Application of Akkermansia muciniphila Akk09 in preparing element absorption accelerator and its products

Abstract

The invention discloses an application of Akkermansia muciniphila Akk09 in preparing an element absorption promoter and a product thereof, belonging to the field of microbial technology. The invention provides Akkermansia muciniphila ( Farmhouse mucinophila )The invention discloses an application of Akk09 in preparing an element absorption promoter and a drug for treating element deficiency, and further provides Akk09 containing Akk09 and a drug for treating element deficiency based on the application. Akk09 of the present invention can effectively promote the transport of iron ions, zinc ions and selenium ions, and enhance the absorption of iron, zinc and selenium ions.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to application of Akkermansia muciniphila Akk09 in preparing element absorption accelerators and products thereof. Background Art

[0002] Akkermansia muciniphila ( Akkermansia muciniphila Akk bacteria) is an oval, strictly anaerobic, Gram-negative bacterium. It is a resident bacterium in the human intestine. It can grow in the intestinal mucus layer and use the mucin secreted by the host as "food". It settles in the intestine and protects the intestine from pathogens through competitive exclusion. Many studies have shown that Akk is associated with many metabolic diseases in humans. In the study of epithelial tumors, a team from the Gustave Roussy Cancer Institute in Paris reported the role of Akk bacteria in improving patients' immune responses. In addition, studies have also reported the role of Akk in improving inflammatory bowel disease, autism, epilepsy, hypertension and other diseases. Because it is closely related to the physiological state of the body's intestinal ecology, metabolism, nerves, etc., it has a good intestinal prebiotic effect and has a broad development prospect and promotion market. Therefore, many scholars call it the next generation of probiotics.

[0003] However, most of the isolation and screening procedures for Akk strains are cumbersome, the culture medium is complex, and the culture conditions are harsh. Therefore, using a simple and effective method is conducive to obtaining more strains and providing more reserve resources for the functional development and market application of Akk.

[0004] There is currently no report in this field that Akk bacteria can effectively promote the absorption of iron, zinc and selenium. Summary of the invention

[0005] In order to fill the blank of providing a new use of Akk bacteria in promoting the absorption of iron, zinc and selenium, the present invention provides the use of Akkermansia muciniphila Akk09 in preparing element absorption promoters and its products.

[0006] The technical solution of the present invention is as follows:

[0007] Akkermansia muciniphila ( Akkermansia muciniphila ) The use of Akk09 in preparing an element absorption promoter, characterized in that the deposit number of the Akkermansia muciniphila Akk09 is CCTCC NO: M 20242401.

[0008] The element is selected from: iron, zinc, and selenium.

[0009] The invention discloses an application of Akkermansia muciniphila Akk09 for preparing a medicine for treating element deficiency, wherein the deposit number of Akkermansia muciniphila Akk09 is CCTCC NO: M 20242401.

[0010] The element is selected from: iron, zinc, and selenium.

[0011] An element absorption accelerator comprises an active ingredient, wherein the active ingredient includes Akkermansia muciniphila Akk09 with a deposit number of CCTCC NO: M 20242401.

[0012] The element absorption promoter further comprises: an auxiliary agent.

[0013] The element is selected from: iron, zinc, and selenium.

[0014] A medicine for treating element deficiency, comprising a pharmaceutically active ingredient, wherein the pharmaceutically active ingredient comprises Akkermansia muciniphila Akk09 with a deposit number of CCTCC NO: M 20242401.

[0015] The medicine for treating element deficiency also contains: pharmaceutically acceptable excipients.

[0016] The element is selected from: iron, zinc, and selenium.

[0017] The beneficial effects of the present invention are as follows:

[0018] The present invention provides Akkermansia muciniphila with a deposit number of CCTCC NO: M 20242401 ( Akkermansia muciniphila ) The invention discloses an application of Akk09 to prepare an element absorption promoter and a drug for treating element deficiency, and further provides a drug containing Akkermansia muciniphila Akk09 and a drug for treating element deficiency based on the application. Experiments have shown that the Akkermansia muciniphila Akk09 of the present invention has physiological and biochemical characteristics such as utilizing D-galactose, D-glucose, D-fructose, D-mannose, N-acetyl-glucosamine, maltose, lactose, and positive contact enzyme test. The inhibition rate of Akk09 on the adhesion of Escherichia coli to Caco-2 cells reaches 30.15%, the inhibition rate on the adhesion of Staphylococcus aureus to Caco-2 cells reaches 20.50%, and the in vitro inhibition rate of Akk09 on hyaluronidase reaches 29.76%. At the same time, Akk09 is sensitive to multiple antibiotics and is safe for the human body. Most importantly, Akk09 was confirmed to significantly increase the iron, zinc, and selenium ion transport in the Caco-2 monolayer cell model over time compared with the control group, indicating that the Akk09 of the present invention can effectively promote the transport of iron, zinc, and selenium ions and enhance the absorption of iron, zinc, and selenium ions. The deposit information of Akkermansia muciniphila Akk09 of the present invention is as follows:

