inflammatory pathway in the colon tissue of mice in each group.

[0200] Fig. 22 is the relative expression level of caspase-9 mRNA in each group of mice.

[0201] Fig.23 is the relative expression level of Bid mRNA in each group of mice.

[0202] Fig.24 is the relative expression level of Bim mRNA in each group of mice.

[0203] In the figure above, Normal is the normal group, Control is the AOM / DSS-induced colorectal cancer model group, Aspirin is the aspirin (67 mg / kg) treatment group, and AKK PROBIO-L (AKKP-L) is the low-dose AKK PROBIO treatment group (10 8 CFU / mouse), AKK PROBIO-H (AKKP-H) is the high-dose AKK PROBIO treatment group (10 9 CFU / each).

[0204] In the above figure, different letters in ac indicate significant differences according to the Duncan test (p<0.05). DETAILED DESCRIPTION

[0205] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0206] Interleukin 1β (IL-1β): It is produced by activated macrophages in the form of proprotein and processed into active form by caspase-1 (CASP1 / ICE). IL-1β is an important mediator of inflammatory response and participates in a variety of cellular activities, including cell proliferation, differentiation and apoptosis.

[0207] Interleukin 6 (IL-6): It is an interleukin that can act as both a pro-inflammatory cytokine and an anti-inflammatory myokine. It is currently a commonly used indicator for detecting infectious diseases in clinical practice.

[0208] Interferon-γ (IFN-γ): is a cytokine, a protein produced by T lymphocytes, natural killer cells and some other immune cells. IFN-γ plays an important role in the immune system, enhancing the body's immunity and resisting the invasion of pathogens.

[0209] Inducible NO synthase (iNOS): It is a type of enzyme that uses nitric oxide's oxidative stress (free radicals) to assist macrophages in fighting pathogens in the immune system. iNOS is a messenger molecule that can produce nitric oxide (NO). When DSS causes inflammation in the body, a large amount of NO expression will aggravate the inflammation and promote the high expression of iNOS.

[0210] Nuclear factor κB (NF-κB): Participates in the response of cells to external stimuli, such as cytokines, radiation, heavy metals, viruses, etc. NF-κB plays a key role in the process of cell inflammation and immune response. NF-κB is composed of five transcription factors, which regulate the expression of various genes involved in a variety of biological processes. These processes include inflammation, cell development and differentiation, cell cycle progression, cell migration, etc.

[0211] The Akkermansia muciniphila in the present invention is AKK PROBIO, abbreviated as AKKP.

[0212] In the following examples, the AOM / DSS mouse model established by combining the mutagen azoxymethane (AOM) with the inflammatory agent dextran sodium sulfate (DSS) can simulate the whole process of the mouse colon from normal mucosa to inflammation, and then to tumor. The model tumor location is mostly located in the distal colon, first growing in a polyp-like shape, and its pathological characteristics are similar to those of human CRC (Snider AJ, Bialkowska AB, Ghaleb AM, et al. Murine model for colitis-associated cancer of the colon [J]. Methods Mol Biol, 2016, 1438: 245-254.), which can reflect the development pattern of human colon inflammation progressing to tumor.

[0213] Example 1

[0214] 1.1 Experimental Materials

[0215] Azoxymethane, purchased from Sigma-Aldrich, catalog number A5486;

[0216] Dextran sulfate sodium, purchased from MP Biomedicals, catalog number 02160110-CF;

[0217] Enteric-coated aspirin tablets were purchased from Bayer Healthcare Co., Ltd., catalog number 83141248;

[0218] RNase-Free water, purchased from Beijing Solebow Technology Co., Ltd.;

[0219] qPCR SYBR Green Master Mix was purchased from Shanghai Yisheng Biotechnology Co., Ltd.;

[0220] Trizol Reagent and RevertAid First Strand cDNA Synthesis Kit were purchased from ThermoFisher Scientific;

