Akkermansia muciniphila, and product and use thereof

Abstract

Provided are an Akkermansia muciniphila strain, and a product and a use thereof. The Akkermansia muciniphila strain is AKK PROBIO, and the preservation number thereof is CGMCC No. 20955. The AKK PROBIO has good tolerance, high safety, a wide range of indications and a good treatment effect, and can prevent and treat diseases such as colitis, colorectal cancer, Alzheimer's disease, and gouty arthritis.

Description

Akkermansia muciniphila and its products and applications

[0001] This invention claims priority to the Chinese patent application filed with the China Patent Office on August 18, 2023, with application number 2023110476140 and invention name “A Akkermansia muciniphila and its products and applications”, the entire contents of which are incorporated by reference into the application. Technical Field

[0002] The invention belongs to the field of microorganisms, and particularly relates to Akkermansia muciniphila and products and applications thereof. Background Art

[0003] The sum of all microbial life in or on a specific part of the human body is called the human microbiome. DNA sequencing technology has facilitated a deeper analysis of the microbiome of the intestine, skin, urogenital tract, and lungs, revealing the role of microorganisms that reside in symbiosis with the host's mucosal surfaces. The composition and activity of the microbiome have significant metabolic, nutritional, and immune effects on the host. The microbiome evolves within the host from birth, constantly fine-tuning to maintain a steady-state balance with the host's immune system. This evolution is influenced by host factors (such as adaptive and innate immune responses), external factors (such as diet, drug and toxin exposure), and disease.

[0004] The types and quantities of gastrointestinal microorganisms occupy a large proportion of the human body, of which approximately 100 trillion microorganisms reside in the intestine. These microorganisms are highly adapted to survive in a complex community structure, and the microbiome has large differences in different individuals. When patients with diseases (such as inflammatory bowel disease) are compared with healthy individuals, this variability is even more obvious, indicating that the imbalance of certain microorganisms in the microbiome (i.e., dysbiosis) may lead to abnormal inflammation and metabolic reactions. By regulating the composition of gastrointestinal microorganisms, some diseases can have a certain therapeutic effect. For example, certain probiotics such as Lactobacillus and Bifidobacterium have been used orally to treat respiratory inflammation in a mouse model.

[0005] Akkermansia muciniphila (A. muciniphila), abbreviated as AKK and sometimes also called Akkermansia muciniphila, is an oval, Gram-negative bacterium isolated from human feces. It is a commensal in the human gut and can survive on the mucins in the intestinal mucus layer. Commensal bacteria in the human body can provide beneficial effects in various ways, from aiding in food digestion to supporting a healthy immune system. As a commensal in the gut, Akkermansia muciniphila has been found to have multiple functions.

[0006] For example, Chinese patent application number CN202310386672.X discloses a tumor-preventing Akkermansia muciniphila product and its application. The Akkermansia muciniphila product comprises at least one or both of AM06 and AM02. Numerous experiments have demonstrated that live, inactivated, and cell lysates of Akkermansia muciniphila have tumor-preventing effects, and that, when combined with other immune checkpoint inhibitors, such as PD-1 or CTLA-4 antibodies, the strain significantly enhances the anti-tumor effects. However, the strain's tumor-suppressing effect is modest, with a low tumor inhibition rate.

[0007] For example, the patent application number JP2022568622 discloses a new Akkermansia muciniphila AK32 strain and a composition containing the strain for preventing or treating intestinal damage. The Akkermansia muciniphila AK32 strain can not only increase the height of the intestinal crypts and the number of goblet cells in animal models, but also promote the expression of genes related to intestinal epithelial cell proliferation, and is effective in the regeneration and growth of intestinal tissue and organoids. Therefore, it is effective for the production of food, medicine, intestinal regulation, probiotics, and probiotics. However, its actual improvement is low, and its scope of clinical application is narrow.

[0008] The prior art also discloses a variety of other indications of Akkermansia muciniphila, such as obesity, diabetes, cardiovascular metabolic diseases, amyotrophic lateral sclerosis, cognitive dysfunction, etc. In some prior art, the mechanism of Akkermansia muciniphila in treating related diseases has also been studied. However, as mentioned above, the therapeutic effect of Akkermansia muciniphila in the prior art on related diseases still has a lot of room for improvement, and the continuous expansion of the germplasm resource library of related strains is also conducive to the subsequent research on the expansion of disease types. In addition, the functions of the strains in the prior art are relatively single. Screening out Akkermansia muciniphila that combines multiple therapeutic effects can make it better and more convenient to serve the clinic.

[0009] Summary of the Invention

[0010] To solve the above problems, the present invention obtained a strain of Akkermansia muciniphila by isolating and screening healthy human fecal specimens, conducted multiple performance tests on the strain, and carried out research in multiple application directions in order to expand the microbial germplasm resource library.

[0011] In one aspect, the present invention provides an Akkermansia muciniphila.

[0012] [Article 91 of the Detailed Rules, 05.09.2024] The Akkermansia muciniphila described is AKK PROBIO, with the deposit number CGMCC No.20955, and was deposited in the China General Microbiological Culture Collection Center on October 26, 2020.

[0013] The Akkermansia muciniphila of the present invention is isolated through a mucin culture medium.

[0014] The 16S sequence of Akkermansia muciniphila is SEQ ID NO. 1;

[0015] SEQ ID NO.1:

[0016] The Akkermansia muciniphila was cultured anaerobically in BHI medium at 37°C for 7 days, producing pale yellow, round colonies on plates with a moist, translucent surface and neat edges. Microscopic examination of the bacteria after anaerobically cultured in BHI medium at 37°C for 4 days revealed rod-shaped cells measuring 0.5-0.9 μm x 0.7-3.8 μm, arranged singly or in pairs, and Gram-negative.

[0017] The AKK PROBIO of the present invention is also referred to as AKK.pro or AKKP in some descriptions.

[0018] In another aspect, the present invention provides a method for culturing the aforementioned Akkermansia muciniphila.

[0019] The culture method at least comprises inoculating the aforementioned Akkermansia muciniphila into a culture medium for culture.

[0020] Specifically, the inoculation method includes but is not limited to: any one or more of the plate streaking method, slant inoculation method, pouring culture method, puncture inoculation method, and liquid inoculation method.

[0021] The inoculation may be performed at an inoculum size of 0.1% to 20%, or in some cases at a higher or lower inoculum size. Specifically, the inoculum size may be 1% to 20%, 2% to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 1% to 8%, 5% to 15%, 5% to 10%, 5% to 8%, 8% to 10%, 8% to 15%, 5% to 12%, or 2% to 7%.

[0022] Specifically, the culture medium can be a solid culture medium, a semi-solid culture medium or a liquid culture medium, more specifically, it can be any suitable culture medium type disclosed in the prior art, or it can be a culture medium that is further improved on the culture medium type disclosed in the prior art to improve the performance of the strain, or it can be a culture medium that is not disclosed in the prior art but can be used for the aforementioned Akkermansia muciniphila culture.

[0023] The culture medium includes, but is not limited to, one or more of brain heart infusion, mucin, amino acids or their salts, glucose or its derivatives, vitamins, calcium chloride, magnesium chloride, disodium hydrogen phosphate, sodium chloride, dipotassium hydrogen phosphate, sodium bicarbonate, sodium sulfide, yeast extract, beef extract, peptone, bile, and animal blood.

[0024] The animal blood includes but is not limited to cow blood, sheep blood, pig blood, chicken blood, and duck blood; preferably sheep blood.

[0025] Preferably, the culture medium is BHI culture medium, which can be in solid or liquid form, and can be a finished culture medium or a self-prepared culture medium, the ingredients of which can be adjusted as needed.

[0026] When the BHI medium is a solid medium, it includes agar and also includes, but is not limited to, any one or more of the following ingredients: brain heart infusion, peptone, dehydrated calf brain extract powder, dehydrated buffalo heart extract powder, glucose, and inorganic salts; preferably, the inorganic salts can be any one or any two of sodium chloride or disodium hydrogen phosphate.

[0027] When the BHI medium is a liquid culture medium, it includes but is not limited to any one or more of the following ingredients: brain heart infusion, peptone, dehydrated calf brain extract powder, dehydrated buffalo heart extract powder, glucose, and inorganic salts; preferably, the inorganic salts can be any one or any two of sodium chloride and disodium hydrogen phosphate.

[0028] The culture medium may further include mucin.

[0029] In some expressions, the culture medium may include a basal culture medium and additives. The basal culture medium may be a common general culture medium in the prior art, such as BHI culture medium. The additives may be other common or uncommon components in the art, such as animal blood. The volume ratio of animal blood to culture medium may be 1%-10%, specifically 1%-5%, 5%-10%, 1%-8%, 2%-10%, 4%-8%, 5%-8%, preferably 5%-10%, and more preferably 5% or 10%. In some embodiments of the present invention, the culture medium includes BHI culture medium and sheep blood. The volume ratio of sheep blood to BHI culture medium may be 1%-10%, specifically 1%-5%, 5%-10%, 1%-8%, 2%-10%, 4%-8%, 5%-8%, preferably 5%-10%, and more preferably 5% or 10%.

[0030] The culture conditions of Akkermansia muciniphila provided by the present invention include temperature and oxygen content, and in some cases may also include light, carbon dioxide content, pH, humidity, stirring conditions, ventilation conditions, shaking conditions, culture medium replacement conditions, culture days, resistance conditions, etc.

[0031] Preferably, the temperature in the culture conditions may be 35-40°C, specifically 35-39°C, 35-38°C, 35-37°C, 35-36°C, 36-40°C, 36-39°C, 36-38°C, 36-37°C, 37-40°C, 37-39°C, 37-38°C, 38-40°C, 38-39°C, 39-40°C, 36.5-38.5°C, 35.5-37.5°C, 37-38.5°C or 37-37.5°C. More preferably, it is 36-38°C, and further preferably 37°C.