[0019] Deposit number: CCTCC NO: M 20242401;

[0020] Classification name: Akkermansia muciniphila Akk09;

[0021] Latin name: Akkermansia muciniphila Akk09;

[0022] Depository: China Center for Type Culture Collection;

[0023] Address of depository: Wuhan University, Wuhan, China;

[0024] Date of preservation: October 31, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph of the Akk09 antibiotic sensitivity experiment of Experimental Example 1 of the present invention.

[0026] Figure 2 It is a bar graph of the Akk09 gastrointestinal fluid tolerance test results of Experimental Example 4 of the present invention.

[0027] Figure 3 This is a bar graph showing the experimental results of Akk09 inhibiting pathogen adhesion to Caco-2 cells in Experimental Example 6 of the present invention.

[0028] Figure 4 It is a bar graph of the test results of Akk09 promoting zinc transport and absorption in Experimental Example 9 of the present invention.

[0029] Figure 5 It is a bar graph of the test results of Akk09 promoting iron transport and absorption in Experimental Example 9 of the present invention.

[0030] Figure 6 It is a bar graph of the test results of Akk09 promoting selenium transport and absorption in Experimental Example 9 of the present invention. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below with reference to experimental examples, but the embodiments of the present invention are not limited thereto.

[0032] 1. Sources of biological materials

[0033] (I) The Akkermansia muciniphila Akk007 of Experimental Example 1 of the present invention is derived from the patented strain ZL202311791739.4 and is deposited in the China Center for Type Culture Collection.

[0034] (ii) The Akkermansia muciniphila ATCC BAA-835 of Experimental Example 4 of the present invention was purchased from the China Industrial Microbiological Culture Collection Center.

[0035] (III) Pathogens of Experimental Example 6 of the present invention: Escherichia coli (strain number ATCC25922), Staphylococcus aureus (strain number ATCC25923), and Salmonella (strain number ATCC14028) were purchased from Guangdong Microbiological Culture Collection Center.

[0036] (IV) The human colorectal adenocarcinoma cells Caco-2 used in Experimental Examples 6 and 9 of the present invention were purchased from China Center for Type Culture Collection.

[0037] 2. Reagents and Culture Medium

[0038] Mucin was purchased from Sigma Company's type II porcine gastric mucin, M2378.

[0039] Brain heart infusion broth (BHI) was purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0040] Antibiotic susceptibility tablets were purchased from Hangzhou Binhe Microbiological Reagent Co., Ltd.

[0041] Fetal bovine serum, MEM medium, MEM-α medium, penicillin, streptomycin, 0.25% trypsin, Glutamax, non-essential amino acids, and sodium pyruvate were all purchased from Gibco.

[0042] Group 1 Example: Application of Akk09 in Preparations of the Present Invention

[0043] This group of embodiments provides Akkermansia muciniphila ( Akkermansia muciniphila )Application of Akk09 in preparing element absorption promoter. All embodiments in this group have the following common features: the deposit number of Akkermansia muciniphila Akk09 is CCTCC NO: M 20242401.

[0044] In a specific embodiment, the element is selected from: iron, zinc, and selenium.

[0045] Any Akkermansia muciniphila having the deposit number of the present invention as CCTCC NO: M 20242401 ( Akkermansia muciniphila ) Akk09 is used to promote element absorption, prepare element absorption enhancers, and prepare element supplements. Akkermansia muciniphila ) The act of placing Akk09 into a product packaging box marked with element absorption purpose falls within the protection scope of the present invention.

[0046] Based on the teachings of the present invention, a person skilled in the art may convert Akkermansia muciniphila ( Akkermansia muciniphila) Akk09 is used for the preparation of element absorption promoters and element supplements other than iron, zinc and selenium, for example, the elements include but are not limited to trace elements and essential elements for the human body, the trace elements include but are not limited to: copper, manganese, cobalt, molybdenum, chromium, nickel, vanadium, fluorine, iodine, silicon, tin, etc.;

[0047] The essential elements for the human body include but are not limited to: calcium, sodium, potassium, magnesium, carbon, hydrogen, oxygen, sulfur, nitrogen, phosphorus, chlorine, etc.