[0221] Interleukin 1β (IL-1β) ELISA kit, Shanghai ELISA Biotechnology Co., Ltd., catalog number ml001814;

[0222] Interleukin 6 (IL-6) ELISA kit, Shanghai ELISA Biotechnology Co., Ltd., catalog number ml002293;

[0223] Interferon-γ (IFN-γ) ELISA kit, Shanghai ELISA Biotechnology Co., Ltd., catalog number ml002277;

[0224] Inducible NO synthase (iNOS) ELISA kit, Shanghai ELISA Biotechnology Co., Ltd., catalog number ml001216;

[0225] Nuclear factor κB (NF-κB) ELISA kit, Shanghai ELISA Biotechnology Co., Ltd., catalog number ml000331;

[0226] The remaining reagents were domestically produced or analytically pure.

[0227] 1.2 Experimental instruments

[0228] 6D45415 upright microscope, Olympus Instrument Co., Ltd., Japan;

[0229] Bioprep-24 biological sample homogenizer, Nano-300 micro-spectrophotometer, Hangzhou Aosheng Instrument Co., Ltd.;

[0230] A200 gradient PCR instrument, Hangzhou Langji Scientific Instrument Co., Ltd.;

[0231] VLBL0TD1 Multi-function Microplate Reader, StepOnePlus Real-Time PCR System, Thermo Fisher Scientific (Suzhou) Co., Ltd.

[0232] 1.3 Experimental methods

[0233] 1.3.1 Animal grouping and treatment

[0234] SPF male C57BL / 6 mice, 6 weeks old, were purchased from Chongqing Labite Biotechnology Co., Ltd. They were kept in a standardized laboratory with a room temperature of 25±2℃, a relative humidity of 50±5%, and 12h light / 12h dark. After 1 week of adaptive feeding, they were randomly divided into 5 groups: normal group, model group, aspirin positive control group, AKK PROBIO low dose (AKKP-L) group and AKK PROBIO high dose (AKKP-H) group, with 8 mice in each group.

[0235] During the experiment, mice were allowed to eat and drink freely. On the first day of the experiment, the model group, aspirin group, AKKPROBIO low-dose group and AKK PROBIO high-dose group were injected intraperitoneally with 10 mg / kg azoxymethane (AOM), and were fed with 2.5% dextran sodium sulfate (DSS) aqueous solution at the 2nd, 5th and 8th weeks, respectively.

[0236] The mice in the normal group and the model group were gavaged with sterile saline every day; the mice in the aspirin group were gavaged with 67 mg / kg aspirin solution; the mice in the AKK PROBIO low-dose group and the AKK PROBIO high-dose group were gavaged with 1×10 8 CFU / cell and 1×10 9 CFU / mouse was administered by gavage for 10 weeks.

[0237] During the experiment, the diet, activity, spirit, hair condition and feces of each group of mice were observed, and the weight of the mice was weighed and recorded every week. After the experiment, blood was collected from the eyeballs and the mice were killed by spinal dissection. The large intestine, spleen, liver and kidney tissues of the mice were dissected and tested.

[0238] 1.3.2 Detection indicators

[0239] 1.3.2.1 Determination of organ indicators

[0240] After the mice were dissected, the large intestine, spleen, liver, and kidney tissues were removed and weighed, and the mouse organ index was calculated using the formula (organ index = liver weight (mg) / body weight (g)). At the same time, the colorectal length of the mice was measured and the number of colorectal tumors in the mice was recorded.

[0241] 1.3.2.2 Pathological observation of rectal tissue

[0242] The mouse rectum was fixed in tissue fixative for 24 hours, then transferred to 95% ethanol for dehydration, and then placed in xylene to displace the alcohol in the tissue to make the rectal tissue transparent. It was then embedded in paraffin, sliced ​​with a microtome, stained with H&E and fixed to make mouse rectal pathological tissue sections with a thickness of 2-3 μm, and observed under an upright microscope.

[0243] 1.3.2.3 Number of colorectal tumors

[0244] After the mice were autopsied, the number of colorectal tumors in the mice was recorded.