[0032] Preferably, the culture is anaerobic culture, and the oxygen content in the culture conditions can be 0-8%, specifically 0-7%, 0-6%, 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 0-5.5%, 0-3.5%, 0-2.5%, 0-0.5%, 1%-8%, 1%-5%, 1%-2% or 0.5%-2%.

[0033] In still another aspect, the present invention provides the aforementioned preparation of Akkermansia muciniphila.

[0034] The preparation includes, but is not limited to, one or more of live bacteria, dead bacteria, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, freeze-dried powder, cell lysate, and secretions.

[0035] The preparation can be obtained by preparing the culture obtained by the aforementioned culture method.

[0036] As an example, the live bacteria can be living cells obtained from the aforementioned culture by centrifugation, filtration or other conventional means.

[0037] As an example, the dead bacteria may be inactivated bacteria, and the inactivation method includes but is not limited to: any one or more of high temperature inactivation, radiation inactivation or chemical inactivation.

[0038] Specifically, the high-temperature inactivation can be dry heat inactivation or wet heat inactivation; the radiation inactivation can be light radiation or ionizing radiation, the light radiation can be ultraviolet or infrared radiation, and the ionizing radiation includes but is not limited to microwave radiation; the chemical inactivation includes but is not limited to gas sterilization or liquid sterilization, the gas sterilization is carried out using a gaseous sterilant, such as ozone and formaldehyde, and the liquid sterilization is carried out using a liquid sterilant.

[0039] As an example, the fermentation broth can be obtained by the aforementioned culture method, which, according to general knowledge in the art, includes inoculating the bacterial strain into a liquid culture medium and culturing under certain conditions to obtain a liquid culture, namely the fermentation broth.

[0040] As an example, the fermentation broth supernatant or fermentation broth precipitate is obtained by centrifuging the fermentation broth. In some cases, the fermentation broth supernatant or fermentation broth precipitate includes Akkermansia muciniphila cells. The centrifugation conditions can be high-speed or low-speed, and can be room temperature, low temperature, or high temperature.

[0041] As an example, the lyophilized powder is obtained by freeze-drying the aforementioned culture solution. The lyophilized powder generally also includes a lyoprotectant. The lyoprotectant includes, but is not limited to, pH buffers, fillers, sugars, nonionic surfactants, ligands, and the like. The pH buffer includes, but is not limited to, any one or more of Tris, amino acids or their salts, citric acid or its salts, acetic acid or its salts. The filler includes, but is not limited to, any one or more of mannitol, glycine, and bovine serum albumin. The sugar can be a disaccharide, such as any one or more of sucrose or trehalose. The nonionic surfactant includes, but is not limited to, Tween, which can be Tween-20, Tween-60, Tween-80, and the like. The lyoprotectant can also include antioxidants, and the lyoprotectant can specifically include albumin, polyethylene glycol, and the like.

[0042] As an example, the cell lysis solution can be obtained by lysing the bacterial cells obtained from the aforementioned culture. The lysis can be physical or chemical. Physical lysis includes, but is not limited to, grinding, ultrasonic disruption, etc. Chemical lysis includes, but is not limited to, chemical reagent lysis and enzymatic hydrolysis, where the enzymatic hydrolysis can be performed using hydrolases or oxidases. Lysis can also be performed by increasing intracellular pressure to cause the cells to rupture.

[0043] As an example, the secretion may be a protein, a carbohydrate or a lipid.

[0044] In another aspect, the present invention provides the use of the aforementioned Akkermansia muciniphila or its preparation in the preparation of food, medicine, health care product, microbial agent, food additive, health care product additive or pharmaceutical raw material.

[0045] The medicine can be used for preventing or treating tumors or their complications.

[0046] The tumor includes but is not limited to solid tumors or non-solid tumors.

[0047] The solid tumors 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.

[0048] Preferably, the tumor includes but is not limited to any one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, and melanoma.

[0049] The medicine can also be used to prevent or treat obesity, diabetes, cardiovascular disease, inflammation, amyotrophic lateral sclerosis, Alzheimer's disease and other diseases or complications of the above diseases.

[0050] The inflammation includes, but is not limited to, acute inflammation or chronic inflammation, and includes, but is not limited to, any one or more of arthritis, gastroenteritis, hepatitis, fasciitis, nephritis, stomatitis, cystitis, pelvic inflammatory disease, cervicitis, keratitis, conjunctivitis, rhinitis, otitis media, pneumonia, and tracheitis.

[0051] In some categories, the drug can be used to prevent or treat cognitive impairment, such as Alzheimer's disease as described above.

[0052] The health care product can be used to maintain the balance of intestinal flora, maintain body shape, maintain blood pressure, maintain blood lipids, protect memory, resist oxidation, regulate metabolism, and regulate immunity.

[0053] In yet another aspect, the present invention provides a product comprising the aforementioned Akkermansia muciniphila or preparation.

[0054] The products include but are not limited to food, medicine, health care products, microbial agents, food additives, health care product additives or pharmaceutical raw materials.

[0055] Preferably, the drug is a medicine, which further comprises pharmaceutically acceptable excipients.

[0056] The pharmaceutically acceptable excipients include, but are not limited to, any one or more of excipients, stabilizers, diluents, adhesives, preservatives, lubricants, and antioxidants.

[0057] Preferably, the medicine is in the form of an internal dosage form or an external dosage form.

[0058] The internal dosage form includes but is not limited to any one of tablets, powders, granules, capsules, oral liquids, powders, pills, syrups, and effervescents.

[0059] The external dosage form includes but is not limited to any one of emulsions, ointments, sprays, liniments, suppositories, lotions, foams, and gels.

[0060] Preferably, the viable count of Akkermansia muciniphila in the drug is not less than 1×10 8 CFU / g or 1×10 8 CFU / mL.

[0061] More preferably, the viable count of Akkermansia muciniphila in the drug is not less than 1×10 9 CFU / g or 1×10 9 CFU / mL. Specifically, the viable count of Akkermansia muciniphila is not less than 1×10 9 CFU / g or 1×10 9 CFU / mL, if not less than 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 9CFU / g, etc. Other point values ​​within this numerical range can be selected.

[0062] Preferably, the viable count of Akkermansia muciniphila in the drug is 1×10 8 -1×10 12 CFU / g or 1×10 8 -1×10 12 CFU / mL. Specifically, it can be: 1×10 8 -1×10 9 CFU / g (CFU / mL), 1×10 8 -1×10 10 CFU / g (CFU / mL), 1×10 8 -1×10 11 CFU / g (CFU / mL), 1×10 9 -1×10 12 CFU / g (CFU / mL), 1×10 9 -1×10 11 CFU / g (CFU / mL), 1×10 9 -1×10 10 CFU / g (CFU / mL), 1×10 10 -1×10 12 CFU / g (CFU / mL), 1×10 10 -1×10 11 CFU / g (CFU / mL), 1×10 11 -1×10 12 CFU / g (CFU / mL), 5×10 8 -1×10 12 CFU / g (CFU / mL), 8×10 8 -1×10 12 CFU / g (CFU / mL), 7×10 9 -2×10 11 CFU / g (CFU / mL) or 9×10 10 -6×10 11 CFU / g (CFU / mL).

[0063] More preferably, the viable count of Akkermansia muciniphila in the drug is 1×10 9 -1×10 12 CFU / g or 1×10 9 -1×10 12 CFU / mL.

[0064] Those skilled in the art can combine the Akkermansia muciniphila or its preparation provided by the present invention with adjuvants in related fields according to actual applications. This can be achieved by routine screening by those skilled in the art.

[0065] Beneficial effects of the present invention:

[0066] The present invention obtains an Akkermansia muciniphila strain through screening, which is AKK PROBIO. The hydrophobicity of the strain reaches more than 30% after 60 minutes; the self-aggregation property tends to be stable after 20 hours and remains at about 52%; the strain has good gastrointestinal fluid tolerance and bile salt tolerance, with a survival rate of about 85% in gastric fluid after 240 minutes and a survival rate of more than 80% in intestinal fluid after 240 minutes.

[0067] AKK PROBIO has good safety and can prevent and treat a variety of diseases.

[0068] AKK PROBIO has a significant regulatory effect on colorectal cancer in mice. It can alleviate AOM / DSS-induced weight loss, shortened colorectal length, and increased spleen and liver indexes in mice to a certain extent, effectively reducing the number of tumors and the degree of rectal tissue damage in CRC mice; 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 genetic level, it can significantly downregulate the mRNA expression levels of p50, p52, p65 and IκBβ in colon tissue, and upregulate the mRNA expression levels of caspase-9, Bid and Bim in colon tissue, reducing the degree of intestinal inflammation and affecting the occurrence and development of colorectal cancer.

[0069] AKK PROBIO has been shown to alleviate Alzheimer's disease in mice. After four consecutive days of Morris water maze training, mice taking AKK PROBIO experienced a shorter escape latency and a significantly shorter path to the platform. By upregulating levels of the Aβ-degrading enzyme IDE and the anti-inflammatory factors IL-4 and IL-10, AKK PROBIO clears Aβ, thereby alleviating Alzheimer's disease in APP / PS1 double-transgenic mice.

[0070] In a gouty arthritis mouse model, AKK PROBIO can treat gouty arthritis at a low dose, and the therapeutic effect is better at a high dose.

[0071] The above results indicate that AKK PROBIO of the present invention has good tolerability, high safety, broad indications and good therapeutic effects, and has a promising prospect for clinical application.