[0048] Second group of examples: pharmaceutical application of Akk09 of the present invention

[0049] This group of embodiments provides the use of Akkermansia muciniphila Akk09 for preparing a drug for treating element deficiency. All embodiments in this group have the following common features: the deposit number of Akkermansia muciniphila Akk09 is CCTCC NO: M 20242401.

[0050] In a specific embodiment, the element is selected from: iron, zinc, and selenium.

[0051] Any Akkermansia muciniphila having the deposit number of the present invention as CCTCC NO: M 20242401 ( Akkermansia muciniphila ) Akk09 is used as a drug for the treatment of element deficiency, and Akkermansia muciniphila ( Akkermansia muciniphila ) The act of placing Akk09 into a product packaging box marked with the purpose of treating element deficiency falls within the scope of protection of the present invention.

[0052] Element deficiencies include but are not limited to: iron deficiency, zinc deficiency, selenium deficiency, copper deficiency, manganese deficiency, cobalt deficiency, molybdenum deficiency, chromium deficiency, calcium deficiency, potassium deficiency, sodium deficiency, etc.

[0053] Based on the teachings of the present invention, a person skilled in the art may convert Akkermansia muciniphila ( Akkermansia muciniphila ) Akk09 is used for the preparation of drugs for treating deficiency of elements other than iron, zinc and selenium, for example, the elements include but are not limited to trace elements and essential elements for the human body, the trace elements include but are not limited to: copper, manganese, cobalt, molybdenum, chromium, nickel, vanadium, fluorine, iodine, silicon, tin, etc.;

[0054] The essential elements for the human body include but are not limited to: calcium, sodium, potassium, magnesium, carbon, hydrogen, oxygen, sulfur, nitrogen, phosphorus, chlorine, etc.

[0055] The third group of examples, element absorption promoters of the present invention

[0056] This group of embodiments provides an element absorption promoter. All embodiments in this group have the following common features: the element absorption promoter comprises an active ingredient, and the active ingredient comprises Akkermansia muciniphila Akk09 with a deposit number of CCTCC NO: M20242401.

[0057] In a further embodiment, the element absorption enhancer further comprises: an auxiliary agent.

[0058] In a specific embodiment, the element is selected from: iron, zinc, and selenium.

[0059] In a more specific embodiment, the excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0060] According to the content of the present invention, for different needs in actual production applications, combined with conventional technical means in the field of preparation preparation (for example, "Encyclopedia of Preparation Technology", "Pharmaceutical Preparation Technology", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned excipients, and prepare Akkermansia muciniphila Akk09 with a deposit number of CCTCC NO: M20242401 into different dosage forms, such as powders, tablets, injections, oral liquids, suppositories, gels, patches, sprays, lotions, granules, etc.

[0061] In a specific embodiment, the dosage form of the product is selected from: one or more of powder, tablet, liquid, and capsule.

[0062] The fourth group of embodiments, the drug for treating element deficiency of the present invention

[0063] This group of embodiments provides a drug for treating element deficiency. All embodiments in this group have the following common features: the drug for treating element deficiency contains a pharmacologically active ingredient, and the pharmacologically active ingredient includes Akkermansia muciniphila Akk09 with a deposit number of CCTCC NO: M 20242401.

[0064] In a further embodiment, the drug for treating element deficiency further comprises: pharmaceutically acceptable excipients.

[0065] In a specific embodiment, the element is selected from: iron, zinc, and selenium.

[0066] In a more specific embodiment, the pharmaceutically acceptable excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0067] According to the content of the present invention, for different needs in actual production applications, combined with conventional technical means in the field of drug preparation (for example, "Encyclopedia of Preparation Technology", "Drug Preparation Technology", "Research and Application of Microbial Agent Technology", etc.), those skilled in the art can select and formulate the above-mentioned pharmaceutically acceptable excipients, and prepare Akkermansia muciniphila Akk09 with a deposit number of CCTCC NO: M 20242401 into different dosage forms, such as powders, tablets, injections, oral liquids, suppositories, gels, patches, sprays, granules, etc.

[0068] In a specific embodiment, the dosage form of the drug is selected from: one or more of powder, tablet, liquid, and capsule.