[0245] 1.3.2.4 Determination of levels of inflammatory factors in mouse serum

[0246] Whole blood was collected from the eyeball, centrifuged at 4000 r / min, 4°C for 10 min, and the upper serum was separated. According to the instructions of the ELISA kit, the levels of IL-1β, IL-6, IFN-γ, iNOS and NF-κB in mouse serum were determined.

[0247] 1.3.2.5 Determination of the expression levels of inflammation- and apoptosis-related genes in mouse colon tissue

[0248] (1) Take 50 mg of colon tissue, wash it with physiological saline, and place it in a homogenizer tube containing 6 zirconium oxide beads. Add 500 μL Trizol reagent to extract RNA from the tissue. The purity and concentration of RNA are measured by micro-spectrophotometer. A260 / A280 is an indicator value for nucleic acid detection. A ratio between 1.8-2.0 indicates that the RNA purity is high and subsequent tests can be carried out.

[0249] (2) After generating cDNA by reverse transcription, prepare a reaction system containing 1 μL cDNA. The remaining reagents include 10 μL qPCRSYBR Green Master Mix, 7 μL sterile distilled water, and 1 μL each of upstream and downstream primers. qRT-PCR amplification reaction conditions: 95°C, 10 s, 60°C, 30 s, 40 cycles; 95°C, 15 s, 60°C, 60 s, 95°C, 15 s. With Eef2 as the internal reference gene, according to Formula 2 -△△Ct Calculate the relative mRNA expression of each target gene. Table 1 below shows the primer sequences used in this experiment.

[0250] Table 1.

[0251]

[0252] 1.4 Data Analysis

[0253] All the results in the experiment were expressed as mean ± standard deviation, and the one-way ANOVA in IBM SPSS27.0 software was used to test whether there were significant differences among the experimental results of each group at the p < 0.05 level.

[0254] 1.4.1 Effect of AKK PROBIO on the body weight of colorectal cancer model mice

[0255] The mice in the normal group were healthy, moved freely, had shiny black fur, ate and drank water normally, and had no diarrhea or blood in the stool. The mice in the other four groups had loose stools, rectal prolapse, blood in the stool, and weight loss. Figure 1 The figure shows the statistical graph of the weight changes of mice in each group. During the period of administration of 2.5% DSS, the weight of mice in each modeling group showed a downward trend, and after cessation, the weight of mice in each group showed a gradual increasing trend. At the end of the experimental period, the weight of mice in each modeling group was significantly lower than that of mice in the normal group, among which the weight loss of mice in the model group was the lowest, while the weight of mice in the aspirin and AKK PROBIO intervention groups recovered. Compared with the normal group, the weight of the remaining four groups of mice at the end of the experimental period was significantly different (p<0.05). The results show that AOM / DSS can cause weight loss in mice, and aspirin and AKK PROBIO intervention can alleviate the weight loss of mice to a certain extent.

[0256] 1.4.2 Effects of AKK PROBIO on the colorectum of colorectal cancer model mice

[0257] like Figure 2 The following table shows the changes in the colorectum of each group of mice. When the colorectum of mice is inflamed, the length of the colorectum of the mice is shortened to a certain extent. The colorectal inflammation can be analyzed from the colorectal length. Compared with the normal group, the colorectal length of the other four groups was shorter (P<0.05). Compared with the model group, the colorectal length of the aspirin group and the AKKP group was longer (P<0.05). As shown above, AKKP intervention can alleviate the shortening of the colorectum of mice.

[0258] like Figure 3 As shown in the figure, the ratio of the colorectal weight to the colorectal length of mice in the normal group was 2.81±0.34, the model group was 4.34±0.35, the aspirin group was 3.17±0.50, the AKKP-L group and the AKKP-H group were 3.18±0.31 and 2.97±0.18, respectively. The model group was significantly different from the normal group, the aspirin group, and the AKKP group (p<0.05). This shows that AKKP intervention can reduce the ratio of the colorectal weight to the colorectal length of mice.