[0072] Preservation Instructions:

[0073] Strain ID: AKK PROBIO;

[0074] Taxonomic nomenclature: Akkermansia muciniphila;

[0075] Deposit number: CGMCC No.20955;

[0076] Deposit date: October 26, 2020;

[0077] [Article 91, 05.09.2024] Depositary: China Center for the Collection of Common Microbiological Cultures;

[0078] [Article 91, 05.09.2024] Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] In the attached figure, the names of the groups refer to the following meanings:

[0080] Normal = normal group, Control = AOM / DSS-induced colorectal cancer model group, Aspirin = aspirin (67 mg / kg) treatment group, AKKP-L = low concentration of Akkermansia muciniphila treatment group (10 8 CFU / kg, AKKP-H = high concentration Akkermansia muciniphila treated group (10 9 CFU / kg), mAKK PRO=inactivated AKK PRO group, Blank=blank control group, DSS=DSS-induced ulcerative colitis model group (DSS model group).

[0081] Figure 1 shows the colony morphology of AKK PROBIO (top) and microscopic morphology (bottom).

[0082] FIG2 is a phylogenetic tree of AKK PROBIO (neighbor joining method, NJ method).

[0083] Figure 3 shows the effects of AKK PROBIO on physiological parameters of mice with DSS-induced ulcerative colitis, including: (a) body weight, (b) food intake, (c) DAI score, (d) liver index, and (e) colon length.

[0084] Figure 4 shows the effect of AKK PROBIO on the body weight of mice bearing colorectal cancer.

[0085] Figure 5 shows the effects of AKK PROBIO on colorectal tissues of mice with colorectal cancer, including colorectal length, colorectal weight to length ratio, and tumor number; different letters in the ad indicate significant differences according to the Duncan test (p<0.05).

[0086] Figure 6 shows the effects of AKK PROBIO on mouse organs, including spleen index, liver index, and kidney index. Different letters in the letters a and b indicate significant differences according to the Duncan test (p<0.05), and NS indicates no significant differences among the groups (p>0.05).

[0087] Figure 7 is a pathological section of mouse rectal tissue.

[0088] FIG8 shows the levels of cytokines (IL-1β, IL-6, IFN-γ, iNOS and NF-κB) in mouse serum. Different letters in ad indicate significant differences according to the Duncan test (P<0.05).

[0089] Figure 9 shows the relative mRNA expression levels of genes related to the inflammatory pathway in mouse colon tissue (p50, p52, p65, IκBβ). Different letters ac indicate significant differences according to the Duncan test (P<0.05).

[0090] Figure 10 shows the relative mRNA expression levels of genes related to the apoptosis pathway in mouse colon tissue cells (caspase-9, Bid, and Bim). Different letters ac indicate significant differences according to the Duncan test (P<0.05).

[0091] Figure 11 shows the hydrophobicity verification results of AKK PROBIO.

[0092] Figure 12 shows the self-aggregation verification results of AKK PROBIO.

[0093] FIG13 shows the survival rate of AKK PROBIO in artificial gastric fluid.

[0094] FIG14 shows the survival rate of AKK PROBIO in artificial intestinal fluid.

[0095] FIG15 shows the results of changes in body weight, food intake, and blood glucose in mice during the acute toxicity experiment.

[0096] FIG16 shows the test results of triglyceride (TG), cholesterol (TC), and bile acid (TBA) content in mice in an acute toxicity experiment.

[0097] FIG17 shows the results of glucose (GLU) level detection in mice in an acute toxicity experiment.

[0098] FIG18 shows the results of total protein (TP) level detection in mice in acute toxicity experiments.

[0099] FIG19 shows the results of the mouse alanine aminotransferase (GPT) enzyme activity test in the acute toxicity experiment.

[0100] FIG20 shows the results of the aspartate aminotransferase (GOT) activity test in mice in the acute toxicity experiment.

[0101] FIG21 shows the results of the detection of mouse creatinine (GRE) content in the acute toxicity experiment.

[0102] FIG22 shows the results of the detection of urea nitrogen (BUN) concentration in mice in the acute toxicity experiment.

[0103] FIG23 shows the pathological sections of the liver and kidney of mice in the acute toxicity experiment.

[0104] FIG24 shows the changes in blood glucose in mice subjected to a subchronic toxicity test.

[0105] FIG25 shows the bile acid levels in the liver of mice in the subchronic toxicity test.

[0106] FIG26 shows the results of Morris water maze test analysis of APP / PS1 double transgenic mice (Alzheimer's disease). DETAILED DESCRIPTION

[0107] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0108] Example 1 Strain Isolation

[0109] 1. Reagent Preparation

[0110] Mucin medium is prepared from solution 1 and solution 2, wherein:

[0111] Solution 1: 1.1 g calcium chloride, 1.0 g magnesium chloride, 5.3 g disodium hydrogen phosphate, 3 g sodium chloride, 4 g dipotassium hydrogen phosphate, 2 g sodium bicarbonate, 2.5 g sodium sulfide, dilute to 100 mL, and sterilize by high-pressure steam.

[0112] Solution 2: Mucin (Sigma, Product No. M1778, CAS No. 84082-64-4) solution.

[0113] Preparation of mucin liquid culture medium: Place 200 mL of sterile distilled water in an autoclaved wide-mouth bottle. Add 2 mL of solution 1 and 30-40 mL of solution 2 in a clean bench. Use an electric pipette to aspirate 5 mL of the liquid into 15 mL centrifuge tubes. Loosen the tube caps and place in an anaerobic jar for 3 days, then tighten the caps and store sealed at 4°C.

[0114] Preparation of mucin solid culture medium: Add 20 g / L agar powder to the mucin liquid culture medium; pour into sterile plates, 20 mL per plate; after cooling and solidification, place in a sterile plastic bag and store sealed at 4°C.

[0115] Pour brain heart infusion (BHI, OXOID, catalog number CM1032B) solid culture medium into a sterile dish, place it in a sterile plastic bag, and store it at 4°C in a sealed container.

[0116] 2. Sample dilution and enrichment culture

[0117] Collect 1-2 g of fecal specimens from healthy individuals and immediately inoculate them into 9 mL of normal saline, which is recorded as 10 -1 ; Continue diluting 5 gradients downward, record as 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 ; from 10 -2 Draw 500 μL from the dilution tube and add it to 4.5 mL of mucin liquid culture medium; -3 -10 -6 Mucin liquid culture medium was inoculated in the same manner, with 2 tubes in each gradient, and the enrichment tubes were placed in an anaerobic constant temperature incubator at 37°C for 3 days.

[0118] 3. Solid plate separation and culture

[0119] Select the turbid enrichment tube; select the relatively uniform, suspended, and free of precipitation, agglomeration, or filamentous material in the turbid liquid tube for separation and culture of the solid plate, dilute 10 -1 -10 -6 , respectively, spread on mucin solid culture medium and BHI solid culture medium, spread 3 plates for each dilution, place the solid plates in a 37℃ constant temperature anaerobic incubator and culture for 5 days.

[0120] IV. Physiological and biochemical identification and 16S preservation

[0121] (1) Pass Physiological and biochemical identification of anaerobic bacteria was performed using ANC identification cards (bioMérieux China, Cat. No. 21347). The results are shown in Table 1.

[0122] Table 1 Physiological and biochemical characteristics

[0123] Explanation of symbols: In the table, “+” represents positive; “-” represents negative.

[0124] (2) Take out the cultured solid plate and pick out milky white, sticky colonies with no bacteria around them, less than 1 mm in diameter, under strong light. Inoculate them into 9 mL of BHI liquid culture medium using aseptic operation and place them in an anaerobic constant temperature incubator at 37°C for 48 hours. Simultaneously perform 16S identification:

[0125] The identification primers were conventional primers, the forward primer was 27F (5'→3'): SEQ ID NO. 2, and the reverse primer was 1492R (5'→3'): SEQ ID NO. 3;

[0126] SEQ ID NO. 2: AGAGTTTGATCCTGGCTCA.

[0127] SEQ ID NO. 3: GGTTACCTTGTTACGACTT.

[0128] Identification PCR system: 27F primer solution 1 μL, 1492R primer solution 1 μL, 2× PCR MasterMix (premix) 12 μL, sterile water 10 μL, template 1 μL;

[0129] PCR reaction conditions: 95°C, 5 min; 30 cycles: 95°C, 30 s; 55°C, 30 s; 72°C, 2 min; 72°C, 10 min.

[0130] [Article 91, 05 / 09 / 2024] A new mucinophilic strain, AKK PROBIO, was screened and identified, with its 16S sequence as SEQ ID NO. 1. After comparison with NCBI, it was classified and named Akkermansia muciniphila. It was deposited with the China General Microbiological Culture Collection on October 26, 2020, under the accession number CGMCC No. 20955. Comparisons with some existing strains are shown in Table 2:

[0131] Table 2 Comparison results with some existing strains

[0132] A phylogenetic tree of 16S rDNA sequences of AKK PROBIO and related species is shown in Figure 2. The phylogenetic tree of 16S rDNA sequences of AKK PROBIO and related species was displayed using MEGA software using the neighbor-joining method. Similarity was calculated 1000 times. Only nodes with bootstrap values ​​greater than 50% are displayed in the figure. The superscript "T" indicates the type strain.

[0133] The fermentation broth was frozen and stored in 25% glycerol tubes.

[0134] AKK PROBIO was cultured anaerobically in BHI medium at 37°C for 7 days. Plate morphology revealed pale yellow, round colonies with moist, translucent surfaces and neat edges. Microscopic examination of AKK PROBIO in BHI medium at 37°C for 4 days revealed rod-shaped cells measuring 0.5-0.9 μm x 0.7-3.8 μm, arranged singly or in pairs, and Gram-negative. (See Figure 1).