[0069] Experimental Example 1: Isolation and Screening of Akkermansia muciniphila

[0070] A total of 100 stool samples were collected from healthy infants and young children in Hubei Province, mixed evenly with 30% sterilized glycerol water, numbered, and stored in a -80°C refrigerator for later use.

[0071] Vancomycin and streptomycin were selected as antibacterial agents and added into Akk's basal medium (BHI + 0.2% mucin) at a dosage of 0.5-10 µg / mL as screening medium.

[0072] After thawing, the frozen samples were diluted 10 times and inoculated into the screening medium containing antibiotics BHI + 0.2% mucin at a 2% inoculum, and cultured at 37°C for 2 days under anaerobic conditions. Akk-specific primers (the primer sequences are recorded in the article "QiyeWang, Zhaobin Wang, Bohao Shang, et al. Journal of Functional Foods (2024), 1756-4646. Tea polyphenols improve lipid deposition via modulation of gutmicrobiota in rats and Ningxiang pigs") were used to preliminarily screen and identify the enriched fecal samples. The presence or absence of the target band can be used to preliminarily determine whether the enriched samples contain Akk strains, and the enriched tubes identified as positive by PCR are selected for separation and culture on solid plates.

[0073] Dilute and separate the enrichment tubes with positive identification results, and select 10 -1 Up to 10 -5 The gradient was plated on a solid screening medium containing antibiotics and BHI + 0.2% mucin, and cultured at 37°C under anaerobic conditions for 5-7 days. Single colonies suspected of Akk in the antibiotic plate were selected, numbered, identified again using Akk-specific primers, and the positive strains were preserved.

[0074] The present method uses Akk-specific primers to preliminarily screen and identify 100 stool samples enriched in antibiotic culture medium, and obtains 29 positive enrichment tubes. The positive enrichment tubes are diluted and separated, and then plated on a solid screening culture medium containing antibiotic BHI + 0.2% mucin, and single colonies suspected of Akk growing in the antibiotic plate are selected. After identification using PCR-specific primers, 27 Akk strains are obtained, and the strains are numbered and preserved. The efficiency of isolating Akk strains from positive enrichment tubes by this method reaches 93%, which greatly improves the isolation efficiency of Akk strains compared with ordinary strain isolation methods.

[0075] Experimental Example 2: Identification and preservation of Akk09 strain

[0076] The preserved target strains were cultured in liquid, the bacteria were collected, the genomic DNA was extracted, and the universal primers 27F and 1492R recorded in Chinese invention patent 202210478937.4 were used for PCR amplification reaction. The PCR amplification procedure was: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 90 seconds, for a total of 35 cycles, and finally extension at 72°C for 10 minutes. The content and purity of the PCR amplification products were then tested. After passing the test, they were sent to Wuhan Jinkairui Bioengineering Co., Ltd. for sequencing. According to the sequencing results, the BLAST tool in the NCBI database was used for homology comparison, and the 16S rRNA gene sequence homology ≥99% was used as the identification standard. One of the strains was found to have excellent physiological and biochemical characteristics, resistance to gastric juice, resistance to bile salts and cell adhesion, as well as a strain that promotes the transport and absorption of the nutrients iron, zinc and selenium. It was identified as Akkermansia muciniphila ( Akkermansia muciniphila ), named it Akk09 and sent it to China Center for Type Culture Collection for preservation. Its preservation information is as follows:

[0077] Deposit number: CCTCC NO: M 20242401;

[0078] Classification name: Akkermansia muciniphila Akk09;

[0079] Latin name: Akkermansia muciniphila Akk09;

[0080] Depository: China Center for Type Culture Collection;

[0081] Address of depository: Wuhan University, Wuhan, China;

[0082] Date of preservation: October 31, 2024.

[0083] Experimental Example 3: Akk09 Physiological and Biochemical Experiment

[0084] The physiological and biochemical experimental process of Akk09 strain can refer to the operating steps recorded in patent ZL202210317985.5 "Use of Bacillus coagulans BC99 in preparing products to relieve colitis or immunomodulatory products". The experimental results are as follows: Akk09 strain can utilize D-galactose, D-glucose, D-fructose, D-mannose, N-acetyl-glucosamine, maltose, and lactose. The catalase test is positive.

[0085] Experimental Example 4: Akk09 artificial gastric juice and bile salt tolerance test

[0086] Simulated artificial gastric juice: prepare 0.5% sodium chloride solution, adjust the pH to 3.0 with 1 mol / L HCl, add 0.3% pepsin, and filter with a 0.22 μm microporous filter membrane to sterilize after fully dissolving.