[0259] The experimental results are as follows Figure 4 As shown in the figure, compared with the normal group, the number of colorectal tumors in the other four groups was significantly higher (p<0.05), and the number of colorectal tumors in the normal group was 0. Compared with the model group, the number of colorectal tumors in the aspirin group and the AKK PROBIO group was lower (p<0.05). The results show that AKK PROBIO intervention can effectively reduce the number of intestinal tumors in mice.

[0260] 1.4.3 Effects of AKK PROBIO on spleen, liver and kidney indexes in colorectal cancer model mice

[0261] Organ index is one of the basic indicators in biomedical research. Figure 5-Figure 7 The following are statistical graphs of the spleen, liver, and kidney indexes of mice in each group. Compared with the normal group, the spleen weight and liver weight of mice in the model group increased significantly (p<0.05), and the difference was statistically significant. Compared with the model group, the spleen weight and liver weight of the aspirin group and the AKK PROBIO group were reduced, and the difference was statistically significant (p<0.05). The kidney tissue structure of the 5 groups of mice was normal, and there was no significant difference between the groups (p>0.05). It can be seen that AKK PROBIO can effectively alleviate the increase in spleen and liver indexes of colorectal cancer caused by AOM / DSS.

[0262] 1.4.4 Effect of AKK PROBIO on the pathological morphology of rectal tissue in colorectal cancer model mice

[0263] like Figure 8-Figure 12 As shown in the figure, the normal group mice had intact rectal mucosal epithelial cells, normal crypts, neatly arranged glands, and no ulcers. Severe erosion of the rectal mucosa was observed in the model group, almost all crypts were destroyed, goblet cells decreased sharply, inflammatory cell infiltration occurred, glandular disorder, and severe ulcers were observed. No obvious erosion was observed in the rectal mucosa of mice in the aspirin group, AKK PROBIO low-dose group, and AKK PROBIO high-dose group, the crypts were relatively intact, the glands were neatly arranged, and the goblet cells were relatively intact. Histological results showed that AKK PROBIO effectively reduced the histopathological damage of the rectum in AOM / DSS-induced mice.

[0264] 1.4.5 Effect of AKK PROBIO on serum inflammatory factor levels in colorectal cancer model mice

[0265] Cytokines are a class of small molecule proteins synthesized by immune cells and some non-immune cells, which can bind to corresponding receptors to regulate immune responses, mediate inflammation and participate in tissue repair. Neutrophil infiltration of colonic mucosa and macrophages secrete a large number of pro-inflammatory cytokines and inhibit the secretion of anti-inflammatory cytokines during inflammation, thereby exacerbating the inflammatory state. Figure 13-17 Shown are the results of measuring the levels of related proinflammatory cytokines IL-1β, IL-6, IFN-γ, iNOS and NF-κB in the serum of CRC model mice.

[0266] The IL-1β content in the serum of mice in the model group was the highest, and the IL-1β content in the serum of mice in the normal group, aspirin group, and AKKP group was lower than that in the model group, and there was a significant difference (P<0.05). This shows that AKKP can significantly reduce the level of IL-1β in the serum of CRC mice.

[0267] Compared with the normal group, the IL-6 content in the serum of the model group mice was the highest, and there was a significant difference between the two (P<0.05). The IL-6 content in the serum of the aspirin group and the AKKP group mice was reduced, but there was no significant difference between the groups (P>0.05). This shows that AKKP can significantly reduce the inflammatory factors in the serum of CRC mice.

[0268] Compared with the normal group, the IFN-γ content in the serum of mice in the model group was the highest, and there was a significant difference between the two (P<0.05). The IFN-γ content in the serum of mice in the aspirin group and AKKP group was lower than that in the model group (P<0.05), and the content in the AKKP-H group was similar to that in the normal group, with no significant difference between the groups (P>0.05).