[0135] Example 2 Cultivation of AKK PROBIO

[0136] (1) Using solid plate culture, the medium composition is: BHI + 5% sheep blood. The medium preparation method is as follows: weigh a certain volume of BHI powder required according to the finished product requirements; add agar powder at a ratio of 20g / L; dilute to the corresponding volume, and sterilize at 121°C for 20 minutes; after the sterilization is completed and cooled to 53°C (not hot to hold), add 5% sterile defibrinated sheep blood, shake well, and pour 15-20mL into the plate for use;

[0137] The culture temperature of AKK PROBIO was set at 37°C, and the culture conditions were completely anaerobic.

[0138] (2) Liquid culture (fermentation broth culture) was used, the culture medium was BHI, the culture temperature was 37°C, and the culture conditions were complete anaerobic culture with 8 layers of gauze, specifically including the following steps:

[0139] Level 1: Take a 2 mL glycerol tube and inoculate it into a 9 mL BHI test tube at 10% inoculum, and incubate anaerobically at 37°C for 24-48 hours;

[0140] Secondary: Take the first-stage fermentation test tube and inoculate it into a flask containing 90 mL of BHI at a 5% inoculum size. Incubate anaerobically at 37°C for 24 h.

[0141] 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% inoculum size. Incubate anaerobically at 37°C for 13 h and store in a 20% glycerol tube at -80°C.

[0142] Example 3 AKK PROBIO improves dextran sulfate sodium (DSS)-induced ulcerative colitis in mice

[0143] Animal Care: Male C57BL / 6J mice (6-10 weeks old, weighing 18-24 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. All procedures involving mice were performed in strict accordance with international animal care standards and approved by the Institutional Animal Care and Management Committee of Jilin Agricultural University. Mice were acclimated for 1 week and maintained at room temperature (25°C ± 1°C) under a 12-h light and 12-h dark cycle.

[0144] Animal experiment design: 5.0% (wt / vol) DSS (molecular weight 36,000-50,000) was added to the drinking water of mice for 3 days to induce colitis. Starting from the first day of DSS administration, Akkermansia muciniphila (live or inactivated, at a concentration of 1×10 10 CFU / mL) for 9 days. C57BL / 6J mice were randomly divided into four groups: control group, DSS model group (DSS), AKK PRO group, and inactivated AKK PRO group (mAKK PRO). Physiological indicators of mice in each group were observed.

[0145] Physiological indicators: Physiological indicators were used to assess the health status of mice. The individual scores of each mouse were combined to generate the disease activity index (DAI), including weight change, food intake, and fecal consistency. The scoring criteria are shown in Table 3. After the mice were euthanized, the colon length of each group of mice was measured, the liver weight of each group of mice was measured, and the immune organ index was calculated according to the following formula: Liver index = liver weight (mg) / body weight (g) [Wang, X., et al., Troxerutin Improves Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice. J Agric Food Chem, 2021. 69(9): p. 2729-2744.].

[0146] Table 3 Disease Activity Index (DAI) score table

[0147] Statistical analysis:

[0148] GraphPad was used for statistical analysis, and all data were expressed as mean ± standard deviation (SD). The statistical significance between two groups was determined by t-test, and multiple groups were compared by two-way analysis of variance (ANOVA). P < 0.05 indicated a significant difference.

[0149] Result analysis:

[0150] (1) Ingestion of Akkermansia muciniphila significantly alleviated the physiological damage of DSS-induced colitis in mice

[0151] To determine whether Akkermansia muciniphila has an ameliorating effect on colitis, acute colitis was induced with 5.0% DSS with or without the addition of Akkermansia muciniphila.

[0152] As shown in Figure 3:

[0153] Mice treated with both DSS and Akkermansia muciniphila showed less weight loss (a) and food intake (b) than mice in the model group, and had lower DAI scores (c);

[0154] Meanwhile, the increase in immune organ index and the decrease in colon length indicated increased inflammation. The results showed that the immune organ index increased significantly in the DSS group (P < 0.05), but decreased significantly after Akkermansia muciniphila treatment (d);

[0155] The colon length of mice in the DSS group was significantly decreased (P < 0.05), but it was significantly increased after the ingestion of Akkermansia muciniphila (P < 0.05) (e).

[0156] The above results indicate that Akkermansia muciniphila can significantly improve the physiological damage of colitis mice.

[0157] Example 4 Effect of AKK PROBIO on AOM / DSS-induced colorectal cancer in mice

[0158] The sources of some experimental reagents used in this example are as follows:

[0159] Azoxymethane (AOM) was purchased from Sigma-Aldrich; dextran sulfate sodium (DSS) was purchased from MP Biomedicals; enteric-coated aspirin tablets were purchased from Bayer HealthCare Co., Ltd.; ELISA kits for interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-γ (IFN-γ), inducible nitric oxide synthase (iNOS), and nuclear factor-κB (NF-κB) were purchased from Shanghai ELISA Biotechnology Co., Ltd.; RNase-free water was purchased from Beijing Solebao Technology Co., Ltd.; qPCR SYBR Green Master Mix was purchased from Shanghai Yisheng Biotechnology Co., Ltd.; Trizol Reagent and RevertAid First Strand cDNA Synthesis Kit were purchased from Thermo Fisher Scientific. All other reagents were domestically produced or of analytical grade.

[0160] Some of the instruments and equipment used in this example are as follows:

[0161] A 6D45415 upright microscope was used (Olympus Instruments, Japan); a Bioprep-24 biological sample homogenizer and a Nano-300 micro-volume spectrophotometer were used (Aosheng Instruments, Hangzhou); an A200 gradient PCR instrument was used (Longji Scientific Instruments, Hangzhou); and a VLBL0TD1 multifunctional microplate reader and a StepOnePlus Real-Time PCR System were used (Thermo Fisher Scientific, Suzhou).

[0162] Experimental animals and group treatment:

[0163] SPF male C57BL / 6 mice, 6 weeks old, were purchased from Chongqing Labite Biotechnology Co., Ltd. They were housed in a standardized laboratory at room temperature of 25±2℃, relative humidity of 50±5%, and 12h light / 12h dark. After one week of adaptive feeding, they were randomly divided into 5 groups: normal group, model group, aspirin positive control group, AKK PROBIO low concentration (AKKPL) group, and AKK PROBIO high concentration (AKKPH) group. During the experiment, mice had free access to food and water. On the first day of the experimental modeling, the model group, aspirin group, and AKKP group were intraperitoneally injected with AOM at 10 mg / kg, and fed with 2.5% DSS aqueous solution on the second, fifth, and eighth weeks, respectively. Mice in the normal and model groups were gavaged with sterile saline every day; mice in the aspirin group were gavaged with aspirin solution at 67 mg / kg; mice in the AKKPL group and AKKPH group were gavaged with 1×10 8 CFU / kg and 1×10 9 The mice were gavaged at a dose of 10 CFU / kg for 10 weeks. During the experiment, the mice in each group were observed for their diet, activity, mental state, coat condition, and fecal characteristics. Their weights were measured and recorded weekly. After the experiment, blood was drawn from the eyeballs and the mice were sacrificed by spinal dissection. The large intestine, spleen, liver, and kidney tissues were dissected and analyzed.

[0164] (1) Determination of organ indicators

[0165] After autopsy, the large intestine, spleen, liver, and kidney tissues were removed and weighed. The organ index (OI) was calculated using a formula. The length of the colorectum and the number of colorectal tumors were also measured. Organ index = organ weight (mg) / mouse body weight (g).

[0166] (2) Rectal histopathological observation

[0167] 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. The rectal tissue was then embedded in paraffin, sliced ​​with a microtome, stained with H&E, and fixed to prepare mouse rectal pathological tissue sections with a thickness of 2-3 μm, which were then observed under an upright microscope.

[0168] (3) Determination of inflammatory-related factor levels in mouse serum

[0169] Whole blood was collected from the eyeball and centrifuged at 4000 rpm and 4°C for 10 min to separate the upper serum layer. The levels of IL-1β, IL-6, IFN-γ, iNOS, and NF-κB in mouse serum were determined according to the ELISA kit instructions.

[0170] (4) Determination of expression levels of inflammation- and apoptosis-related genes in mouse colon tissue

[0171] About 50 mg of colon tissue was taken and washed with physiological saline, and then placed in a homogenate tube containing 6 zirconium oxide beads. 500 μL of Trizol reagent was added to extract RNA from the tissue. The purity and concentration of RNA were measured by a microspectrophotometer. A260 / A280 is an indicator value for nucleic acid detection. Its ratio between 1.8 and 2.0 indicates that the RNA purity is high and subsequent tests can be performed. Then, after generating cDNA by reverse transcription, a reaction system containing 1 μL of cDNA was prepared. The remaining reagents included 10 μL of qPCR SYBR Green Master Mix, 7 μL of sterile distilled water, and 1 μL each of upstream and downstream primers. qRT-PCR amplification reaction conditions: 95°C, 10s, 60°C, 30s, 40 cycles; 95°C, 15s, 60°C, 60s, 95°C, 15s. Eef2 was used as the internal reference gene, according to formula 2 -△△Ct Calculate the relative mRNA expression of each target gene. Table 4 shows the primer sequences used in this experiment.

[0172] Table 4 Primer sequences

[0173] In the table, "F" represents the forward primer and "R" represents the reverse primer.

[0174] (5) Data Analysis

[0175] All the results in the experiment were expressed as mean ± standard deviation, and the one-way ANOVA method in IBM SPSS27.0 software was used to test whether there were significant differences between the experimental results of each group at the P < 0.05 level.