[0087] Bile salt solution: Weigh 0.2% sodium thioglycolate and 0.3% porcine bile salt and add them to BHI liquid culture medium. After fully dissolved, sterilize in an autoclave at 115°C for 20 min for later use.

[0088] Akkermansia muciniphila Akk09 was cultured in BHI + 0.2% mucin liquid medium at 37°C overnight. The activated Akk09 bacterial solution was centrifuged, the supernatant was discarded, the cells were collected, and the bacterial solution concentration was adjusted to 10 8 CFU / mL. Take 10mL of bacterial suspension and centrifuge. After collecting the bacteria, add them into 10mL of prepared simulated gastric juice and bile salt solution with pH 3.0, mix them, keep them at 37℃, and take the suspension at 0h and 3h to detect the number of live bacteria and calculate the survival rate.

[0089] The survival rate of the strain was calculated according to the following formula:

[0090] ;

[0091] Where N0 represents the number of viable bacteria (CFU / mL) of the strain at 0 h, and Nt represents the number of viable bacteria (CFU / mL) of the strain after 3 h of treatment.

[0092] The experimental results are as follows Figure 2 As shown in the results, after treatment at 37℃ for 3h in gastric juice pH 3.0, the survival rate of live bacteria of Akkermansia muciniphila Akk09 was as high as 98.1%, and after treatment at 37℃ for 3h in bile salt solution, the survival rate was 63.5%, which was much higher than that of the standard strain Akkermansia muciniphila ATCC BAA-835. This indicates that Akkermansia muciniphila Akk09 has a strong tolerance to artificial gastric juice and bile salt solution, can pass through the gastrointestinal tract smoothly, and can effectively play a probiotic function after entering the body.

[0093] Experimental Example 5: Study on the surface characteristics of Akk09 cells

[0094] 1. Take the Akk09 glycerol tube strain and activate the second generation, then inoculate it into the anaerobic tube at a 5% inoculation rate, culture it anaerobically at 37℃ for 16-18h, centrifuge it at 10000r / m for 5min, wash it twice with PBS, adjust the OD600 value of the bacterial solution to 1, and test the OD600 again, and record the OD600 value A0; take 3mL of the prepared bacterial suspension and 1mL of xylene and vortex mix them for 3min respectively, and the buffer solution is the blank control; after standing for 20min, measure the OD600 value of the lower water layer A1.

[0095] 2. The activated Akk09 bacterial suspension was treated at 121℃ for 20min to inactivate, centrifuged and washed with PBS as mentioned above, and the OD600 value of the bacterial suspension was adjusted to 1; the adjusted OD was tested again, and the OD600 value A0 was recorded; 3mL of the prepared bacterial suspension and 1mL of xylene were vortexed and mixed for 3min, and the buffer solution was used as a blank control; after standing for 20min, the OD600 value of the lower water layer was measured to be A1.

[0096]

[0097] The stronger the surface hydrophobicity of the strain, the stronger the adhesion ability of the bacteria to the intestinal mucosa, the more effective it can colonize the intestine and exert its probiotic effect. As shown in Table 1, the hydrophobicity of live Akk09 cells is 86.20%, and the hydrophobicity of the inactivated Akk09 strain reaches 81.32%, indicating that the Akk09 strain can not only colonize in the intestinal epithelial cells in an active state, but also the inactivated Akk09 bacteria can adhere to the intestinal mucosa, reducing the adhesion of pathogens from the perspective of steric hindrance, and playing a role in protecting the body.

[0098] Table 1. Akk09 hydrophobicity experimental results

[0099] Akk09 Hydrophobicity (%) Active strains 86.20±1.51 Inactivated strains 81.32±1.33

[0100] Experimental Example 6: Experiment on the inhibition of pathogen adhesion of Akk09 to Caco-2 cells

[0101] Active material preparation:

[0102] Cell culture: The revived Caco-2 cells were inoculated into 12-well cell culture plates supplemented with DMEM medium supplemented with 10% (v / v) fetal bovine serum. The cell addition amount was 2.4×10 cells per well. 5 cell / mL, 5% CO2, 37℃ constant temperature culture until 80% of the cells adhered to the wall, washed with PBS three times and then set aside.