[0269] The iNOS content in the serum of mice in the model group was the highest. The iNOS content in the serum of mice in the normal group, aspirin group and AKKP group was lower than that in the model group, and there were significant differences between them (P<0.05).

[0270] The NF-κB content in the serum of mice in the model group was the highest. The NF-κB content in the serum of mice in the normal group, aspirin group and AKKP group was lower than that in the model group (P<0.05), and the content in the AKKP-H group was similar to that in the aspirin group, with no significant difference among the groups (P>0.05).

[0271] 1.4.6 Effect of AKK PROBIO on mRNA expression levels of related factors in colon tissue of colorectal cancer model mice

[0272] 1.4.6.1 mRNA expression levels of inflammatory pathway-related factors in colon tissue

[0273] Members of the NF-κB family include RelA / p65, RelB, c-Rel, NF-κB1 (p50 / p105) and NF-κB2 (p52 / p100). Studies have shown that under the induction of AOM / DSS, the NF-κB signaling pathway of mice is activated, which is manifested as increased levels of inflammation in the body. Therefore, by inhibiting the NF-κB signaling pathway, mice can be protected from inflammatory stimulation and the occurrence and development of CRC can be intervened.

[0274] Figure 18-21 is the mRNA expression level of p50, p52, p65 and IκBβ.

[0275] The mRNA expression levels of p50 and IκBβ in the colon tissue of mice in the model group were the highest. The relative mRNA expression levels of p50 and IκBβ in the colon tissue of mice in the aspirin group and AKKP-H group were significantly decreased (P<0.05), and there was no significant difference in the relative expression levels of p50 and IκBβ in the colon tissue of mice in the high-concentration group and the normal group (P>0.05).

[0276] The mRNA expression levels of p52 and p65 in the colon tissue of mice in the model group were the highest. The relative mRNA expression levels of p52 and p65 in the colon tissue of mice in the normal group, aspirin group and AKKP group were significantly decreased (P<0.05), and there was no significant difference in the relative expression levels of p52 and p65 in the colon tissue of mice in the AKKP-H group and the aspirin group (P>0.05).

[0277] 1.4.6.2 mRNA expression levels of apoptosis pathway-related factors in colon tissue

[0278] Caspases are central to the apoptotic machinery, as they are both initiators (caspase-2, caspase-8, caspase-9, and caspase-10, which are primarily responsible for the initiation of the apoptotic pathway) and executioners (caspase-3, caspase-6, and caspase-7, which are responsible for the definitive cleavage of cellular components) of cell death. The intrinsic pathway is tightly regulated by the B-cell lymphoma 2 (Bcl-2) family of intracellular proteins. Bid is a pro-apoptotic member of the Bcl-2 family that is directly regulated by miR-20a in CRC cells to induce mitochondrial apoptosis. Bim-mediated intrinsic apoptosis regulates apoptosis in CRC cells.

[0279] Figure 22-Figure 24 The relative expression of caspase-9, Bid and Bim in the colon of mice in the model group was the lowest, and the relative expression of caspase-9, Bid and Bim in the colon tissue of mice in the normal group, aspirin group and AKK PROBIO group was significantly increased (p<0.05), and the mRNA expression in the AKK PROBIO group showed a concentration-dependent trend. With the increase of AKK PROBIO gavage concentration, the mRNA expression of caspase-9, Bid and Bim in the colon of mice in the AKK PROBIO high-dose group was significantly increased (p<0.05). This shows that gavage of AKK PROBIO to CRC mice can increase the mRNA expression of caspase-9, Bid and Bim in colon tissue, thereby reducing the degree of intestinal damage.