[0176] (6) Experimental results

[0177] 6.1 Effect of AKK PROBIO on Body Weight in Colorectal Cancer Model Mice:

[0178] The mice in the normal group were healthy and mobile, with shiny dark fur. They ate and drank normally, and did not experience diarrhea or bloody stools. The mice in the other four groups experienced loose stools, rectal prolapse, bloody stools, and weight loss. Figure 4 shows a statistical chart of weight changes in each group of mice. During the administration of 2.5% DSS, the weight of the mice in each modeling group showed a downward trend. After the administration of DSS, the weight of the mice in each group gradually increased. At the end of the experimental period, the weight of the mice in each modeling group was significantly lower than that of the mice in the normal group. The model group had the lowest weight loss, while the weight of the mice in the aspirin and AKKP intervention groups recovered. Compared with the normal group, the weight of the mice in the other four groups at the end of the experimental period was significantly different (P < 0.05). The results show that AOM / DSS causes weight loss in mice, and aspirin and AKKP intervention can alleviate the weight loss in mice to a certain extent.

[0179] 6.2 Effects of AKK PROBIO on the Colorectum of Colorectal Cancer Model Mice:

[0180] Figure 5 shows the changes in the colorectum of each group of mice. When colorectal inflammation occurs in mice, the length of the colorectum shortens to a certain extent. Colorectal length can be used to analyze the extent of colorectal inflammation. 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 in mice.

[0181] The ratio of colorectal weight to colorectal length in the normal group was 2.81±0.34, in the model group it was 4.34±0.35, in the aspirin group it was 3.17±0.50, and in the AKKPL and AKKPH groups it was 3.18±0.31 and 2.97±0.18, respectively. The model group showed significant differences compared with the normal, aspirin, and AKKPH groups (P<0.05). This suggests that AKKP intervention can reduce the ratio of colorectal weight to colorectal length in mice.

[0182] Compared with the normal group, the other four groups had significantly more colorectal tumors (P < 0.05). Compared with the model group, the aspirin and AKKP groups had fewer colorectal tumors (P < 0.05). These results suggest that AKKP intervention can effectively reduce the number of intestinal tumors in mice.

[0183] 6.3 Effects of AKK PROBIO on the spleen, liver, and kidney indices of colorectal cancer model mice:

[0184] Organ index is one of the basic indicators of biomedical research and an important basis for research. As shown in Figure 6, it is a statistical graph of the spleen and liver indices of mice in each group. Compared with the normal group, the spleen weight and liver weight of mice in the model group were significantly increased (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 AKKP 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 AKKP can effectively alleviate the increase in spleen and liver indices of colorectal cancer caused by AOM / DSS.

[0185] 6.4 Effect of AKK PROBIO on Rectal Histopathological Morphology in Colorectal Cancer Model Mice

[0186] As shown in Figure 7, the rectal mucosal epithelial cells of the normal group mice were intact, the crypts were normal, the glands were neatly arranged, and there were no ulcers. Severe erosion of the rectal mucosa was observed in the model group, with almost all crypts destroyed, a sharp decrease in goblet cells, inflammatory cell infiltration, glandular disorder, and severe ulcers. No obvious erosion was observed in the rectal mucosa of mice in the aspirin, AKKPL, and AKKPH groups, with relatively intact crypts, neatly arranged glands, and relatively intact goblet cells. Histological results showed that AKKP effectively reduced the histopathological damage to the rectum in mice induced by AOM / DSS.

[0187] 6.5 Effect of AKK PROBIO on Serum Inflammatory Factor Levels in Colorectal Cancer Model Mice

[0188] Cytokines are a class of small molecule proteins synthesized by immune cells and certain non-immune cells. They can bind to corresponding receptors to regulate immune responses [Minxuan X, Sun Y, Dai X, et al. Fisetin attenuates high fat diet-triggered hepatic lipid accumulation: A mechanism involving liver inflammation overload associated TACE / TNF-αpathway[J]. Journal of Functional Foods, 2019, 53, 7-21.], mediate inflammation and participate in tissue repair [Bamias G, Arseneau KO, Cominelli F. Cytokines and Mucosal Immunity. Curr. Opin[J]. Gastroenterol, 2014, 30, 547–552.]. Neutrophil infiltration of the colonic mucosa and macrophages secretes a large amount of proinflammatory cytokines and inhibits the secretion of anti-inflammatory cytokines during inflammation, thereby exacerbating the inflammatory state [Shukla PK, Chaudhry KK, Mir H, et al. Chronic Ethanol Feeding Promotes Azoxymethane and Dextran Sulfate Sodium-Induced Colonic Tumorigenesis Potentially by Enhancing Mucosal Inflammation [J]. BMC Cancer 16, 2016.]. Figure 8 shows the results of the measurement of the levels of related proinflammatory cytokines IL-1β, IL-6, IFN-γ, iNOS, and NF-κB in the serum of CRC model mice.

[0189] As a pro-inflammatory factor, IL-1β can promote the activation and aggregation of inflammatory cells, increase the permeability of epithelial and endothelial cells, aggravate inflammation of the intestinal mucosa, and mediate hyperalgesia in inflammation [Park JS, Choi J, Hwang SH, et al. Cottonseed Oil Protects against Intestinal Inflammation in Dextran Sodium Sulfate-Induced Inflammatory Bowel Disease[J]. Med. Food 2019, 22, 672-679.]. The IL-1β content in the serum of mice in the model group was the highest, while 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.

[0190] IL-6 is a multifunctional inflammatory regulator. As the most representative member of the interleukin family, it was first discovered in leukocytes as a factor that transmits cell signals. It participates in inflammatory responses, immune regulation, and cancer development [Zeng Xinyu. Study on the Effect of Bifidobacterium Preparation on Postoperative Recovery of Elderly Colorectal Cancer Patients under ERAS Pathway [D]. Hunan Normal University, 2021.]. Compared with the normal group, the IL-6 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 IL-6 content in the serum of mice in the aspirin group and the AKKP group was reduced, but there was no significant difference between the groups (P>0.05). This shows that AKKP can significantly reduce inflammatory factors in the serum of CRC mice.

[0191] INF-γ is a dimeric glycoprotein that can severely affect the structure and function of intestinal epithelial cells and increase intestinal mucosal permeability [Zingoni A, Sornasse T, Cocks BG, et al. Cross-talk between activated human NK cells and CD4 T cells via OX40-OX40 ligand interactions. [J]. Immunol, 2004, 173: 3716-3724.]. Studies have shown that IFN-γ levels increase in intestinal inflammation, thereby playing an immunomodulatory role [Ferrier L, Mazelin L, Cenac N, et al. Stress-induced disruption of colonic epithelial barrier: role of interferon-gamma and myosin light chain kinase in mice [J]. Gastroenterology 125 795-804. 2003]. 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 groups (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 AKKPH group was similar to that in the normal group, with no significant difference between the groups (P>0.05).

[0192] 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 inflammation and promote the high expression of iNOS [Pan Y, Ning Y, Hu J, et al. The Preventive Effect of Lactobacillus plantarum ZS62 on DSS-Induced IBD by Regulating Oxidative Stress and the Immune Response [J]. Oxid Med Cell Longev, 2021.]. 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 compared with the model group (P < 0.05).

[0193] NF-κB is one of the indicators of the occurrence and development of colorectal cancer. It participates in the response of cells to stimulation. Its abnormally elevated level is associated with multiple factors such as inflammation, cancer, and autoimmune diseases, and can reflect the condition of CRC patients [Shi Xinpeng, Luo Xiaoyong, Li Chaoping, et al. Effects of cetuximab combined with chemotherapy on the short-term efficacy and serum NF-κB, EGFR, and HER-2 levels in patients with drug-resistant advanced colorectal cancer [J]. Practical Clinical Integration of Traditional Chinese and Western Medicine, 2021, 21(19): 92-93.]. 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 AKKPH group was similar to that in the aspirin group, with no significant difference between the groups (P>0.05).

[0194] 6.6 Effect of AKK PROBIO on mRNA Expression Levels of Related Factors in Colon Tissue of Colorectal Cancer Model Mice

[0195] 1) mRNA expression levels of inflammatory pathway-related factors in colon tissue

[0196] NF-κB is composed of five transcription factors that regulate the expression of various genes involved in a variety of biological processes. These processes include inflammation, cell development and differentiation, cell cycle progression, and cell migration [Wei H, Prabhu L, Hartley AV, et al. Gene Expression and Regulation in Mammalian Cells—Transcription from General Aspects. Intech Open; London, UK: 2018. Methylation of NF-κB and its Role in Gene Regulation; p. 291.]. 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 AOM / DSS-induced activation of the NF-κB signaling pathway in mice is manifested by increased levels of inflammation. Therefore, inhibiting the NF-κB signaling pathway could protect mice from inflammatory stimuli and intervene in the development and progression of CRC.

[0197] FIG9 shows the mRNA expression levels of p50, p52, p65, and IκBβ.

[0198] 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 AKKPH 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 between the high-concentration group and the normal group (P>0.05).

[0199] 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 between the AKKPH group and the aspirin group (P>0.05).