[0103] Preparation of Akk09 bacterial suspension: The secondary activated Akkermansia muciniphila Akk09 was inoculated into liquid BHI + mucin medium at a 5% inoculum volume, and cultured overnight at 37°C to obtain bacterial suspension. 1 mL of bacterial suspension was collected by centrifugation at 10,000 rpm for 3 min at room temperature, and the cells were washed twice with an equal amount of sterile PBS. DMEM medium was added to the precipitate after centrifugation to adjust the cell concentration to 10 9 CFU / mL to obtain the standby bacterial suspension.

[0104] Preparation of pathogenic bacteria liquid: Take the cultured Staphylococcus aureus, Escherichia coli, and Salmonella liquid, centrifuge at 5000r / m for 5 min at room temperature to collect the bacteria, wash them twice with sterile PBS, and finally resuspend them in DMEM medium and adjust the bacterial concentration to 10 8 cfu / mL;

[0105] Experiment on inhibiting pathogen adhesion to Caco-2 cells:

[0106] 1. Competition test: remove the prepared Caco-2 cell monolayer culture medium, add PBS buffer to rinse twice, remove the buffer, add 0.8 mL of DMEM to each well, and then add 0.1 mL of Akk09 bacterial suspension (1×10 8 cfu / well) and 0.1 mL of pathogen suspension (1×10 7 cfu / well), mixed and incubated for 90 min at 5% CO2 and 37°C. The control group used 0.1 mL of DMEM instead of probiotics. After the incubation, the Caco-2 monolayer cells were washed three times with 1 mL of sterile PBS solution to remove the bacteria that were not bound to the cells. The cells were then digested with EDTA trypsin solution, and the digested cells were graded diluted. The viable bacteria of the pathogens were counted using LB medium and cultured for 48 hours.

[0107] 2. Rejection test: Remove the culture medium of the Caco-2 cell monolayer, add PBS buffer to rinse twice, remove the buffer, add 0.9 mL of DMEM to each well, and then add 0.1 mL of probiotic suspension (10 8 cfu / well), after mixing, incubate at 5% CO2 and 37°C for 90 minutes, wash twice with 1 mL of PBS to remove the non-adherent probiotics, add 0.9 mL of DMEM to each well, and then add 0.1 mL of pathogen suspension, mix well, incubate at 5% CO2 and 37°C for 90 minutes, and replace the probiotics with 0.1 mL of DMEM in the control group, and other operations remain unchanged. After the incubation, wash the Caco-2 monolayer cells three times with 1 mL of sterile PBS solution to remove the bacteria that are not bound to the cells, and then digest the cells with trypsin solution containing EDTA, dilute the digested cells in a gradient manner, and use LB medium to count the viable bacteria of the pathogens, and culture for 48 hours;

[0108] 3. Displacement test: The steps are the same as the rejection test, except that in the displacement test, the pathogens adhere first and then the probiotics adhere;

[0109] Calculation formula:

[0110] like Figure 3As shown in the data, when Akk09 was subjected to competition experiment, exclusion experiment and displacement experiment with three different pathogens, the exclusion experiment had the best effect. Among them, the inhibition rate of Akk09 on the adhesion of Escherichia coli to Caco-2 cells reached 30.15%, and the inhibition rate of Akk09 on the adhesion of Staphylococcus aureus to Caco-2 cells reached 20.50%, indicating that Akkermansia muciniphila Akk09 can effectively inhibit the adhesion of Staphylococcus aureus and Escherichia coli to intestinal epithelial cells and reduce the invasion of pathogens on the intestine.

[0111] Experimental Example 7: Akk09 inhibits hyaluronidase activity in vitro

[0112] Strain activation: Activate the Akk strain according to the method of Experimental Example 5 for later use.

[0113] In vitro inhibition of hyaluronidase activity experiment: add 0.1mL 2.5mmol / L CaCl2 and 0.5mL 600U / mL hyaluronidase to the test tube, treat at 37℃ for 20min; add 0.5mL of test bacteria suspension, treat at 37℃ for 20min; add 0.5mL 0.5mg / mL sodium hyaluronate solution, treat at 37℃ for 30min; let stand at room temperature for 5min, add 0.5mL acetylacetone solution and 0.1mL NaOH solution, boil in water bath for 15min, and immediately ice bath for 5min; add 1mL Ehrlich reagent (weigh 0.8g of p-dimethylaminobenzaldehyde dissolved in 15mL concentrated hydrochloric acid and 15mL anhydrous ethanol), develop at room temperature for 20min, and measure its absorbance at a wavelength of 530nm.