[0280] In summary, AKK PROBIO has a significant regulatory effect on mouse colorectal cancer. It can alleviate the weight loss, shortened colorectal length, and increased spleen and liver indexes induced by AOM / DSS in mice to a certain extent, effectively reducing the number of tumors in CRC mice and the degree of damage to rectal tissues; it can significantly reduce the expression levels of proinflammatory factors IL-1β, IL-6, IFN-γ, iNOS, and NF-κB in mouse serum; at the gene level, it can significantly downregulate the mRNA expression levels of p50, p52, p65, and IκBβ in colon tissues, and upregulate the mRNA expression levels of caspase-9, Bid, and Bim in colon tissues, reduce the degree of intestinal inflammation, and affect the occurrence and development of colorectal cancer. AKK PROBIO has a good intervention effect on colorectal cancer.

[0281] Comparative Example

[0282] Referring to the experimental method in Example 1, the strain was replaced with the ATCC standard strain (ATCC BAA 835). The dose of ATCC standard strain group mice was 1×109 CFU / piece, and the rest were the same as in Example 1.

[0283] The experimental results are shown in the following table:

[0284] Table 2.

[0285] Group Number of tumors ATCC standard strain group 10

[0286] As can be seen from the above table, the number of colorectal tumors in the ATCC standard strain is less than that in the model group, but the effect is not as good as that of the AKK PROBIO strain of the present invention.

[0287] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0288] Application Example 1: AKK PROBIO live bacteria preparation method

[0289] AKK PROBIO live bacteria culture method

[0290] (1) Culture medium

[0291] Solid plate culture: BHI (Brain Heart Infusion, OXOID, Catalog No.: CM1032B)) + 5% sheep blood

[0292] 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.

[0293] (2) Culture conditions

[0294] Culture temperature: 37°C;

[0295] Culture conditions: completely anaerobic;

[0296] Liquid fermentation broth culture: BHI;

[0297] Culture temperature: 37°C;

[0298] Culture conditions: Completely anaerobic culture on 8 layers of gauze.

[0299] (3) Expansion culture of bacterial strains

[0300] 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.

[0301] 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.

[0302] 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.

[0303] (4) Fermentation tank culture

[0304] 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.

[0305] Application Example 2: AKK PROBIO dead bacteria preparation method

[0306] The wet bacteria prepared in Example 1 are killed by chemical or physical methods to obtain dead bacteria;

[0307] The chemical method may be gas sterilization and / or liquid sterilization;

[0308] Specifically, the gas sterilization may be ozone, ethylene oxide, formaldehyde, propylene glycol, glycerin or peracetic acid vapor;

[0309] The liquid sterilization agent is a chemical reagent, such as ethanol;

[0310] The physical method may be heat sterilization, light sterilization and / or microwave sterilization;

[0311] Specifically, the thermal sterilization method may be a burning method, a dry baking method, a boiling method and / or a pressure steam sterilization method;

[0312] The light sterilization can be sunlight exposure, ultraviolet irradiation and / or ionizing radiation.

[0313] Application Example 3: An oral medicine containing AKK PROBIO

[0314] 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 freeze drying, spray drying, tableting, etc.;

[0315] 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.

[0316] Application Example 4: A yogurt containing AKK PROBIO.

[0317] Raw materials: wet bacteria prepared in Example 1, milk, yogurt, soy milk or milk powder (fresh), yogurt starter (Lactobacillus bulgaricus and Streptococcus thermophilus);

[0318] Take milk preparation as an example:

[0319] Mix fresh milk, yogurt starter and wet bacteria in a certain proportion, stir evenly, cover with plastic wrap and ferment in a warm place (preferably 30-40℃) for 6-8 hours (or overnight), and refrigerate after fermentation.

Claims

1. Akkermansia muciniphila The use of the compound in the preparation of a drug or a pharmaceutical raw material for treating and / or assisting in the treatment of colorectal cancer is characterized in that: The Akkermansia muciniphila The deposit number is CGMCC No.20955; The drug or pharmaceutical raw material 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 an oral preparation.

3. The use according to claim 1, characterized in that: The drug or pharmaceutical raw material Akkermansia muciniphila The number of viable bacteria is 1×10 8 -1×10 12 CFU / g or 1×10 8 -1×10 12 CFU / mL.

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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