[0200] 2) mRNA expression levels of apoptosis pathway-related factors in colon tissue

[0201] The mechanism of apoptosis is complex and involves many signaling pathways. Apoptosis can be triggered in cells through extrinsic or intrinsic pathways mediated by caspases, which are mainly executed by a family of proteases called caspases (cysteine, aspartate-specific proteases) [Li J, Yuan J.Caspases in apoptosis and beyond. Oncogene. 2008; 27: 6194-6206.]. Caspases are at the core of the apoptosis mechanism because they are both initiators of cell death (caspase-2, -8, -9 and -10, which are primarily responsible for the start of the apoptosis pathway) and executioners (caspase-3, -6 and -7, which are responsible for the clear cleavage of cellular components) [Thomberry NA, Laxebnik Y.Caspases:enemies within. Science. 1998; 281: 1312-1316.]. The intrinsic pathway is closely regulated by the intracellular protein B-cell lymphoma 2 (Bcl-2) family. Bid is a pro-apoptotic member of the Bcl-2 family and has been found to be directly regulated by miR-20a in CRC cells, thereby inducing mitochondrial apoptosis [Huang G, Chen X, Cai Y, Wang X, Xing C. miR-20a-directed regulation of BID is associated with the TRAIL sensitivity in colorectal cancer. Oncol. Rep. 2017; 37: 571-578.]. Bim-mediated intrinsic apoptosis regulates apoptosis in CRC cells [Drury LJ, Wendt MK, Dwinell MB. CXCL12 chemokine expression and secretion regulates colorectal carcinoma cell anoikis through Bim-mediated intrinsic apoptosis. PLoS One. 2010; 5: e12895.].

[0202] Figure 10 shows the mRNA expression levels of caspase-9, Bid, and Bim. The relative mRNA expression levels of caspase-9, Bid, and Bim in the colon of mice in the model group were the lowest. The relative mRNA expression levels of caspase-9, Bid, and Bim in the colon tissue of mice in the normal, aspirin, and AKKP groups were significantly increased (P < 0.05). The mRNA expression levels in the AKKP group showed a concentration-dependent trend. With increasing AKKP concentrations, the mRNA expression levels of caspase-9, Bid, and Bim in the colon of mice in the AKKPH group were significantly increased (P < 0.05). This suggests that oral administration of AKKP to CRC mice can increase the mRNA expression of caspase-9, Bid, and Bim in colon tissue, thereby reducing the degree of intestinal damage.

[0203] These results demonstrate that AKK PROBIO has a significant regulatory effect on colorectal cancer in mice. It can, to a certain extent, alleviate AOM / DSS-induced weight loss, shortened colorectal length, and increased spleen and liver indexes in mice, effectively reducing the number of tumors and the extent of rectal tissue damage in CRC mice. It can also significantly reduce the expression levels of proinflammatory cytokines IL-1β, IL-6, IFN-γ, iNOS, and NF-κB in mouse serum. At the genetic level, it can significantly downregulate the mRNA expression levels of p50, p52, p65, and IκBβ in colon tissue, while upregulating the mRNA expression levels of caspase-9, Bid, and Bim in colon tissue, reducing the degree of intestinal inflammation and impacting the development and progression of colorectal cancer. In summary, AKK PROBIO has a significant interventional effect on colorectal cancer.

[0204] Example 5 Safety Evaluation of AKK PROBIO (AKK.pro)

[0205] (1) Hydrophobicity

[0206] Based on the ability of bacteria to adhere to hydrocarbons, the cell surface hydrophobicity of the strain AKK PROBIO was determined. The bacterial adhesion test to hydrocarbons (BATH) was performed using xylene with some modifications. Specifically, the strain was cultured overnight at 37°C in BHI medium. The cells were collected by centrifugation (4°C; 8000rpm; 10min), washed twice, and resuspended in phosphate buffered saline (PBS, pH 7), and the absorbance (OD600 nm) was adjusted to 0.5 to standardize the number of bacteria. Then, an equal volume of xylene was added and vortexed for 5min to fully mix the two-phase system. After incubation at room temperature for 15, 30 and 60 minutes, the aqueous phase was carefully removed and its absorbance at 600nm was measured using a spectrophotometer. The affinity (hydrophobicity) for hydrocarbons was calculated using the following formula:

[0207] Among them, P0 and P1 are the absorbance values ​​before and after extraction with hydrocarbons, respectively.

[0208] The results are shown in FIG11 , which show that the hydrophobicity of AKK PROBIO increases with time, reaching more than 30% at 60 min.

[0209] (2) Self-aggregation

[0210] Prepare a cell suspension of strain AKK PROBIO as described above and incubate at 37°C. Adjust the absorbance (OD600nm) to 0.5. Then, incubate the bacterial suspension at 37°C and measure the absorbance at 600nm at 4, 8, 12, 16, 20, and 24 hours. Calculate the percentage of autoaggregation using the following formula:

[0211] The results are shown in FIG12 , which show that the self-aggregation of the strain increases with time and tends to be stable at around 52% at 20 h.

[0212] (3) Gastrointestinal fluid tolerance

[0213] Gastric fluid (5M HCl, 2g / L NaCl, 3.2g / L pepsin, pH 2.0) and intestinal fluid (6.8g / L KH2PO4, 10g / L pancreatin, 0.1M NaOH, pH 8.0) were prepared and subsequently sterilized by filtration using a 0.22μm sterile filter. Akk Probio, activated twice in 5mL of BHI liquid medium, was centrifuged at 4000rpm for 10min to collect the cells, washed twice with sterile saline, and resuspended in 5mL of saline. The bacterial solution was mixed with sterile artificial gastric or intestinal fluid at a ratio of 1:1 (v / v), shaken, and then incubated in a constant temperature incubator at 37°C. The viable bacterial count was determined at 0h and 3h, respectively, and the survival rate of Akk Probio in artificial gastric or intestinal fluid was calculated according to formula (1).

[0214] The results are shown in Figures 13-14. Figure 13 shows that the survival rate of the strain in gastric juice showed a downward trend with time, and the survival rate was around 85% at 240 minutes; Figure 14 shows that the survival rate of the strain in intestinal fluid showed an overall downward trend, and the survival rate was above 80% at 240 minutes.

[0215] (4) Biofilm formation ability

[0216] AKK PROBIO was cultured overnight in tryptic soy broth (TSB, Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd., catalog number: HB4114) at 37 °C. Then, bacterial cells were harvested by centrifugation at 8000 rpm for 10 min at 4 °C, washed twice with PBS, and resuspended in the following media: TSB medium without glucose and TSB medium supplemented with 0.25%, 1%, and 2.5% glucose. Three 200 μL aliquots of each bacterial suspension were placed into 96-well plates, and the uninoculated medium served as a negative control. The plates were incubated at 37 °C for 24 h. The medium in each well was aspirated and washed three times with sterile saline to remove unattached cells. The remaining attached cells were fixed with 200 μL of methanol per well. After 15 min, the liquid in the wells was aspirated and air-dried. Then, each well was stained with 200 μL of 2% crystal violet for 5 min. Then, it was washed three times with sterile saline to remove excess dye. After air-drying the plates, the adherent cells were resuspended in 160 μL of 33% (v / v) glacial acetic acid, and the absorbance at 600 nm was measured. The critical value (ODC) was defined as the mean OD value of the negative control. Based on the OD values, the strains were classified as non-biofilm producers (OD ≤ ODC), weak (OD0 < OD ≤ 2 × ODC), moderate (2 × ODC < OD ≤ 4 × ODC), or strong biofilm producers (4 × ODC < OD).

[0217] The results are shown in Table 5:

[0218] Table 5 Biofilm formation of AKK PROBIO

[0219] The data in Table 5 indicate that after incubation at 37 °C for 24 h in TSB containing different concentrations of glucose (0%, 0.25%, 1%, and 2.5%). The critical value (ODC) was defined as the mean OD value of the negative control. Based on the OD values, the strains were classified as non-adherent NA (OD ≤ ODC), weakly adherent WA (ODC < OD ≤ 2 × ODC), moderately adherent MA (2 × ODC < OD ≤ 4 × ODC), and strongly adherent SA (4 × ODC < OD). By measuring the biofilm-forming ability of the strains, the biofilm-forming ability of this strain was determined to be weak.

[0220] (5) Toxicological evaluation

[0221] 5.1 Bacterial reverse mutation test

[0222] The bacterial strains used were Salmonella typhimurium TA97a (National Type Culture Collection Center NTCC, 506391), TA98 (National Type Culture Collection Center NTCC, 506392), TA100 (National Type Culture Collection Center NTCC, 506395), TA1535 (National Type Culture Collection Center NTCC, 506396), and Escherichia coli WP2 uvrA (Qi's Biotechnology Co., Ltd., Catalog No.: 6-1705). To prepare the dosing formulation, a suspension (100 mg / mL) of the test item was prepared using distilled water as a carrier. The test item (strain AKK PROBIO) has the potential to affect the test results in various ways, thereby invalidating the study. Therefore, bacteria in the test item samples were removed by mechanical means. During the mixing process of the formulation, any soluble components of the test item were dissolved in the carrier, and residual bacteria were removed by centrifugation (4°C; 8000 rpm; 10 min). After centrifugation, the supernatant was removed and sterile filtered through a 0.22 μm filter before preparing lower concentration preparations and treating the test bacterial strains.

[0223] Based on the results of the preliminary experiments performed, initial mutation studies (plate incorporation method) and confirmatory mutation studies (pre-incubation procedure) were performed. Confirmatory mutation studies were performed at 15.81, 50, 158.1, 500, 1581, and 5000 μg / plate, with the highest concentration being the maximum concentration recommended by current regulatory guidelines. Positive controls included dixazine, sodium azide, methyl methanesulfonate, and 2-aminofluorene dissolved in distilled water or dimethyl sulfoxide. The viability of the test bacterial cells was checked by plating in each experiment. The test item was considered mutagenic if there was a dose-related increase in the number of revertants and / or; a reproducible, biologically relevant positive reaction occurred in at least one strain with or without metabolic activation for at least one dose group. An increase was considered biologically relevant if the number of reversals was more than twofold higher than the reversal rate of the negative control in Salmonella typhimurium strains TA98, TA100, and Escherichia coli WP2 uvrA; and more than threefold higher than the reversal rate of the negative control in Salmonella typhimurium strains TA97a and TA1535.