[0114] Calculation formula:

[0115] Wherein: A is the absorbance of the control group (acetate buffer replaces the bacterial suspension); B is the absorbance of the blank group (acetate buffer replaces the enzyme solution and bacterial suspension); C is the absorbance of the experimental group; D is the absorbance of the sample blank group (acetate buffer replaces the enzyme solution).

[0116] Hyaluronic acid exists naturally in the human body as a biopolymer, mainly in connective tissues such as joints and skin. It can lubricate joints, promote fluid movement and reduce bone friction to protect joint health. In the treatment of osteoarthritis, the synergistic effect of hyaluronic acid can relieve pain, improve joint function, reduce tendon lesions and inflammation, and even promote the repair of meniscus lesions. Excessive activity of hyaluronidase can lead to the degradation of hyaluronic acid, which leads to the occurrence of inflammation. The degradation of hyaluronic acid was evaluated by the hyaluronidase inhibition rate as an indicator. The greater the hyaluronidase inhibition rate, the stronger the anti-inflammatory activity. As shown in Table 2, the in vitro inhibition rate of Akk09 on hyaluronidase reached 29.76%, indicating that the inhibitory effect of Akk09 strain on hyaluronidase reduced the degradation of hyaluronic acid, thereby protecting bone and joint health, relieving joint pain, improving joint function, and enhancing the rehabilitation of damaged muscles and bones.

[0117] Table 2. In vitro inhibition rate of hyaluronidase

[0118] strain Inhibition rate (%) Akk09 29.76±1.28

[0119] Experimental Example 8: Antibiotic Sensitivity Test

[0120] The antibiotic sensitivity test was performed using the drug sensitivity paper agar diffusion method. After the bacteria to be tested were activated, a bacterial suspension was prepared and the concentration of the bacterial suspension was adjusted to 10 8 CFU / mL, take 200μL of bacterial suspension on the surface of BHI solid plate, evenly spread the bacterial solution on the plate with a sterile cotton swab, stick the antibiotic sensitive paper on the BHI solid medium with sterile tweezers, mark it, and culture it at 37℃ for 120h. Use a vernier caliper to measure the diameter of the inhibition zone. The edge of the inhibition zone is defined as the boundary when Akk09 cannot be seen to the naked eye. The sensitivity results of Akkermansia muciniphila Akk09 strain to 8 commonly used antibiotics are shown in Table 3. Akkermansia muciniphila Akk09 is sensitive to all 8 antibiotics, indicating that the strain is safe and has the potential to be used in biological products or dietary supplements.

[0121] Table 3. Susceptibility of Akkermansia muciniphila Akk09

[0122] Serial number antibiotic Diameter of inhibition zone (mm) Sensitivity 1 Erythromycin 18.33±1.23 S 2 Streptomycin 0.00±0.00 R 3 penicillin 52.00±0.50 S 4 Ampicillin 53.00±0.87 S 5 Polymyxin B 33.00±0.92 S 6 Ceftriaxone 41.67±1.99 S 7 tetracycline 43.20±0.90 S 8 Vancomycin 12.67±0.58 R 9 Ceftriaxone 30.66±0.67 S 10 Oxacillin 16.49±0.45 S

[0123] Experimental Example 9: Iron, zinc, and selenium transport and absorption test

[0124] Caco-2 monolayer cell model was established by using complete cell culture medium containing 77% MEM, 20% fetal bovine serum, 1% double antibody, 1% Glutamax, 1% non-essential amino acids and 1% sodium pyruvate (all ratios are volume fractions) at 37°C and 5% CO2 concentration. When the cells grew to 80%, 2×10 5The cells were inoculated into the upper chamber of a 12-well transwell plate at a density of 0.5 mL / mL. The upper chamber was supplemented with 1.5 mL of complete medium. The culture was continued for 21 days, and the medium was changed every 2 days until the cells formed a complete membrane. The integrity of the cell monolayer was determined by measuring the transmembrane resistance. When the resistance exceeded 500 cm 2 When , the monolayer membrane was used for transport experiments.

[0125] Iron, zinc, and selenium transport assay: After culturing Caco-2 cells for 21 days, the culture medium was discarded, and the monolayer cells were immediately washed twice with 37°C HBSS (balanced salt without iron and zinc), then transferred to a new 12-well plate and incubated with HBSS in an incubator for 30 minutes. The two groups of samples were added to the upper layer of the transwell chamber respectively. For the iron test group, 100 μg / mL FeSO4 solution was added to each well. 9 CFU / mL Akk09 mixed solution 0.5mL; zinc test group: add 100μg / mL ZnSO4 solution resuspended 10 9 CFU / mL Akk09 mixed solution 0.5mL; Selenium test group: add 10 CFU / mL sodium selenite solution resuspended in each well 9 CFU / mL Akk09 mixed solution 0.5mL; iron control group: 100µg / mL FeSO4 solution 0.5mL; zinc control group: 100µg / mL ZnSO4 solution 0.5mL; selenium control group: 20µg / mL sodium selenite solution 0.5mL. At 60, 90, and 120min, 0.5mL HBSS buffer was collected from the lower chamber to measure the concentrations of iron ions, zinc ions, and selenium ions, and 0.5mL fresh HBSS buffer was added to maintain a constant volume. The concentrations of iron ions, zinc ions, and selenium ions were determined using an atomic absorption spectrophotometer.

[0126] The formula for calculating the transport rate is:

[0127] Iron is an important component of hemoglobin, myoglobin, cytochrome and respiratory enzymes in animals. Iron deficiency can lead to anemia, decreased immune function and other problems. Zinc is one of the essential trace elements for the human body and an important nutrient. More than 100 enzymes in the human body contain zinc. It plays an extremely important role in the growth and development of the human body. It is hailed as the "flower of life" and "source of intelligence" and plays an important role in maintaining the health of the body. Zinc deficiency can cause corresponding diseases, such as picky eating, anorexia, hair loss, and decreased immunity. Selenium is an essential trace element for maintaining normal growth and metabolism of life. Selenium cannot be stored for a long time in the human body and cannot be synthesized. Selenium must be obtained through food to meet the needs of the body. Appropriate amounts of selenium can delay aging, inhibit cancer and fight cancer, and remove excessive reactive oxygen free radicals in the body. The spontaneous differentiation of the Caco-2 cell line shows the typical structural and functional characteristics of human small intestinal epithelial cells. The Caco-2 cell model is widely used in simulated food and drug absorption research. The present invention adds Akk09 and FeSO4, ZnSO4, and Na2O3Se mixtures to the upper layer of the transwell chamber, and measures the content of iron, zinc, and selenium in the lower layer. The wells with only FeSO4, ZnSO4, and Na2O3Se added are used as controls to evaluate the ability of Akk09 to promote the absorption of iron, zinc, and selenium. The transport experiment results of the Caco-2 monolayer cell model are shown in Figure 2. Figure 4 , Figure 5 , Figure 6 As shown in the figure, as the transport time increases, the transport amount of iron, zinc and selenium ions in the control group and the experimental group increases. At 60min and 90min, the zinc transport rate in the experimental group was significantly higher than that in the control group ( P <0.05), and at 120 minutes, the zinc transport rate of the experimental group was still higher than that of the control group. Similarly, Akk09 also showed a significant promoting effect on iron ions at 60 minutes and 90 minutes ( P <0.05), and it tended to be flat at 120 minutes. Compared with the control, the transport amount of the test group was higher than that of the control group. The selenium test group also showed the same trend. This shows that Akk09 can promote the transport of iron ions, zinc ions, and selenium ions and enhance the absorption of iron, zinc, and selenium ions.

Claims

1. Akkermansia muciniphila Akkermansia muciniphila )Application of Akk09 in preparing element absorption promoter, characterized in that: The deposit number of the Akkermansia muciniphila Akk09 is CCTCC NO: M 20242401; the element is selected from: iron, zinc, and selenium.

2. The use of Akkermansia muciniphila Akk09 for preparing a drug for treating element deficiency, characterized in that: The deposit number of the Akkermansia muciniphila Akk09 is CCTCC NO: M 20242401; the element is selected from: iron, zinc, and selenium.

3. An element absorption accelerator, characterized in that: The invention comprises active ingredients, wherein the active ingredients include Akkermansia muciniphila Akk09 with a deposit number of CCTCC NO: M 20242401; and the elements are selected from the group consisting of iron, zinc and selenium.

4. The element absorption promoter according to claim 3, characterized in that: Also contains: adjuvants.

5. A drug for treating element deficiency, characterized in that: The invention contains pharmacologically active ingredients, wherein the pharmacologically active ingredients include Akkermansia muciniphila Akk09 with a preservation number of CCTCC NO: M 20242401; and the elements are selected from the group consisting of iron, zinc and selenium.

6. A drug for treating element deficiency according to claim 5, characterized in that: Also contains: pharmaceutically acceptable excipients.

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