[0224] The results are shown in Table 6.

[0225] Table 6

[0226] Note: The drugs for positive controls 1-4 are: dixon, methyl methanesulfonate, sodium azide, and 2-aminofluorene.

[0227] The results of bacterial reverse mutation test showed that the strain was not mutagenic.

[0228] 5.2 Acute toxicity

[0229] To evaluate the acute toxicity of strain AKK PROBIO, SPF Kunming mice (7 weeks old, half male and half female, weighing 10-18 g) were randomly divided into four groups (n=8). Each group was given 1×10 9 , 5×10 10 or 5×10 11 Animals were treated with a suspension of AKK PROBIO (0.5 mL, in saline) containing 100 CFU / day of the strain (CFU / day) or 0.5 mL / day of saline for one day, designated as the sodium chloride solution group (Saline), the low-dose group (Low Dosage), the medium-dose group (Medium Dosage), and the high-dose group (High Dosage). General condition and body weight were observed daily for 7-10 days. At the end of the experimental period, blood samples were collected for hematological and serum biochemical analyses (see Example 4). Histopathological examinations of major organs and tissues were performed on all animals in each group (see Example 4).

[0230] Figure 15 shows changes in mouse weight, food intake, and blood glucose levels. The results indicate that no mice died after acute oral administration of high, medium, or low doses of AKK.pro. There was no significant difference in daily weight change between the test mice and the saline control group. During the acute oral administration period, there was no significant difference in daily food intake between the test mice and the saline control group. There was also no significant difference in daily blood glucose levels between the test mice and the saline control group.

[0231] The results of mouse blood routine tests are shown in Table 7:

[0232] Table 7 Blood routine of mice gavaged with high, medium and low doses of AKK.pro

[0233] The data in Table 7 show that there were no significant differences in the blood parameters between the acute gavage group and the control saline group.

[0234] The results of the mouse triglyceride (TG), cholesterol (TC), and bile acid (TBA) levels are shown in Figure 16. The results showed that liver triglyceride levels in the test mice were significantly lower than those in the control saline group; serum triglyceride levels did not differ significantly between the test and control mice. There were no significant differences in cholesterol and bile acid levels in the serum or liver between the test and control mice.

[0235] There was no significant difference in the glucose (GLU) levels in the serum and liver between the test mice and the control mice, see Figure 17. There was no significant difference in the total protein (TP) levels in the serum and liver between the test mice and the control mice, see Figure 18. There was no significant difference in the alanine aminotransferase (GPT) enzyme activity in the serum and liver between the test mice and the control mice, see Figure 19. There was no significant difference in the aspartate aminotransferase (GOT) enzyme activity in the serum and liver between the test mice and the control mice, see Figure 20. There was no significant difference in the creatinine (GRE) content in the serum between the test mice and the control mice, see Figure 21. There was no significant difference in the urea nitrogen (BUN) concentration in the serum between the test mice and the control mice, see Figure 22.

[0236] The comparison results of organ indexes showed that there was basically no significant difference in the weights of major organs such as the heart, liver, spleen, kidney, thymus, brain, testicles, lungs, stomach and intestines between the test mice and their control group mice.

[0237] Pathological sections of the mouse liver and kidney are shown in Figure 23. There was no significant pathological damage in the liver and kidney of the test mice and the control group mice.

[0238] 5.3 Subchronic toxicity test

[0239] The animals were randomly divided into 4 groups: Group 1 (half male and half female) (normal saline, 0 CFU / kg body weight / day), Group 2 (half male and half female) (9.2×10 8 CFU / kg body weight / day), group 3 (half male and half female) (27.6×10 8 CFU / kg body weight / day) and group 4 (half male and half female) (92×10 8 CFU / kg body weight / day). A fresh bacterial suspension was prepared daily. Oral gavage was performed for 90 consecutive days at a dose volume of 5 mL / kg body weight. Clinical signs were recorded daily, and detailed clinical observations were performed once a week. Body weight, food consumption, and water intake were recorded once a week. At the end of the experimental period, blood samples were collected for hematological and serum biochemical analysis (see Example 4). All animals were autopsied, and the weights of major organs were recorded. Histopathological examinations of major organs and tissues of all animals in each group were performed (see Example 4).

[0240] Mouse glucose tolerance test method:

[0241] Before the experiment begins, mice need to fast for 12-16 hours, but still have access to water. Glucose solution preparation: Prepare glucose solution, generally using 2g / kg body weight of 25% glucose solution. Calculate the amount of glucose required based on the weight of the mouse.

[0242] Determine basal blood glucose: Before the start of the experiment, measure the basal blood glucose level of the mouse using a glucometer or other blood glucose determination method. Usually, the blood is measured in the tail vein of the mouse.

[0243] Glucose loading: Glucose solution was administered to mice via oral gavage.

[0244] Blood glucose determination: At certain time intervals after administration, at 0, 15, 30, 60, 90 and 120 minutes, blood was collected from the tail vein of the mice to measure the blood glucose level of the mice using a blood glucose meter.

[0245] Mouse insulin tolerance test method:

[0246] Insulin Solution Preparation: Prepare an insulin solution of appropriate concentration, typically using insulin injection. Calculate the required insulin dosage for each mouse based on 0.5 U / kg body weight. Insulin Injection: Administer the insulin solution subcutaneously to the experimental group mice. The control group mice do not receive this treatment. Blood Glucose Measurement: Blood glucose levels are measured using a glucometer at intervals of 0, 15, 30, 60, 90, and 120 minutes after administration.

[0247] The results of the experiment showed that no mice died after being orally gavaged with high, medium, and low doses of AKK.pro for 90 consecutive days. There was no significant difference in weekly weight change between the test group and the control saline group. During the continuous gavage period, there was no significant difference in weekly food intake between the test group and the control saline group. During the continuous gavage period, there was a significant difference in weekly blood glucose changes between the mice gavaged with the medium dose and the control saline group, while there were no significant differences between the other groups and the control group (see Figure 24). There were no significant differences in any blood markers between the gavage groups and the control saline group. There were no significant differences in triglyceride and cholesterol levels in the liver and serum between the test mice and the control group. There was a significant difference in bile acid levels in the liver between the mice gavaged with the medium dose and the control group (Figure 25), while there were no significant differences in serum or liver between the other groups and the control group. There were no significant differences in the glucose levels, total protein content, alanine aminotransferase activity, and aspartate aminotransferase activity in the serum and liver between the test mice and the control mice. There were no significant differences in the creatinine content and urea nitrogen concentration in the serum between the test mice and the control mice. There were basically no significant differences in the weights of major organs such as the heart, liver, spleen, kidney, thymus, brain, testicles, lungs, pancreas, stomach, and intestine between the test mice and the control mice. There was no significant pathological damage in the liver and kidneys of the test mice and the control mice. There was no significant difference in the blood sugar regulation ability (glucose tolerance and insulin tolerance) between the test mice and the control mice.

[0248] (6) Drug sensitivity analysis

[0249] The drug susceptibility of AKK PROBIO was determined according to the Clinical and Laboratory Standards Institute (CLSI) method [CLSI. Performance Standards for Antimicrobial Susceptibility Testing; 26th ed. CLSI supplement M100S. Wayne, PA, USA: Clinical and Laboratory Standards Institute; 2016.].

[0250] The quality control strain was Bacteroides fragilis ATCC 25285.

[0251] Main reagents and culture medium:

[0252] Brucella agar and Brucella broth were purchased from BD; anaerobic gas generation bags were purchased from Mitsubishi; sheep blood was purchased from Shanghai Kangrun Biotechnology Co., Ltd.; hemin was purchased from Sinopharm; vitamin K1 was purchased from Sigma; ampicillin, ceftriaxone, cefotaxime, meropenem, and tetracycline were purchased from China National Institute for Food and Drug Control; moxifloxacin and chloramphenicol were purchased from European Pharmacopoeia.

[0253] The susceptibility of AKK PROBIO to ampicillin, ceftriaxone, cefotaxime, meropenem, tetracycline, moxifloxacin, and chloramphenicol was determined using the agar dilution method recommended by CLSI, and the quality control strain Bacteroides fragilis ATCC 25285 was tested using the broth microdilution method recommended by CLSI.

[0254] Results of drug sensitivity test:

[0255] As shown in Table 8, the tested strain AKK PROBIO was sensitive to ampicillin, ceftriaxone, cefotaxime, meropenem, tetracycline, moxifloxacin, and chloramphenicol.

[0256] Table 8 Drug sensitivity test results

[0257] Example 6: Effect of AKK PROBIO on Alzheimer's Disease in APP / PS1 Double Transgenic Mice

[0258] Twenty 12-month-old APP / PS1 double-transgenic mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., batch MJ0324-058) were randomly divided into two groups, APP and APP+AKK, with 10 mice in each group. Ten wild-type C57 BL / 6J mice from the same littermate served as control mice (WT group). All mice were gavage-treated and injected daily for 30 days starting at 12 months of age (gavage conditions are shown in Table 9). After completion of all experiments, blood was collected from the orbital vein and the mice were sacrificed by cervical dislocation. Partial brain, liver, and colon tissues were collected and dissolved in 4% paraformaldehyde. The remaining tissues were stored in a -80°C freezer.

[0259] Table 9 Oral gavage groups and doses

[0260] DDW: double distilled water. Preparation of AKK PROBIO bacterial suspension: take 5mL of AKK PROBIO frozen bacterial suspension for activation culture, centrifuge at 4000r / min for 10min, wash twice, and add 5mL of physiological saline after washing to form a concentration of 1*10 9 CFU / mL of bacterial suspension.

[0261] (1) After the administration, a 4-day Morris water maze experiment was first conducted. The water maze consists of a circular pool (120 cm in diameter) and is equally divided into four quadrants. During the experiment, the mice were moved into the laboratory two hours in advance to familiarize themselves with the environment in advance. The water temperature was adjusted to around 21-22°C, and milk was poured into the pool to make it milky white. Days 1-3 were the training period. The mice were gently placed in the water from the first, second, third, and fourth quadrants facing the direction of the pool wall prompts. The swimming trajectory was recorded for 1 minute. Mice that did not find the platform within the specified time needed to be guided to the platform. Each mouse was trained once in each quadrant per day for 3 consecutive days. Day 4 was the experimental period. Each mouse randomly selected a quadrant and placed it in the pool. The time required to find the platform within 60 seconds, i.e., the escape latency, was recorded. The swimming trajectory of each mouse was recorded using a video acquisition system. If the mouse failed to find the platform within 60 seconds after entering the water, the escape latency was recorded as 60 seconds.

[0262] (2) Determination of Aβ, inflammatory factors, and chemokine levels in hippocampal tissue of AD mice

[0263] Analysis of Aβ protein expression in hippocampal tissue:

[0264] Hippocampal tissue was homogenized in 1 mL of RIPA (Thermofisher, Waltham, MA, USA) and 10 μL of PMSF (Thermofisher) and centrifuged at 12,000 × g for 5 min at 4°C. Protein was quantified using a BCA protein assay kit (Thermofisher). Protein samples were mixed with sample buffer (Thermofisher) in a 4:1 ratio and heated at 95°C for 5 min. The samples were then spotted onto SDS-PAGE gel wells and run at 100 V. Bands from the SDS-PAGE gel were transferred to a PVDF membrane, which was blocked with 5% skim milk for 1 h and then incubated with a primary antibody (Thermofisher) overnight at 4°C. After washing, the membrane was incubated with the primary antibody (Thermofisher) for 1 hour. Chemiluminescence was performed using a Western ECL substrate (Thermofisher) and images were acquired on an iBright (Thermofisher) for subsequent analysis.

[0265] Determination of inflammatory cytokines and chemokine levels:

[0266] Hippocampal tissue was homogenized with PBS, and the supernatant was removed for the experiment. Elisa assay kit instructions were used to measure the levels of IL-4 (interleukin-4, Nanjing Jiancheng Bioengineering Institute, Catalog No. H005-1-2), IL-10 (interleukin-10, Nanjing Jiancheng Bioengineering Institute, Catalog No. H009-1-2), CCL2 (chemokine ligand 2, Nanjing Jiancheng Bioengineering Institute, Catalog No. H318-1), and CXCL1 (CXC chemokine ligand 1, Nanjing Jiancheng Bioengineering Institute, Catalog No. H306-1) in hippocampal tissue.

[0267] (3) Data Analysis

[0268] The measurements of the tissue samples were performed in parallel for three times, and the mean values ​​were calculated. Data were averaged and analyzed using SPSS software (SPSS v.25 for Windows, IBM Software Group, Chicago, IL, USA). Differences between the mean values ​​of the groups were assessed by one-way analysis of variance using Duncan's multiple range test. Differences with p < 0.05 were considered statistically significant.

[0269] (4) Experimental results

[0270] Analysis of Morris water maze results is shown in Figure 26. After four consecutive days of Morris water maze training, the escape latency of mice in each group showed a decreasing trend. Compared with the APP group, mice in the APP+AKK PROBIO group had a shorter escape latency and a significantly shorter path to the platform.

[0271] Analysis of inflammatory markers in AD mice is shown in Table 10. The WT group had the lowest levels of CXCL1 and CCL2 in the hippocampus, while the highest levels of IL-4 and IL-10. The APP group had the lowest levels of IL-4 and IL-10, while the highest levels of CXCL1 and CCL2. In the APP+AKK group, CXCL1 and CCL2 levels decreased compared to the APP group, while IL-4 and IL-10 levels increased compared to the APP group. This suggests that AKK PROBIO can eliminate Aβ by upregulating the levels of the Aβ-degrading enzyme IDE and the anti-inflammatory factors IL-4 and IL-10, thereby alleviating Alzheimer's disease in APP / PS1 double-transgenic mice.

[0272] Table 10 Contents of inflammatory indicators in AD mice

[0273] ad, the same lowercase letters indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups (p<0.05).

[0274] Example 7 Functional Verification of AKK PROBIO in a Gouty Arthritis Mouse Model

[0275] Experimental animals:

[0276] Fifty 6-week-old male BALB / c mice (20-22 g) were purchased from Shanghai Slake Laboratory Animal Co., Ltd.

[0277] Drugs and reagents:

[0278] Sodium urate (2,6,8-trihydroxypurine) and glucosamine sulfate were purchased from Sigma, USA; IL-6, IL-10, IL-1β, and TNF-α ELISA kits were purchased from Thermo Fisher, USA; myeloperoxidase (MPO), superoxide dismutase (SOD), and reduced glutathione (GSH) activity detection kits and malondialdehyde (MDA) content determination kits were purchased from Nanjing Jiancheng Technology Co., Ltd.

[0279] Experimental grouping and sample processing:

[0280] Mice were randomly divided into five groups: normal group, model group, glucosamine sulfate (positive control) group, Akk low concentration experimental group (Akk-L) group, and Akk high concentration experimental group (Akk-H) group, with 10 mice in each group. Mice in the normal group and model group were gavaged with distilled water at a dose of 0.1 mL / 10 g.bw daily, mice in the glucosamine sulfate group were gavaged with glucosamine sulfate at a dose of 195 mg / kg.bw, and mice in the Akk-L and Akk-H groups were gavaged with 10 8 CFU / kg.bw and 10 9 CFU / kg.bw Akk experimental bacteria were gavaged for 7 days. Then all experimental mice were anesthetized with isoflurane, and 50μL of PBS solution was injected into the right tibiotarsal joint (ankle joint) of the normal group of mice. 50mg of sodium urate was added to 1mL of PBS buffer to prepare a suspension. Except for the blank group, 50μL of the suspension was injected into the right tibiotarsal joint (ankle joint) of the other mice to induce gouty arthritis. After the mice woke up, they continued to gavage the corresponding samples for 7 days according to the dose of the samples gavaged in the previous 7 days.

[0281] (1) Determination of oxidative status of ankle joint tissue in mice:

[0282] 0.1 g of mouse ankle joint tissue was weighed and added to liver tissue containing 0.9 mL of normal saline. The mixed tissue sample was then centrifuged at 4000 rpm for 10 minutes to obtain the supernatant. Next, MPO, SOD, GSH, and MDA assay kits were used to measure the relevant indicators in the tissue homogenate supernatant. The results are shown in Table 11:

[0283] Table 11 MPO, SOD, GSH enzyme activities and MDA levels in the ankle joint tissues of mice

[0284] ac, the same lowercase letters indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups (p<0.05).

[0285] (2) Determination of inflammatory cytokines in mouse serum:

[0286] Whole blood samples were collected from mice and centrifuged at 4000 rpm for 10 minutes at 4°C to obtain the upper serum layer. Serum-related inflammatory markers were measured using ELISA kits for IL-6, IL-10, IL-1β, and TNF-α. The results are shown in Table 12:

[0287] Table 12 Cytokine levels of IL-6, IL-10, IL-1β, and TNF-α in mouse serum

[0288] ad, the same lowercase letters indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups (p<0.05).

[0289] The above results show that AKK PROBIO can treat gouty arthritis at low doses, and the therapeutic effect is better at high doses.

[0290] The above embodiments are only used to illustrate the technical solutions of the present invention and are not used to limit the present invention. Those skilled in the art can make substitutions and improvements based on the above disclosed technical solutions without departing from the spirit of the present invention, which are all within the scope of protection of the present invention.

Claims

1. Akkermansia muciniphila, characterized in that The deposit number is CGMCC No.20955.

2. The method for cultivating Akkermansia muciniphila according to claim 1, characterized in that: The method comprises inoculating the Akkermansia muciniphila according to claim 1 into a culture medium for culturing.

3. The culture method according to claim 2, characterized in that The culture medium is a solid culture medium, a semi-solid culture medium or a liquid culture medium.

4. The culture method according to claim 3, characterized in that The culture medium is BHI culture medium; the culture condition is 35-40° C., anaerobic culture.

5. The culture method according to claim 4, characterized in that The culture conditions are 37° C. and anaerobic culture.

6. The preparation of Akkermansia muciniphila according to claim 1, characterized in that The preparation is one or more of live bacteria, dead bacteria, fermentation liquid and freeze-dried powder, and the freeze-dried powder includes the Akkermansia muciniphila bacteria.

7. Use of Akkermansia muciniphila according to claim 1 or the preparation according to claim 6 in the preparation of food, medicine, health care product, microbial preparation or pharmaceutical raw material.

8. A product comprising Akkermansia muciniphila according to claim 1 or a preparation according to claim 6, characterized in that It is food, medicine, health care product, bacterial agent or pharmaceutical raw material.

9. The product according to claim 8, characterized in that The drug further comprises pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include: any one or more of excipients, stabilizers, diluents, adhesives, preservatives, lubricants, and antioxidants.

10. The product according to claim 9, characterized in that The dosage form of the medicine is any one of tablets, powders, granules, capsules, oral liquids, powders, pills, syrups, effervescents, emulsions, pastes, sprays, liniments, suppositories, lotions, foams, and gels.

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